>> MY NAME IS ALAN SCHECHTER AND I'D LIKE TO WELCOME YOU ALL TO THIS YMPOSIUM THAT WAS DESIGNED TO ACCOMPANY THE RECENT OPENING OF AN EXHIBIT DEDICATED TO THE LIFE AND WORK OF CHRIS ANFINSEN. THAT EXHIBIT IS IN THE MAIN CORRIDOR OP SIEVE OPPOSITE THE BOOK STORE AND I HOPE ALL OF YOU WILL VISIT THAT SOMETIME THIS AFTERNOON OR IN THE NEAR FUTURE. BUT WE TRIED TO -- FOR THE DAY, HOWEVER, WE'VE TRIED TO HAVE SCIENTIFIC PRESENTATIONS SHOWING THE CONTINUED IMPORTANCE OF CHRIS ANFINSEN'S WORK, AND YOU'LL HEAR MORE ABOUT THAT IN THE MAIN TALKS IN THE EARLY PART OF THIS AFTERNOON. BUT BEFORE, I'D LIKE TO THANK GRIFF RODGERS, WHO'S DIRECTOR OF NIDDK, THE PRIME SPONSORING INSTITUTION FOR THIS EVENT AND FOR THE EXHIBIT TO MAKE SOME REMARKS WELCOMING. GRIFF IS AN ALUMNUS OF CHRIS ANFINSEN'S LABORATORY. >> THANK YOU, ALAN, AND I'M CERTAINLY PLEASED TO BE HERE TODAY TO WELCOME YOU ON BEHALF OF NIH AND NIDDK, AS ALAN MENTIONED, I'M THE DIRECTOR OF NIDDK, IT'S ADMINISTERED BY NHLBI, AND I SAY THAT FOR A PURPOSE THAT WILL BECOME OBVIOUS TO YOU IN A MOMENT. I'M PLEASED TO BE HERE TO MARK THE THIS SPECIAL ACHIEVEMENT AND COMMEMORATION FOR CHRIS ON HIS SCIENTIFIC LIFE AND ALL OF HIS ACHIEVEMENTS. OBVIOUSLY CHRIS WAS A WINNER OF THE NOBEL PRIZE IN CHEMISTRY IN 1972, AND I WANT TO THANK OUR COLLEAGUES FROM NHLBI, WHERE DR. ANFINSEN WORKED, FROM 1950 TO 1962 FOR CO-SPONSORING THIS EVENT. AS DR. SCHECHTER MENTIONED, EARLIER THIS YEAR WE HELD A DEDICATION OF A HISTORICAL EXHIBIT OF ANFINSEN'S CAREER. IT'S ON DISPLAY IN THE CENTRAL CORRIDOR, NOT VERY FAR FROM HERE, SO AS DR. SCHECHTER MENTIONED, I HOPE THAT YOU'LL HAVE AN OPPORTUNITY WHILE YOU'RE HERE TO VISIT THE THAT VISIT THAT DISPLAY RIGHT AFTER TODAY'S IMPORTANT SYMPOSIUM. I FIRST MET CHRIS IN THE SPRING OF 1981, WHEN I WAS A SECOND-YEAR MEDICAL RESIDENT AT WASHINGTON UNIVERSITY. I WAS INTERVIEWING FOR A RESEARCH POSITION THE NEXT YEAR TO WORK WITH DR. SCHECHTER BEGINNING IN 1982, AND BACK THEN, AS A PROCESS, NOT ONLY DID YOU MEET WITH THE SECTION CHIEF BUT YOU ALSO HAVE TO PASS MUSTER WITH THE LAB CHIEF. AND AS I RECALL, I HAD MY INTERVIEW WITH CHRIS WHICH LASTED MAYBE 10, AT MOST 15 MINUTES, THE FORMAL INTERVIEW, BUT THEN THE NEXT 30 TO 45 MINUTES, I SAT DOWN WITH CHRIS AND HE GAVE ME SOME RATHER AVUNCULAR ADVICE ON HOW TO SUCCEED IN SCIENCE AS A PHYSICIAN. AND THERE WERE A FEW THINGS THAT CHRIS TAUGHT ME AT THE TIME DURING THAT CONVERSATION, BUT I REALLY LATER BEGAN TO APPRECIATE MORE AND MORE AFTER I FORMALLY JOINED NIDDK IN 1982. IT'S IMPORTANT TO POINT OUT THAT CHRIS WAS VERY INSTRUMENTAL IN THE CREATION OF THE IN-HOUSE FOUNDATION FOR THE ADVANCED EDUCATION IN THE SCIENCES, OR FAES AS WE KNOW IT HERE TODAY. THE TASK AT THE TIME WAS MAKING THE NIH MORE LIKE A UNIVERSITY THAN MOST OTHER GOVERNMENT AGENCIES. THIS WAS PARTICULARLY NEEDED BACK IN THE 60s, GIVEN THE LARGE NUMBER OF PHYSICIANS COMING TO THE NIH AT THAT TIME TO DO BOTH THEIR RESEARCH TRAINING AS WELL AS THEIR CLINICAL TRAINING IN AN ERA BEFORE THERE WAS A GENERAL AVAILABILITY OF MD/PH.D. PROGRAMS. THE SECOND THING I LEARNED WAS THAT CHRIS, WHO LEFT NHLBI IN '62 BUT WAS THEN HIRED BACK TO NIH AND SPECIFICALLY TO NIAMD, WHICH WAS A PRECURSOR OF NIDDK, BY OUR SCIENTIFIC DIRECTOR AT THE TIME, JOSEPH ED RAHL. RAHL AND CHRIS BECAME VERY GOOD FRIENDS, AND THEY BOTH FOSTERED VERY INTERACTIVE ACTIONS THAT SERVED THEM BOTH QUITE WELL IN TERMS OF CONTRIBUTING TO THE STRENGTHS OF THEIR RESEARCH PROGRAMS. THEY BOTH HAD A FOND APPRECIATION FOR MUSIC AND THE ARTS THAT THEY OFTEN SHARED. IN FACT, WHEN THIS EXHIBIT WAS OPENED JUST A FEW MONTHS AGO, IT WAS THE DAY BEFORE THE INAUGURAL JOSEPH ED RAHL CULTURAL EVENT WHICH IS NOW AN ANNUAL EVENT, AND AT THAT EVENT, WE HAD NONE OTHER THAN BARBARA STREISAND TO GIVE THE CULTURAL LECTURE. THIS WAS GREAT FOR ME BECAUSE I GOT A CHANCE TO MEET HER AND SUBSEQUENTLY ACTUALLY THIS MONTH, SHE HAS LENT HER VOICE TO A TOPIC THAT'S OF GREAT INTEREST BOTH TO NIDDK AND NHLBI. SHE HAS AN INTEREST IN THE ADVERSE EFFECTS OF HEART DISEASE, PARTICULARLY IN WOMEN, AND WHICH SHE IS TALKING ABOUT IN THIS CURRENT MONTH IS HEART DISEASE IN WOMEN, PARTICULARLY THOSE WOMEN WITH DIABETES. AND SO AGAIN, ANOTHER THING THAT I CAN DIRECTLY THANK CHRIS FOR. I WOULD SAY ON A WHOLE, CHRIS WAS PROBABLY MORE SUCCESSFUL IN TRAINING PHYSICIANS THAN IN TRAINING BASIC SCIENTISTS. SOME OF WHOM I'VE GOTTEN TO KNOW QUITE WELL SINCE MY TIME HERE, FREDERICK SON, GIL OMAN, HENRY EPSTEIN, DAVID SACHS, BRUCE FEWERY AND, OF COURSE, ALAN SCHECHTER, WHO'S BEEN PAYING IT FORWARD IN A WAY FOR THE LAST 35 YEARS. LET ME ONCE AGAIN THANK YOU, WELCOME YOU TO NIH, NIDDK. PLEASE ENJOY THE SYMPOSIUM, AND NOW LET ME TURN THE PODIUM BACK OVER TO MY COLLEAGUE AND MY MENTOR, DR. ALAN SCHECHTER. THANK YOU. [APPLAUSE] >> THANK YOU, GRIFF. I SHOULD ALSO ADD THAT FRANCIS COLLINS SENT A NICE HANDWRITTEN NOTE EXPRESSING HIS SADNESS NOT BEING HERE, BEING ABLE TO BE HERE. HE HAD ARRANGED TO TALK BEFORE A GENETICS GROUP BEFORE THIS WAS SCHEDULED. THIS IS ONE OF MY FAVORITE PHOTOGRAPHS OF CHRIS ANFINSEN. SOME OF YOU HERE KNEW HIM, SOME OF YOU WERE FORTUNATELY RELATIVES OF HIM, BUT I THINK THE NUMBER OF PEOPLE WHO WORKED WITH HIM AND KNEW HIM IS UNFORTUNATELY -- CONTINUES TO DECREASE AND VERY GLAD THAT WE'RE ABLE TO MAKE THIS SYMPOSIUM NOW, WHEN THERE'S STILL A FAIR NUMBER OF INDIVIDUALS HERE AND AROUND WHO CAN PERSONALLY IDENTIFY WITH HIS PERSONALITY AND HIS SCIENCE. THE EXHIBIT THAT IS THE EXCUSE FOR THIS SYMPOSIUM, AS GRIFF MENTIONED, IS IN THE THE FIRST -- THE MAIN CORRIDOR OF THE FIRST FLOOR, AND AS GRIFF SAID, I HOPE YOU GO PAST IT TODAY. THANKS TO HANK GRASSO, THERE ARE FLOURS SURROUNDING IT RIGHT NOW, IT LOOKS EVEN A LITTLE BETTER, AND THIS EXHIBIT WAS JOINED BY ONE FROM MIKE POTTER WHEN THEY WERE INSTALLED IN LATE SPRING OF THIS YEAR, THEY JOINED THE ONES FROM MARSHALL NURENBURG JUST OUTSIDE THIS AUDITORIUM, ONE FOR MARTY RODBELT AND A FEW OTHERS THAT THE OFFICE OF NIH HISTORY STARTING WITH THE WORK OF VICKIE HARDEN HAVE CREATED TO HONOR NIH'S HISTORY, AND IT'S IN PARTICULAR THE WORK OF HANK GRASSO AND MICHELLE LYONS, TWO STALWARS OF THE HISTORY OFFICE, WHO PUT TOGETHER THE PICTURES AND THE TEXT FOR THIS EXHIBIT. THIS IS THE TITLE THAT I GAVE TO THE SYMPOSIUM TODAY: "THE LEGACY OF CHRISTIAN B. ANFINSEN AND HIS THERMODYNAMIC HYPOTHESIS, FROM TOTAL CHEMICAL SYNTHESIS TO THE FOLDING OF PROTEINS IN AND OUT OF THE CELL." THE POINT THAT I GRADUALLY HAVE BECOME AWARE OF IN THE LAST 20 OR 30 YEARS IS THAT HIS WORK, UNLIKE THAT, TO BE HONEST, OF MANY OTHER NOBEL PRIZE WINNERS, IS CONTINUING TO ATTRACT INTEREST AND DEVELOPMENT OF HIS IDEAS IN NEW SCIENCE, AND WHAT I'VE TRIED TO DO IN THE THREE MAIN TALKS TODAY IS HIGHLIGHT THE NEW SCIENCE THAT HAS BEEN BASED UPON CHRIS ANFINSEN'S WORK OF 60 YEARS AGO. THIS WAS MY SUBJECTIVE IMPRESSION, BUT MY FRIENDS IN THE NIH LIBRARY, JOSH AND CHRIS BETTA, PUT TOGETHER A PLOT FOR ME OF THE CITATIONS TO CHRIS' WORK. THESE ARE THE CITATIONS OF THE FOREMOST CITED PAPERS, AND WHAT'S VERY INTERESTING, TWO PAPERS OF METHODOLOGICAL INTEREST, THIS ONE ON 2D ELECTROPHORESIS AND THIS ONE ON AFFINITY CHROMING TOGRAPHY WERE HEAVILY CITED IN THEIR TIME, AND I REMEMBER THE ENTHUSIASM FOR AFFINITY CHROMING TOGRAPHY AT THAT TIME, BUT CITATIONS TO 1973 NOBLE LECTURE AS PUBLISHED IN SCIENCE HAVE CONTINUED TO INCREASE, COMING DOWN THE LAST COUPLE YEARS TO A VALUE OF CLOSE TO 200 CITATIONS PER YEAR. IF YOU MAKE A COMPARISON TO HALF A DOZEN OTHER NOBEL PRIZE WINNERS FROM THE TIME, THIS IS UNIQUE AS FAR AS I CAN TELL, RARELY ARE PAPERS CITED MORE THAN A FEW TIMES, THEN RAPIDLY DIMINISH AFTER FIVE OR 10 YEARS. I HAVE SOME THEORIES, I'M INTERESTED IN PEOPLE'S THOUGHTS ABOUT THIS. ALSO THE GRAY LINE IS THE FIRST KINETIC PAPER PUBLISHED BY ANFINSEN'S GROUP AND IT'S BEGUN TO INCREASE IN NUMBERS AND I THINK THIS IS THE BASIS OF THE PDI STUDIES THAT YOU'LL HEAR FROM BRUCE FURY. SO MY SUBJECTIVE IMPRESSION THAT THE WORK 60 YEARS AGO STILL LIVES ON AS SCIENCE, I THINK IS CONFIRMED BY THESE DATA IF YOU ACCEPT MY INTERPRETATION. IN ANY CASE, THE SYMPOSIUM TODAY, STEPHEN KENT, UNIVERSITY OF CHICAGO, TALKING ABOUT TOTAL CHEMICAL SYNTHESIS OF PROTEINS, WHICH WAS ONE OF THE PRIME GOALS OF ANFINSEN'S WORK ON REFOLDING IN VITRO, BRUCE FURIE, AN ALUMNUS OF THE LAB, TALKING ABOUT HIS WORK WITH THE PDI ENZYME, AND THEN ARTHUR HORWICH OF YALE, WHO'S DONE VERY ELEGANT WORK ON CHAPERONINS AND HOW THEY RELATE TO THE FOLDING THAT ANFINSEN STUDIED IN VITRO IN THE CELL. THEN WE'LL HAVE HALF A DOZEN COMMENTS BY ALUMNI OF LABORATORY FROM ABOUT 3:15 TO -- AND INCLUDING MYSELF TO ABOUT 4:30, THEN A RECEPTION IN THE TERRACE LOUNGE FOR ALL THOSE WHO ARE ABLE TO JOIN US THERE. BUT LET ME BEGIN NOW BY INTRODUCING DR. KENT. DR. KENT IS A PROFESSOR AT THE UNIVERSITY OF CHEMISTRY AND BIOCHEMISTRY AT THE UNIVERSITY OF CHICAGO. HE'S ORIGINALLY FROM NEW ZEALAND AND WAS TRAINED IN ORGANIC CHEMISTRY AT BERKLEY BUT THEN JOINED BRUCE AT THE ROCKEFELLER UNIVERSITY FOR SIX OR SEVEN YEARS, IN WHICH HE WAS KEY TO THE WORK OF MERRY FIELD ON DEVELOPING SOLID PHASE SYNTHESIS. SUBSEQUENTLY HE WAS ON THE FACULTY AT CAL TECH AS WELL AS SEVERAL BIOTECH COMPANIES BUT IN THE LAST 20 YEARS, HAS BEEN PRIMARILY AT THE UNIVERSITY OF CHICAGO, WORKING ON THE CHEMICAL SYNTHESIS OF PROTEINS. AND I'M VERY PLEASED THAT HE CAN JOIN US TODAY TO TALK ABOUT THESE STUDIES. [APPLAUSE] PERSONALLY MET FACE TO FACE WITH CHRIS ANFINSEN, AT THE ROCKEFELLER UNIVERSITY, I WAS THE FREQUENT RECIPIENT OF PHONE CALLS FROM CHRIS AS HE WORKED ON HIS ATTEMPT AT TOTAL CHEMICAL SYNTHESIS, SO HE WOULD CALL UP AND SAY I TRIED DOING THIS REACTION, THIS IS WHAT HAPPENED, WHAT DO I DO NEXT? I HAD A GOOD TELEPHONIC RELATIONSHIP WITH HIM. SO TODAY, I'M GOING TO TALK ABOUT HOW THE THERMODYNAMIC HYPOTHESIS OF CHRIS ANFINSEN IMPACTED OUR WORK ON THE TOTAL CHEMICAL SYNTHESIS OF PROTEINS. SO ALTHOUGH HIS THERMODYNAMIC HYPOTHESIS IS ACTUALLY QUITE SOPHISTICATED AND QUITE COMPLEX IN ITS DETAILS, IT'S USUALLY REDUCED TO THE STATEMENT THAT, "THE NATIVE, FOLDED STRUCTURE OF A PROTEIN MOLECULE IS DETERMINED BY THE PROTEIN'S AMINO ACID SEQUENCE." AND THAT TURNS OUT TO BE KEY. IF YOU WANTED TO MAKE PROTEINS BY TOTAL CHEMICAL SYNTHESIS, YOU NEEDED TO HAVE CHRIS ANFINSEN COME UP WITH THIS INSIGHT. SO THE TOTAL CHEMICAL SYNTHESIS OF ENZYMES AND OTHER PROTEIN MOLECULES WAS ACTUALLY ONE OF THE GRAND CHALLENGES OF ORGANIC CHEMISTRY IN GENERAL IN THE 20TH CENTURY. SO THE GOAL WAS FIRST ENUNCIATED BY EMIL FIS CHER IN HIS NOBEL PRIZE SPEECH IN 1902 AND HIS OVERWHELMING -- IN THIS EXCERPT FROM A LETTER TO ADOLPH BAEYER WRITTEN IN 1905. FISCHER AND HIS SUCCESSORS WERE ABLE TO MAKE PEPTIDES, FISHER FOUNDED THE SCIENCE OF PEPTIDE CHEMISTRY, BUT HE WAS NEVER ABLE TO ACCOMPLISH THE TOTAL SYNTHESIS OF AN ENZYME. SO AMONGST OTHER ISSUES, HE NEEDED PROTEIN SEQUENCE DATA. SO LET'S QUICKLY REVIEW WHAT WOULD BE INVOLVED IN MAKING AN ENZYME BY TOTAL CHEMICAL SYNTHESIS. SO AT THE TOP HERE, WE HAVE AN ENZYME THAT WILL BECOME -- CARTOON FORM THAT WILL BECOME FAMILIAR TO YOU IN THE NEXT 20 MINUTES OR SO, AND THE PRECURSOR TO THIS FOLDED DISULFIDE CROSS-LINKED ENZYME MOLECULE IS A LINEAR POLYPEPTIDE CHAIN. SO FOR SMALL ENZYMES, THIS MAY BE AS SMALL AS 120 TO 150 AMINO ACIDS. FOR LARGER MORE COMPLEX ENZYMES, IT'S TYPICALLY AROUND 300 AMINO ACIDS. SO QUITE A LONG POLYPEPTIDE CHAIN. BEYOND THE CAPABILITY OF EVEN PEPTIDE CHEMISTS EVEN TODAY TO MAKE IN A STEP WISE ONE AMINO ACID AT A TIME FASHION. SO THE PRECURSOR TO THIS HAS TO BE A SERIES OF SHORTER PEPTIDE SEGMENTS, 30 OR 40 AMINO ACIDS LONG THAT OF HAVE BEEN MADE BY TOTAL CHEMICAL SYNTHESIS. SO IN ORGANIC CHEMISTRY, THIS IS REFERRED TO AS A RETRO SYNTHETIC ANALYSIS, COURTESY OF E.J. CORRIE. LOTS OF NOBEL LORE YACHTS LAUREATES WILL B E MENTIONED TODAY. SO AS I SAID, IF YOU WANT TO TAKE THE TOTAL CHEMICAL SYNTHESIS OF A PROTEIN, THE FIRST THING YOU NEED TO KNOW IS ITS COVALENT STRUCTURE. ALTHOUGH FISCHER AND HOFMEISTER, APPARENTLY INDEPENDENTLY OF ONE ANOTHER BUT AT THE SAME SYMPOSIUM IN, I THINK, 1904, ANYWAY, ENUNCIATED THE PEPTIDE THEORY OF PROTEIN STRUCTURE, NAMELY THE PEPTIDES -- EXCUSE ME -- PROTEINS -- LINEAR SEQUENCES OF AMINO ACIDS LINKED HEAD TO TAIL. THE FIRST SEQUENCE OF A PROTEIN MOLECULE WAS DETERMINED BY FRED SANGER IN THE 1950s. AS SHOWN HERE, ALONG WITH THE PICTURE OF THE ACTUAL INSULIN PROTEIN, AND THIS IS WHAT YOU WOULD SEE COMING AT YOU, AND ESPECIALLY IF YOU WERE THE INSULIN RECEPTOR. AND THIS IS A CARTOON REPRESENTATION. WE'LL SEE A LOT OF CARTOON REPRESENTATIONS DURING THIS SHORT PRESENTATION. SO AFTER THE DETERMINATION OF THE STRUCTURE OF THE -- EXCUSE ME -- THE COVALENT STRUCTURE OF INSULIN, TIM LAR CHEMICAL TECHNIQUES WE USED TO WORK OUT THE AMINO ACID SEQUENCE AND DISULFIDE BOND CONNECTIVITY OF A VARIETY OF OTHER PROTEINS. MOST IMPORTANTLY, THE LYSOSIEM LYSOSIMES. THIS WAS THE PROTOTYPICAL SEQUENCE SHOWN HERE, 129 AMINO ACIDS DETERMINED HERE AT THE NIH. AND MANY OTHER SPECIES OF EGG WHITE LYSOSYME HAD THEIR AMINO ACID SEQUENCE DETERMINED. ULTIMATELY, BY 1971, EVEN THE SEQUENCE OF HUMAN LYSOSYME HAD BEEN DETERMINED. THE HUMAN LYSOSYME IS CARRIED IN -- WITH THIS KIND OF SEQUENCE INFORMATION KNOWN IN THE EARLY 70ss, THE STAGE WAS SET FOR ONE OF THE PRE-EMINENT SYNTHETIC ORGANIC CHEMIST OF HIS ERA, GEORGE KENNER IN THE U.K., TO UNDERTAKE THE TOTAL CHEMICAL SYNTHESIS OF THE ENZYME LYSOSYME USING A SIMPLIFIED CONSENSUS SEQUENCE OF 129 AMINO ACIDS BY COMPARING ALL THE DIFFERENT SEQUENCE INFORMATION THAT WAS AVAILABLE AT THE TIME. SO HIS SYNTHETC SCHEME CONSISTED OF MAKING SMALLER FULLY PROTECTED SEGMENTS AND STITCHING THEM TOGETHER TO GIVE TWO FINAL LARGE CHUNKS OF ROUGHLY 65 AMINO ACIDS EACH THAT WOULD THEN BE CONNECTED. ALL THAT WAS SUCCESSFULLY UNDERTAKEN AND ACHIEVED BY HIS TEAM OF APPROXIMATELY 16 VERY TALENTED SCIENTISTS, MANY OF WHOM WENT ON TO INDEPENDENT CAREERS AND GREAT SUCCESS IN THEIR OWN RITE. UNFORTUNATELY AS WAS THE CASE FORS ALMOST ALL OF THESE ATTEMPTS BY CONVENTIONAL ORGANIC CHEMISTRY WAS NOT SUCCESSFUL. THIS WORK IS SUMMED UP IN THE LECTURE IN 1977 THAT GAVE -- HE WAS ACTUALLY SO DEPRESSED BY THE FAILURE THAT HE COMMITTED SUICIDE. THAT'S NOT GOOD EITHER. SO LET'S DO ONE SLIDE'S WORTH OF WHAT WAS RIGHT AND WHAT WAS WRONG OF THAT CONVERGENT CONVENTIONAL SYNTHESIS. SO THE CONCEPT IS EXCELLENT. THE IDEA IS THAT YOU TAKE BUILDING BLOCKS AND STITCH THEM TOGETHER CORE VER GENTLY, SO NOT STEP WISE, BUT BUILD THE TWO -- OF THE MOLECULE AND THEN STITCH THOSE TOGETHER. YOU CAN THEN PURIFY IMMEDIATE REACTION PRODUCTS. YOU USE MAXIMAL PROTECTION OF OTHER FUNCTIONAL GROUPS PRESENT IN THE AMINO ACID SIDE CHAINS OF THE PROTEIN'S POLYPEPTIDE CHAIN IN ORDER TO PREVENT SIDE REACTIONS. HOWEVER, IT TURNS OUT OH THE PROTECTED PEPTIDES WOULD RATHER INTERACT WITH EACH OTHER THAN WITH A SOLVENT, WHICH TRANSLATES IN REAL LIFE TO THE FACT THAT THEY AREN'T VERY SOLUBLE, THEY PRECIPITATE, EVEN IF YOU CAN KEEP THEM IN SOLUTION AT LOW CONCENTRATIONS, AND YOU GET VERY POOR YIELDS OF THE BIMOLECULAR REACTIONS THAT YOU'RE PERFORMING. THERE ARE OTHER SIDE REACTIONS THAT ALSO ARE INHERENT IN THIS APPROACH. YOU'VE ALSO MASKED ALL THE GROUPS THAT ALLOW YOU TO PURIFY THINGS WELL, AND THAT MAKES ANALYTICAL CONTROL ALSO VERY DIFFICULT. SO A NEW APPROACH WAS NEEDED, AND I WAS FORTUNATE ENOUGH TO COME UP WITH SUCH AN APPROACH THAT'S TURNED OUT QUITE WELL AND IS WIDELY USED THROUGHOUT THE WORLD, AND THAT'S CHEMICAL LIGATION. IT'S A CREATIVE FORM OF CHEATING. WE KNEW WE COULD KEEP UNPROTECTED PEPTIDES, WE COULD THAT THEY WERE PURIFIED, KEEP THEM IN SOLUTION. WHAT WE NEEDED WAS A WAY OF STITCHING THEM TOGETHER. THAT WAS NON-TRIVIAL IN THE FIRST INSTANCE, BUT BY LOOKING AT IT FROM A COMPLETELY DIFFERENT VIEWPOINT, I REALIZED THAT IF YOU WERE NOT WORRIED ABOUT MAKING A PEPTIDE BOND AT THE SITE WHERE YOU JOINED THE PEPTIDES TOGETHER, IT YOU'LLLY RENDERED THE PROBLEM TRIVIAL. SO HERE'S AN EXAMPLE OF HIGH SCHOOL SYNTHETIC ORGANIC CHEMISTRY. TAKE TWO PEPTIDES WITH ALL THEIR FUNCTIONAL GROUPS AND PUT TWO UNIQUE MUTUALLY REACTIVE FUNCTIONALITIES, ONE ON EACH PIECE. THE ONLY THING THERE THEY WILL REACT WITH EACH OTHER BUT NOT WITH ANY OF THE OTHER FUNCTIONALITIES THAT ARE PRESENT, AND THAT'S CALLED A CHEMO SELECTIVE REACTION, AND YOU GENERATE A LIGATION PRODUCT. THE PRICE YOU PAID FOR SIMPLIFYING THE CHALLENGE IS THAT YOU DON'T HAVE AN AMID BOND WHERE YOU'VE STITCHED THE TWO PEPTIDES TOGETHER. USING THIS CHEMISTRY, YOU END UP WITH A THYOESTHER, WHICH WAS A REASONABLE MIMIC BUT AS MANY OF MY COLLEAGUES THROUGHOUT THE WORLD AFTER WE PUBLISHED THIS IN "SCIENCE" WERE MOVED TO COMMENT, YOU KNOW, YOU'RE CHEATING, IT'S NOT A REAL AMID BOND. HOWEVER, THE PROTEINS DIDN'T CARE SO AFTER ALL THE STRUGGLES OF CHRIS TO MAKE STAPH NUCLEASE, HERE'S AN EXAMPLE OF MAKING THE HV1 PROTEASE -- THE CHEMISTRY THAT I SHOWED ON THE PREVIOUS SLIDE, YOU CAN MAKE TENS OF MILLIGRAMS OF FULLY FOLDED ENZYME MATERIALS. SO THIS ALL OF A SUDDEN RENDERED ACCESSIBLE ALL SORTS OF PROTEINS, BUT THERE WAS STILL THIS PROTEST THAT WE WEREN'T ACTUALLY MAKING AMID BONDS, WE WERE MAKING THIOESTHER LINKED PRODUCTS. AND I WAS A VICTIM OF CHAIRING A SESSION IN PUBLIC WHERE WE SET UP ON A STAGE AT A SCIENTIFIC CONFERENCE AND THE PEOPLE IN THE SESSION WERE BUSY SAYING YOU'RE MAKING THIOESTHERS, NOT AMIDST. SO WHILE THEY WERE TALKING, I THOUGHT, WELL, IF WE PUT IN AN EXTRA METHYLENE GROUP AND ON THAT METHYLENE GROUP, WE HAVE A NITROGEN ATOM, AN AMINO GROUP, THEN ONE COULD IMAGINE AN INTRAMOLECULAR KNEW THREE OWE PHILIC ATTACK TO GENERATE AN AMID BOND. SO OUR CHALLENGE BOILED DOWN USING THIS IDEA WAS HOW DO WE MAKE THIS INTO MEDIATE? PHIL DAWSON, NOW DEAN OF GRADUATE STUDIES IN LA JOLLA, PROFESSOR OF CHEMISTRY, WAS A SECOND YEAR GRADUATE STEUBT OF MY STUDENT OF MY LAB AND HE FIGURED OUT HOW TO MAKE THIS VERY EFFICIENTLY. THIS IS THE KEY TO THE WELL-KNOWN NATIVE CHEMICAL LIGATION REACTION. TAKE A PEPTIDE THIOESTHER, A PEPTIDE WITH AN AMINO TERMINAL SUSTAINED RESIDUE, DOESN'T MATTER IF THERE ARE OTHER CYSTINES, THIS FORM OF THIS ATTACKS THE THIOESTER. THIS IS A REVERSIBLE REACTION THAT GENERATES THIS INTO MEDIATE WHICH IRREVERSIBLY IS CONVERT TODAY A NATIVE AMID BOND. THE REACTIONS ARE CARRIED OUT AT MUTUAL PH, AN AQUEOUS SOLUTION, UNPROTECTED PEPTIDES FROM GLOBULAR PROTEINS ARE FREELY SOLUBLE AND TO MAKE SURE THEY STAY IN SOLUTION, WE USE A SOLUBLIZING AGENT, AND AN AERIAL THIOCATALYST THAT PROMOTES THE REVERSIBILITY OF THIS REACTION. THAT'S KEY TO THE -- ACTIVITY. IF YOU HAVE OTHER CYST STAINS, IT DOESN'T MATTER BECAUSE EVENTUALLY THEY'LL END UP IN THIS FORM AND REVERSIBLY BE CONVERTED TO THE DESIRED PRODUCT. SO WE'RE ALMOST THERE. EXCEPT THAT EVEN A LONG POLYPEPTIDE CHAIN IS NOT A PROTEIN. SO IF YOU REMEMBER OUR RETRO SYNTHETIC NAIL CYST, ONLY NOW WE'RE LOOKING AT THE SYNTHETIC DIRECTION, WE KNOW HOW TO MAKE THE PEPTIDES USING OPTIMIZED VERSIONS OF BRUCE MERRIFIELD'S SOLID PHASE SYNTHESIS, WE NOW KNOW HOW TO CHARACTERIZE THEM, WE KNOW HOW TO STITCH THEM TOGETHER USING THE CHEMISTRIES I JUST DESCRIBED. WE NEED TO KNOW HOW TO FOLD THEM. FORTUNATELY, BY THE TIME WE'D FIGURED OUT HOW TO MAKE THE LONG POLYPEPTIDE CHAINS, THE FOLDING PROBLEM IN PRACTICAL TERMS BASED ON CHRIS ANFINSEN'S THERMODYNAMIC HYPOTHESIS HAD BEEN WORKED OUT FOR THE FLEDGLING BIOTECHNOLOGY PHARMACEUTICAL INDUSTRY BETWEEN THE MID 1980s AND MID 1990s. AND THE PRINCIPAL CONTRIBUTOR TO THAT WORK RAINER RUDOLPH IN THE FORMER EAST GERMANY. HIS WORK IN THAT AREA IS SUMMARIZED IN THIS ARTICLE THAT I'VE CITED. THE PRINCIPLES STRAIGHT OUT OF THE CHRIS ANFINSEN PLAYBOOK. IT'S EQUILIBRIUM FOLDING, KEEP THE AGGREGATING MISFOLDED INTERMEDIATES, STOP THEM FROM PRECIPITATING. SO YOU NEED WHAT IS COMMONLY CALLED A DENATURANT, AND TO ENCOURAGE DISULFIDE FORMATION, YOU NEED A REDOX COUPLE, SO THAT IT'S THE EEK LEIB TBREEM FOLDED THERMODYNAMIC MINIMUM STATE OF THE PROTEIN THAT DETERMINES THE DISULFIDES AND THE CORRECT FOLD. ALL STRAIGHT OUT OF CHRIS ANFINSEN'S WORK. SO LET ME ILLUSTRATE THIS WITH HUMAN LYSOZYME. TOTAL SYNTHESIS PERFORMED BY THOMAS DUREK, A POSTDOC IN MY LAB. SO RATHER THAN HAVING A TEAM OF 16 PEOPLE AS IN THE KENNER WORK, I NOW HAVE ONE POSTDOC DOING THIS, ALL MANUALLY, NO MACHINES INVOLVED, EXCEPT FOR THE ANALYTICAL CONTROL. SO THE TARGET IS 130 AMINO ACID POLYPEPTIDE CHAIN WHEN CORRECTLY FOLDED HAS FOUR DISULFIDE BONDS. SO THIS WAS MADE FROM FOUR SYNTHETIC PEPTIDES. ONE, TWO, THREE, FOUR. OVER HERE WE HAVE A NATIVE CHEMICAL LIGATION REACTION. OVER HERE WE HAVE A VARIATION ON THAT WHERE WE LEAVE OUT THE THIOAERIAL CATALYST, AND THEN WE CAN CONDENSE A THIOAERIAL ESTER AND END UP WITH THE OTHER HALF OF THE POLYPEPTIDE CHAIN AS THE THIOESTER. A FINAL NATIVE CHEMICAL LIGATION STEP, THE FULL LENGTH POLYPEPTIDE CHAIN, WHICH IS FOLDED TO GIVE THE CORRECTLY FOLDED ENZYME OF FULL ENZYMATIC ACTIVITY. THIS SHOWS THE LAST STEP IN THAT SYNTHESIS. THE CONDENSATION OF A 64 RESIDUE POLYPEPTIDE CHAIN WITH A 66 RESIDUE POLYPEPTIDE CHAIN. THOMAS OBVIOUSLY WANTED TO GO HOME AND GRAB SOME SLEEP, SO AFTER 12 HOURS, HE HAD A NEAR QUANTITATIVE REACTION. WE USED A SLIGHT EXCESS OF ONE COMPONENT, SO AN ATOM ECONOMY TERMS, 80% OF THE REACTANTS ENDED UP IN THE PRODUCT WHICH IS QUITE EXTRAORDINARY FOR SUCH HIGH MOLECULAR WEIGHT REACT ANTIS. ANTS. SO THIS IS THEN FOLDED UNDER -- CONDITIONS AGAIN SO SLIGHTLY ELEVATED P.H. IN THE PRESENCE OF A REDOX COUPLE GENERATED BY THESE TWO REACT ANTIS. THIS IS THE STARTING MATERIAL, THIS IS THE CRUDE FOLDING REACTION AND THIS IS THE PURIFIED SYNTHETIC PROTEIN. YOU CAN THEY'LL THE FOUR DISULFIDES ARE FORMED FROM THE MASS DIFFERENCE. AND TO CHARACTERIZE THE SYNTHETIC PROTEIN, WE CHECK ITS PURE IT,ITY, THAT IT HAS THE CORRECT MASS, THAT IT HAS A UNIQUE FOLD, USUALLY SHOWN BY MULTIDIMENSIONAL NMR, DETERMINE THE STRUCTURE OF THE FOLDED SYNTHETIC PROTEIN BY X-RAY DIFFRACTION METHODS. THIS SHOWS THE QUALITY OF THE ELECTRON DENSITY MAP FOR THE CHEMICALLY SYNTHESIZED ENZYME. AT THE TIME, 10 YEARS AGO, THIS WAS THE HIGHEST RESOLUTION CRYSTAL STRUCTURE OF HUMAN LYSOZYME. AND THEN FINALLY, AFTER ALL THIS KERIK E.T.IZATION, IT MAKE SENSE TO SEE WHETHER IT ACTUALLY WORKS AS AN ENZYME, AND IN THIS CASE, IT DOES, IT'S DIGESTING THE PROTEOGLYCANS THAT FORM UP THE ARM OF THE BACTERIA AND STOP IT FROM EXPLODING IN G2 OSMOTIC PRESSURE. SO NOW WE HAVE A GENERAL WAY OF MAKING PROTEINS USING CHEMISTRY. WE CAN START WITH AN AMINO ACID SEQUENCE, BOTTLES OF WHITE PO DER, POWDER, MAKE THE FULL LENGTH POLYPEPTIDE CHAIN, FOLDED, BASED ON CHRIS ANFINSEN AS WORK AND THEN CARRY OUT STRUCTURE ACTIVITY RELATIONSHIP STUDIES TO FIGURE OUT WHAT MAKES THE PROTEIN TICK. WHICH IS THE WHOLE POINT. SO I THOUGHT I'D FINISH OFF THIS PRESENTATION BY TALKING ABOUT ONE OF THE REALLY UNUSUAL THINGS YOU CAN DO WITH TOTAL CHEMICAL SYNTHESIS. SO ALL PROTEINS THAT ARE FOUND IN NATURE ON THIS PLANET ANYWAY, ARE MADE UP OF GLYCINE. YOU'VE GOT CHIRAL MODELS WITH YOU, THIS IS THE LEFT HAND, THESE ARE THE RIGHT HAND, THESE ARE CHIRAL OBJECTS, NON-SUPER IMPOSABLE ON THEIR MIRROR IMAGES. IN MOLECULAR TERMS, THE CHIRAL MOLECULES -- SO ALL -- LEFT LANDED THIS IS A RIGHT-HANDED PROTEIN. HOW DO WE GET THERE? WELL, THE KEY IS ANFINSEN'S THERMODYNAMIC HYPOTHESIS. IT IMPLIES -- I'M NOT SURE IF IT WAS EXPLICITLY STATED, IT MAY HAVE BEEN BUT I NEVER FOUND IT, THAT IF YOU USE D AMINO ACIDS, AND THE GLYCINE, THEN YOU WILL GET A MIRROR IMAGE PROTEIN. AND JUST TO BE ABSOLUTELY EXPLICIT ABOUT THIS, HERE WE HAVE D MIRROR IMAGE FORM OF HIV1 PROTEASE ADMIRING ITSELF IN THE MIRROR WHERE IT'S IN THE NATURAL FORM. AND THE WAY THAT WAS GENERATED IS ACTUALLY -- BY THE ORIGINAL HIGH SCHOOL SYNTHETIC ORGANIC CHEMISTRY, SO AS WELL AS MAKING THE L FORM OF THE ENZYME IN ALMOST 50 MILLIGRAM YIELD AND LOW SCALE RESEARCH LAB SYNTHESIS, WE ALSO CARRIED OUT -- WE ENDED UP WITH ROUGHLY THE SAME YIELD OF THE MIRROR IMAGE PROTEIN. AND OF COURSE THE ENZYMES CARRY OUT CHIRAL REACTIONS, IN THIS CASE, THIS ENZYME CHEWS UP PEPTIDES WHICH ARE CHIRAL ENTITIES. THE MIRROR IMAGE ENZYME DOES NOT WORK ON THE NATURAL SUBSTRATE THAT CLEAVES THE MIRROR IMAGE SUBSTRATE WITH EQUAL VELOCITY. INCIDENTALLY, I COULDN'T FIND THE SLIDE BUT WHEN WE FIRST FOLDED THESE SYNTHETIC HIV-1 PROTEASE MOLECULES, WE WENT THROUGH ALL SORTS OF LAB FOLDING PROTOCOLS. THIS DOESN'T HAVE DISULFIDE. IF YOU JUST TAKE THE POLYPEPTIDE AND DELIGHT IT INTO THE ASSAY, YOU IMMEDIATELY GET THE SAME DATA AS YOU GET HERE, SO THE PROTEIN FOLDS LIKE THEM, EITHER UNDER ASSAY CONDITIONS. SO CAN YOU USE MIRROR IMAGE PROTEINS FOR ANYTHING? WELL, YOU CAN USE THEM FOR A COUPLE OF THINGS. THE FIRST THING YOU CAN DO IS HELP THE STRUCTURAL BIOLOGISTS OUT OF A MAJOR PROBLEM OR CHALLENGE IN THEIR X-RAY CRYSTALLOGRAPHY STRUCTURE DETERMINATION OF PROTEINS. SO THANKS TO THE FUNDED STRUCTURAL GENOMICS PROGRAMS, WE HAVE WONDERFUL STATISTICS ON THE SUCCESS RATE FOR GLOBULAR PROTEIN MOLECULES. SO HERE YOU SEE ALMOST 48,000 PURIFIED POLLLY PEPTIDES, PROTEINS OF WHICH ONLY 32% CRYSTALLIZED AND ONLY HALF OF THOSE CRYSTALS OF DEFRACTION QUALITY. THERE'S MUCH MORE SOPHISTICATED DAY THAT NOW, AND IT TURNS OUT THERE'S AN 18% FAILURE RATE, DESPITE ALL THE TRICKS WE KNOW HOW TO PLAY. SO ONE WAY OF OVERCOMING THIS HURDLE TO PROTEIN STRUCTURE DETERMINATION WAS PREDICTED BY TODD YATES IN 1995 IN A THEORETICAL ARTICLE AND THEN INADVERTENTLY REED TO EXPERIMENTAL PRACTICE BY US WHEN WE WERE TRYING TO FIGURE OUT THE STRUCTURE THE ANTIFREEZE PROTEIN YEARS AGO, AND THAT'S TO TAKE A MIRROR IMAGE -- THE TWO MIRROR IMAGES, PREPARED BY CHEMICAL SYNTHESIS, AND MIX THEM 1 TO 1 SO YOU HAVE A RACE MI. C SOLUTION. THIS GREATLY FACILITATES CRYSTALLIZATION, THE FORMATION OF DEFRACTION QUALITY CRYSTALS. IT ALSO GREATLY SIMPLIFIES THE PHASE PROBLEM IN SOLVING THE STRUCTURE BECAUSE ALL PHASES ARE NOW QUANTITY ADVERTISED AND RELATED BY ZERO AND PI RADIANCE SO THIS IS NOW BEING USED IN A LOT OF LABS THROUGHOUT THE WORLD WORLD. WE'VE DONE ABOUT 20 OF THESE RACEMIC STRUCTURES OR ATTEMPTED STRUCTURES AND THIS JUST SHOWS SOME OF THEM. THIS WAS THE ORIGINAL ONE, AND A BUNCH OF OTHER ONES INCLUDING THIS UNDER THE RESTING GUY OVER HERE WHICH IS KEY TO HOW MYCOBACTERIUM TUBERCULOSIS ENTERS PERSISTENT DORMANCY. SO ALMOST ALL OF THESE ARE PROTEINS THAT COULD NOT BE CRYSTALLIZED AS NORMAL L PROTEINS, DESPITE EVERYONE'S BEST EFFORTS. WHAT WE FOUND IS THAT IN THEEG 20 INSTANCES, WE HAD ABOUT AN 85% SUCCESS RATE. AND THIS CORRESPONDS WITH EXPERIENCE IN OTHER LABS THROUGHOUT THE WORLD, SO WE SEEM TO HAVE DRAMATICALLY CHANGED THE ABILITY TO GET DEFRACTION QUALITY CRYSTALS BY THIS USE OF A RACEMIC MIXTURE OF CHEMICALLY SYNTHESIZED PROTEINS. SO WHAT ELSE CAN YOU DO WITH A D PROTEIN? SO THIS IS VASCULAR ENDOTHELIAL GROWTH FACTOR TYPE A. IT'S THE TARGET FOR TWO OF GENENTECH'S EARLY BLOCKBUSTER DRUGS. THIS IS ACTUALLY THE WORK FROM THE PROTEIN GROUP AT GENENTECH BACK IN THE 1990s. THIS WAS THE MOLECULE THAT WAS USED TO DEVELOP THOSE TWO DRUGS. SO IT'S A COVALENT DIMER OF POLYPEPTIDE CHAINS WITH EIGHT CYSTANES. THIS IS A REAL CHALLENGE FOR CHRIS ANFINSEN'S THERMODYNAMIC HYPOTHESIS. SO WE SET OUT TO MAKE THE MIRROR IMAGE PROTEIN, SO THE CONVENTION IN THE FIELD IS THAT LOWER CASE -- D AMINO ACIDS AND THE GLYCINE IS STILL SHOWING IN UPPER CASE. SO WE NEEDED THIS, AND I'LL SHOW YOU WHY IN A MOMENT. WE MADE IT FROM THREE SYNTHETIC PEPTIDE. THE SERIES OF REACTIONS WAS CARRIED OUT WITHOUT ANY PURIFICATION OF INTERMEDIATE PRODUCTS. THIS IS THE TOTAL CRUDE PRODUCT MIXTURE FROM WHICH THE FULL LENGTH 102 RESIDUE POLYPEPTIDE WAS READILY SEPARATED AND PURIFIED, AND HAD THE CORRECT MASS. THIS SHOWS YOU THE RAW MASS SPEC DATA. THE FOLDING WAS CARRIED OUT AGAIN UNDER THE STANDARD CONDITIONS OF A SLIGHTLY ELEVATED P.H. AS A LOW CONCENTRATION TO FACILITATE -- A REDOX MIXTURE AND AS YOU CAN SEE HERE, IT'S ACTUALLY REALLY INTERESTING, THE PROTEIN POLYPEPTIDE CHAIN REALLY DOES KNOW HOW TO GET TO THE FINAL ANSWER, OKAY, SO HERE'S THE BEGINNING, HERE'S AFTER A DAY, WHERE YOU GOT MISFOLDED FORMS AND THEY ALL SHUFFLE DOWN UNDER THESE CONDITIONS TO GIVE YOU AN ALMOST QUANTITATIVE YIELD OF CORRECTLY FOLDED MATERIAL. NOW YOU HAVE A 24 KILODALTON COVALENTLY LINKED PROTEIN MOLECULE. POSTDOC IN MY LAB DID ALL THIS WORK, AND ALSO DETERMINED THE CRYSTAL STRUCTURE, WHICH IT TURNS OUT IS ACTUALLY THE MIRROR IMAGE OF THE RECOMBINANTLY PRODUCED PROTEIN. SO THESE WERE DONE 15 YEARS APART BY TOTALLY DIFFERENT METHODS AND YET THE MIRROR IMAGE SHAPES ARE EVIDENT. SO WHAT DO WE DO WITH THIS MIRROR IMAGE VASCULAR ENDOTHELIAL GROWTH FACTOR? WE CAN TAKE ADVANTAGE OF THE WORK OF THE PETER KIM LAB, THEN AT THE WHITE INSTITUTE AT M.I.T., SO TOM SCHUMACHER, AND THEIR INGENIUS MIRROR IMAGE PEPTIDE DISPLAY TECHNOLOGY. IT'S A WONDERFUL INVENTION. THE IDEA WAS, IF YOU WANTED TO DEVELOP A PEPTIDE THAT WILL WORK, A MIRROR IMAGE PEPTIDE, D PEPTIDE THAT WILL WORK AGAINST AN L PROTEIN TARGET, TAKE THE L PROTEIN TARGET, USE CHEMISTRY TO MAKE IT AN N. A MIRROR IMAGE FORM. A SOMEWHAT TOPICAL SUBJECT, THANKS TO THE NOBEL PRIZE FOR THAT, JUST RECENTLY, AND THEN YOU'RE GOING TO END UP WITH AN L PEPTIDE, WHICH YOU CAN AFFINITY MAX -- MAXIMIZE THE AFFINITY. YOU'VE NOW GOT A WONDERFUL L PEPTIDE AGAINST THE MIRROR IMAGE OF VASCULARLY ENDOTHELIAL GROWTH FACTOR AND THAT WILL BE GOOD FOR TREATING PEOPLE IN THE MIRROR IMAGE WORLD, BUT IN THAT WORLD, YOU NOW NEED TO TAKE THE SEQUENCE INFORMATION FOR THIS PEPTIDE LIGAND AND USE CHEMISTRY TO MAKE IT IN THE MIRROR IMAGE FORM, AND BECAUSE OF THE PRINCIPLES OF SYMMETRY, THIS D PEPTIDE WILL BE A HIGH AFFINITY SPECIFIC LIGAND FOR THE ORIGINAL PROTEIN MOLECULE. AND WITH TORONTO, WE ADAPTED THE MIRROR IMAGE PEPTIDE PHAGE DISPLAY USING VEGF A, MADE IT MIRROR IMAGE FORM, NOW WE'RE SCREENING PROTEIN LIBRARIES, SMALL PROTEIN SCAFFOLD, DEVELOP A HIGH AFFINITY L PROTEIN LIGAND, MAKE IT IN MIRROR IMAGE FORM, AND NOW WE HAVE AN ENGINEERED D PRO PROTEIN SPECIFIC FOR NATURAL VEGF-A AND WE'D LIKE TO KNOW HOW IT BINDS TO THAT AND HOW IT COMPARES TO AVASTIN, WHICH IS AN ANTIBODY, AND LUCEN TI. S, WHICH IS A TRUNK ATED ANTIBODY, COMPARE THOSE BINDING SITES TO WHAT IS GOING ON HERE. SO WE USE RACEMIC CASE TALLOGRAPHY. WHY NOT THE? SO L-VEG IF AND TWO EQUIVALENTS OF THE MIRROR IMAGE FORMS OF THE IDENTIFIED SMALL PROTEIN LIE MIXED TOGETHER AND GREATLY FACILITATED CRYSTAL FORMATION, INK TRON -- X-RAY DIFFRACTION DATA COLLECTED TO 1.6 -- ARE SOLD BY MOLECULAR REPLACEMENT, AND THIS JUST SHOWS YOU THE HETEROCHIRAL PROTEIN COMPLEX IN A SENTRY SEMITIC CRYSTALLINE ARRAY. HERE WE HAVE VEG-F WITH A D PROTEIN BOUND TO EITHER END. AND ITS MIRROR IMAGE D VEG-F HAS AN L PROTEIN BOUND TO EACH END. THE PROTEIN DATA BANK IS KIND ENOUGH TO ADD A FEATURE CALLED STRUCTURE WEIGHT, SO STRUCTURE WEIGHT HERE IS 73 KILODALTONS ALL MADE BY CHEMICAL SYNTHESIS. SO WHAT I WANTED TO EMPHASIZE IN CLOSING IS THAT ALL OF THIS IS BASED ON CHRIS ANFINSEN'S WORK. THE CENTRAL DOGMA OF PROTEIN SCIENCE, MAINLY THAT THE AMINO ACID SEQUENCE HAS INFORMATION THAT CODES FOR THE FOLD FOLDED STRUCTURE AT A THERMODYNAMIC MINIMUM, AND IT'S THAT STRUCTURE THAT DICTATES THE FUNCTION OF THE PROTEIN, IN THIS CASE, HUMAN LYSOZYME. SO THANK YOU VERY MUCH FOR YOUR ATTENTION. [APPLAUSE] >> YOU HAVE AN EXTRA SULFA GROUP AFTER THE LIGATION. DOES THAT NOT INTERFERE WITH FOLDING? >> NO, I SEE WHAT YOU'RE SAYING. WHAT YOU'RE ACTUALLY SAYING IS, WHAT -- IS NOT A NATURAL CYSTANE? YOU'VE JUST CREATED THE CISTANE TO FORM A DISULFIDE. YES. IF YOU DON'T HAVE A NATIVE SUSTAIN -- WHAT YOU WOULD LIKE TO BE, YOU JUST PUT ONE THERE AND NOW YOU'VE GOT AN EXTRA CYSTANE. WITH YOUBUT THE TRICK THERE IS TO PROTECT THE OTHER ONES, YOU CAN DESULFURIZE AND IT BECOMES A -- THEN YOU CAN GO AHEAD AND DEPROTECT. >> OTHER QUESTIONS? [APPLAUSE] >> OUR NEXT SPEAKER, BRUCE FU RI. E, PROFESSOR OF MEDICINE AT HARVARD MEDICAL SCHOOL, CONTINUES THE STORY OF THE CURRENT DEVELOPMENTS FROM CHRIS' WORK. BRUCE WAS AN UNDERGRADUATE AT PRINCETON, WENT TO MEDICAL SCHOOL AT THE UNIVERSITY OF PENNSYLVANIA, THEN AFTER CLINICAL TRAINING, CAME TO NIH TO WORK IN THE ANFINSEN LABORATORY WITH ME FORTUNATELY, AND THEN HAS HAD A VERY DISTINGUISHED CAREER IN ACADEMIC MEDICINE, MOSTLY IN CONJUNCTION WITH RESEARCH EFFORTS BY HIM AND BARBARA, HIS WIFE, WHICH HAVE CONTINUED ALMOST TO THIS DAY. BRUCE WILL TALK ABOUT THE CURRENT APPRECIATION OF THE IMPORTANCE OF THE PROTEIN DISULFIDE ESOMERASE WHICH CHRIS ANFINSEN WAS THE FIRST TO EXPERIMENTALLY DEFINE AND WHICH AS I SHOWED THAT SLIDE OF CITATIONS, CONTINUES TO GENERATE INTEREST TO THIS DAY, IN FACT, INCREASING INTEREST. AND I THINK YOU'LL SEE WHY FROM BRUCE' PRESENTATION. >> THANK YOU VERY MUCH, ALAN. IT'S A SPECIAL PLEASURE TO BE HERE. I WAS ONE OF THOSE ROOKIE BIOCHEMISTS THAT CAME TO THE LAB ALMOST FULLY TRAINED AS A CLINICIAN, BUT PRETTY GREEN AS A SCIENTIST. AND THIS WAS MY FIRST EXPOSURE TO THE WORLD OF FIRST CLASS SCIENCE. IN THE ANFINSEN LABORATORY, THERE'S THE RIBONUCLEASE ERA WHICH WE'RE ACTUALLY CELEBRATING TODAY. I ARRIVED AFTER RIBONUCLEASE WAS IN THE FAR MIRROR, AND I CAME DURING THE STAPH NUCLEASE ERA. I CAME IN THE EARLY 70s. THINGS WERE SIMPLER THEN, RIBONUCLEASE, LIKE STAPH NUCLEASE, IT WAS LIKE A SPRING. FOLDED EXTREMELY EASILY. THAT WAS THE TYPICAL PROBLEMS THAT WERE WORKED ON DURING THIS PERIOD. I WANT TO TALK TO YOU A LITTLE BIT ABOUT THE REGULATION OF PROTEIN ACTIVITY, AND I'M GOING TO REMIND YOU THAT THERE ARE DIFFERENT TYPES OF MODIFICATIONS. TYPE 1, YOU SEE BONFICATION, PHOSPHORYLATION IS A PERFECT EXAMPLE. THE OTHER IS CLEAVAGE OF A PEPTIDE BOND THAT CHANGES THE FUNCTION OF A PROTEIN. MY TOPIC TODAY IS TO TALK ABOUT TYPE 3, CLEAVAGE OF MATURED DISULFIDE BONDS. SO THE TYPE 1 POST TRANSLATION AND MODIFICATION, THIS IS MY OWN EXAMPLE OF PROBABLY -- THE TYPICAL CONOTOXIN FROM SNAIL WHERE NINE OUT OF 13 AMINO ACIDS ARE MODIFIED. HOWGLYCOSYLATED -- HYDROXY PROLINE, AND SO ON. SO WE ALL KNOW THAT MODIFICATION OF PROTEINS BY POST TRANSLATIONAL MODIFICATION CHANGES FUNCTION DRAMATICALLY. THE NIH KEEPS ASKING ME TO JOIN THEIR NETWORK. THE OTHER TYPE OF CHANGE IN PROTEIN STRUCTURE LEADING TO CHANGES IN THE STRUCTURE IS BY LIMITED PRO YOE LYSIS, PROTEOLYSIS, SO THA T IT GOES FROM AN INACTIVE STATE TO AN ACTIVE STATE. I'VE SHOWN YOU A KNOWN SLIDE HERE ABOUT FACTOR 10, ONE OF THE BLOOD CLOTTING PROTEINS, WHICH GOES FROM AN INACTIVE TO ACTIVE STATE. THIS IS TRIPSINOGEN -- BUT THERE'S A TYPE 3 ALLOSTERIC DISULFIDE BONDS WHICH I VENTURE TO SAY MANY OF YOU HAVE NOT HEARD ABOUT. THE PARTY LINE THAT CAME OUT OF THE ANFINSEN WORK WAS THAT PDI, PROTEIN DISULFIDE ISOMERASE LIVED IN THE ENDOPLAS PUBLIC RI TICK LUM, IT STAYED INSIDE THE CELL. AND MATURE PROTEINS ARE STABLE. SO THIS CONCEPT OF ALLOSTERIC DISULFIDE BONDS IS THAT SOME OF THESE DISULFIDE BONDS ARE STRESSED. THEY'RE UNSTABLE, AND THEY CAN BE CLEAVED IN THE MATURE PROTEIN. THAT REDOWX OXIDATION OF DISULFIDE BONDS, THEREFORE -- STRUCTURE AND THE FUNCTION OF THE PROTEIN, AND THAT THESE REDOX REACTIONS ARE CATALYZED BY THIOISOMERASES. NOW I'VE NEVER EVEN THOUGHT ABOUT PROTEIN -- IN THE VINCENT LABORATORY. IT WAS IN THE -- FAR IN THE PAST AND MOVED ON. THIS IS THE WORK OF HAAG FROM SYDNEY, AUSTRALIA, WHO STUDIED ABOUT 2,000 X-RAY COORDINATES AND FOUND THAT IN THOSE, THERE WERE ABOUT 13,000 DISULFIDE BONDS, AND ABOUT 500 WERE STRESSED, POTENTIAL ALLOSTERIC DIE SULFIDES. THAT MEANS THEIR ANGLES WERE SUITABLY STRESSED SO THAT THEY COULD POTENTIALLY BE LABILE. AND THIS SHOWS THE DIE DIHEDRA STRAINED -- INDICATING THAT THESE DISULFIDES ARE INDEED ABNORMAL. AND HE CLASSIFIED THEM INTO THREE CHARACTERISTICS. I WON'T GO INTO THIS IN DETAIL, BUT SUFFICE IT TO SAY THAT THESE ARE THE THREE TYPES OF DISULFIDE BOND FORMATS THAT INDEED LED TO THIS IDEA OF ALLOSTERIC DISULFIDE BONDS. THIS FIELD IS NEW, AND IT'S RATHER INTERESTING IN THAT THESE ARE THE PROTEINS THAT HAVE BEEN STUDIED AND FOUND TO HAVE ALLOSTERIC DISULFIDE BONDS AND ALSO TO CHANGE THEIR FUNCTION ON THE CHANGES IN THOSE DISULFIDE BONDS. I'M NOT GOING TO READ DOWN THE WHOLE LIST, BUT I WILL EMPHASIZE TISSUE FACTOR. TISSUE FACTOR IS ONE OF THE PROTEINS THAT I'VE BEEN INTERESTED IN FOR A LONG TIME BECAUSE IT'S THE WAY BLOOD COAGULATION GETS ACTIVATED. HERE'S TISSUE FACTOR. IN THE BLOOD COAGULATION CASCADE. AND THE WHOLE CASCADE ENDS WITH THE GENERATION OF FIBRIN. SO TISSUE FACTOR IS A PARTICULARLY INTERESTING PROTEIN. IT'S A MEMBRANE PROTEIN ON THE EXTERIOR OF ALMOST ALL MAMMALIAN CELLS, AND IT EXISTS -- IT'S BEEN KNOWN FOR 40 YEARS THAT IT EXISTS IN ENCRYPTED FORM, AN INACTIVE FORM AND CHANGES TO AN ACTIVE FORM. AND NO ONE YET HAS EXPLAINED THE MOLECULAR BASIS FOR THAT SWITCH. WHEN IT WAS PROPOSED THAT TISSUE FACTOR, WHICH -- HERE'S THE CRYSTALLOGRAPHIC STRUCTURE OF THE EXTRA CELLULAR DOMAIN, HAS A DISULFIDE BOND WHICH IS FORMED AND GENERATES AK NIFT TISSUE FACTOR, WHEREAS THE INACTIVE FORM LACKS THE DISULFIDE. I SUDDENLY BECAME INTERESTED IN WORKING ON THIS PROBLEM. I WAS EXCITED ABOUT THIS HYPOTHESIS ABOUT ALOE STEERK DISULFIDE BONDS AND WE THOUGHT WE HAD THE PERFECT TOOLS TO STUDY IT. NOW, PROTEIN DISULFIDE EYE SOME RACE WAS DISCUSS BID CHRIS ANFINSEN. WE NOW KNOW IT IS AN EXAMPLE OF A WHOLE SERIES OF THIOEYE SOME RACES, ALL WITH CHARACTERISTIC ACTIVE SITE CXXC. AND PDI HAS FOUR DOMAINS, TWO OF WHICH HAVE ACTIVE SITES. -- KNOWN TO BE PRESENT IN THE VASCULATURE. NOW WHAT DID I PLAN TO BRING INTO THIS? I WAS TRAINED AS A PROTEIN BIOCHEMIST. BESIDES MY HEMATOLOGY CLINICAL ACTIVITIES. AND IT SEEMS LIKE WE WERE GETTING TO THE POINT THAT THERE WERE 100 PROTEINS THAT WERE ALL INTERACTING WITH EACH OTHER, AND WE COULDN'T CONTINUE TO JUST STUDY THESE PROTEINS IN VITRO FOR YEARS WE AND OTHERS PURIFIED THESE PROTEINS, INTERACTED WITH ANOTHER PROTEIN AND BASICALLY TRIED TO UNDERSTAND HOW THIS COMPLEX SYSTEM WORKED, INCLUDING THE PROTEINS THAT ARE ON PLATELETS, THE PROTEINS THAT ARE ON ENDOTHELIAL CELLS. COAGULATION IS A VERY IMPORTANT PROBLEM CLINICALLY. IT'S THE BASIS FOR HEART ATTACKS, FOR MYOCARDIAL INFARCTION, IT'S A BASIS FOR STROKE, AND IT'S THE SECOND LEADING CAUSE OF DEATH IN CANCER PATIENTS. SO WE FELT WE HAD TO -- I HAD TO SWITCH FROM BEING A BIOCHEMIST TO BEING A PHYSIOLOGIST. I'M A LITTLE EMBARRASSED BY THAT BECAUSE I'M NOT -- I'VE ALWAYS BEEN A MOLECULAR PERSON, AND NOW AS MY WIFE SAYS, WE HAVE 6,000 MICE TO STUDY. SO WHAT WE DECIDED TO DO WAS TO SEE USING OUR SPECTROSCOPY EXPERIENCE, COULD WE DESIGN AN INSTRUMENT THAT WOULD ALLOW US TO STUDY IN VIVO IN A LIVE MOUSE, COULD WE STUDY THROMBUS FORMATION IN REALTIME. I SHOULD TELL YOU JUST AS AN ASIDE, I RECEIVED AN AWFUL LOT OF CRITICISM FROM MY MICROSCOPY COLLEAGUES. THIS CAN'T BE DONE. I DIDN'T BRING MY NIH PINK SHEETS TO TELL YOU THAT THEY TOLD ME THIS COULD NOT BE DONE. AND IN THE END, WE DECIDED TO TRY TO PROCEED OURSELVES. IN ANY CASE, THIS IS THE MOUSE, THE EYES, THE CREMASTER MUSCLE, AND WE'RE LOOKING AT THE ARTERIOLES AND VEEN VENULES IN THE CREAMASTER MUSCLE. WE CAN MAKE AN INJURY IN THOSE VESSELS USING A LASER THAT GOES THROUGH THE MIKE SCOA. THE THE MICROSCOPE. AND THIS IS REALTIME. OUR PLATELETS ARE LABELED RED. FIBRIN BLUE. TISSUE FACTOR GREEN. BLOOD FLOW IS THIS DIRECTION. THIS IS A COMBINATION OF FIBRIN AND PLATELETS. SO THIS ALLOWS US TO ACTUALLY SEE THROM WITH THROMBUS FORMATION. AND WE'VE EXPLOITED THIS TO BASICALLY REVIEW AND REDEFINE, CONFIRM AND RECONFIRM SOME OF THE THINGS THAT EVERYBODY HAD LEARNED FROM DOING THESE EXPERIMENTS IN VITRO. NOW HERE IS A SURPRISE. WE SAW THAT TISSUE FACTOR WAS POTENTIALLY ACTIVATED WITH PDI DOING THE CONVERSION TO THE ACTIVE FORM. TISSUE FACTOR. IN THIS EXPERIMENT, THE THROMBUS IS MADE, AND ONLY PDI IS BEING LABELED. PDI IS GREEN. SO THIS WAS A SURPRISE TO US. I WAS ALWAYS TAUGHT THAT PDI WAS INTRACELLULAR, AND THAT THERE'S A GROWING LITERATURE THAT THEY HAVE MULTIPLE FACTIONS OF ACTIVITIES OUTSIDE, BUT THIS SHOWED THAT P DI. PDI WAS DEFINITELY SECRETED BY ENDOTHELIAL CELLS AND PLATELETS. JUST BECAUSE IT GOT SECRETED DOESN'T NECESSARILY MEAN IT HAD AN ACTIVITY. SO HERE PLATELETS ARE LABELED RED, FIBRIN GREEN, AND THIS IS A CONTROL JUST SHOWING YOU THE FORMATION OF THE THROMBUS, AND HERE WE'VE USED AN ANTIBODY TO PDI TO INHIBIT PDI ACTIVITY, AND WE COMPLETELY BLOCK THROMBUS FORMATION. THIS WAS REALLY A SURPRISE TO US, AND COMPLETELY UNANTICIPATED. BUT IT LED TO THE CONCLUSION THAT PDI IS REQUIRED FOR THROMBUS FORMATION. NOW THERE ARE OTHER THIOISOMERASES THAT HAVE ACTIVITIES TOO. THERE'S PDI, ERP5, ERP57, AND SIMILARLY, THEIR ANTIGENS EXPRESS DURING THROMBUS FORMATION. THE ANTIGEN IS GREEN. WHAT DO THEY DO TO ACTIVITY? THE POINT I WANT TO MAKE IS THAT JUST AS PDI -- INHIBITS THROMBIN FOR MAIG, THAT'S ALSO TRUE FOR OTHER THIOISOMERASES, ERP5 AND ER57, ALTHOUGH PDI IS THE MOST POTENT. WHAT ARE THE THIOISOMERASES DOING, WHAT IS PDI DOING TO THE BLOOD COAGULATION SYSTEM? WHAT IS THE SUBSTRATE, IN OTHER WORDS, OF PDI. SO WE CONVERTED THE ACTIVE SITE OF PDI, WHICH IS NORMALLY -- THE ACTIVE SITE IN THE WILD TYPE CXXC, THEN WE CHANGED IT SO THAT IT AN ALANINE INSTEAD OF A CYSTINE. THIS LEADS TO THE FORMATION OF THE STABLE COMPLEX BETWEEN PDI AND ITS SUBSTRATE. THIS IS THE MECHANISM THAT'S BEEN DEFINED BY OTHERS FROM STUDIES INTRACELLULARLY, AND THIS IS THE MECHANISM FOR THE ACTION OF PDI, WHICH LEADS FROM THE -- FROM REDUCED PDI GOING TO OXIDIZED PDI AND VICE VERSA FOR SUBSTRATE. AND IF YOU MODIFY THIS GROUP, SO THAT IT CAN CLEAVE THE COMPLEX BETWEEN SUBSTRATE AND PDI THE COMPLEX IS FORMED. SO IN OTHER WORDS, WE CAN MAKE A VARIANT OF PDI WHICH REMAINS ATTACHED TO THE -- COVALENTLY THROUGH THE DISULFIDE TO ITS SUBSTRATE. TO FIGURE OUT WHAT THE SUBSTRATE WAS, WE MADE A WHOLE SERIES OF WHAT WE CALL TRAPPING MUTANTS, AND THEY DIFFERENT VARIATIONS IN CHANGES OF THE ACTIVE SITE. THIS PARTICULAR MUTANT IS COMPLETELY INACTIVE BECAUSE IT DOESN'T HAVE CYSTINES, AND THIS IS THE WILD TYPE UP HERE. SO WE MIX PLATELET-RICH PLASMA THAT'S BEEN ACTIVATED WITH THESE DIFFERENT VARIANTS, WE SEE PDI HERE, PDI IS LABELED GREEN. BUT WHEN IT'S ATTACHED TO ITS SUBSTRATES, IT IS OBVIOUSLY OF HIGHER MOLECULAR WEIGHT. AND MY MASS SPEC, ONE CAN IDENTIFY A WHOLE SERIES OF PROTEINS. I WANT TO FOCUS ON -- VITRINECTIN. THESE ARE IMMUNOBLOTS OF VITRINECTIN BOUND TO PDI, AND YOU CAN SEE THAT PDI IS PICK UNDER WITH ANTIBODIES TO THE VITRONECTIN, AND HOW DOES PID MODIFY THESE SUBSTRATES? I'M NOT GOING TO EVEN TRY TO GO INTO THIS BUT THE MASS SPEC INDICATES THAT THERE ARE TWO DISULFIDE BONDS THAT ARE BROKEN IN VITRONECTIN, CYSTINE 137 TO 16 IS, AND CYSTINE 274 TO 453. WE TAKE ENDOTHELIAL CELLS IN THIS CASE, LABELED WITH A GREEN DYE, AND SEE THAT IN THE PRESENCE OF PDI, WE GET BINDING. IN THE ABSENCE OF PDI, WE GET NO BINDING TO ENDOTHELIAL CELLS. IN THE INTRAVITAL SYSTEM, WE SEE VITRONECTIN BINDING TO THE VASCULAR EDGE OF THE ENDOTHELIUM. AND WE AGAIN HERE SHOW THAT IN THE PRESENCE OF ANTIBODY TO PDI, WE BLOCK VITRONECTIN BINDING TO THE ENDOTHELIUM. SO IN SHORT, THIS LITTLE CARTOON SHOWS YOU THAT VITRONECTIN, WHICH IS A SIGNIFICANT PLASMA PROTEIN AT 300-MICROGRAMS PER ML, IS IN OUR PLASMA, AND DURING PLATELET ACTIVATION, IT LEADS TO THE CLEAVAGE OF THE DISULFIDE IN VITRONECTIN, WHICH THEN ALLOWS VITRONECTIN TO INTERACT WITH ONE OF THE IMPORTANT MEMBRANE PROTEINS ON THE ENDOTHELIAL PLATELET SURFACE. SO ONCE WE FORM THIS COMPLEX, IT ALLOWS FOR THROMBUS FORMATION. AND WE DON'T KNOW THE DETAILS YET, BUT VITRONECTIN IS REQUIREED FOR THROMBUS FORMATION. HERE SHOWS YOU KNOCKOUT MICE WITH VITRONECTIN DEFICIENCY, AND THEY CAN'T MAKE A THROMBUS MADE OUT OF PLATELETS, AND THE FIBRIN ACCUMULATION IS MARKEDLY REDUCED AS WELL. SO WHAT IS ALL OF THIS ABOUT? HERE'S PDI, LEADING TO MULTIPLE SUBSTRATES. THE ONLY ONE WE'VE REALLY STUDIED IN DETAIL SO FAR IS VI TROA VITRONECTIN, BUT ON THE LIST ARE FACTOR 5 -- THE WHOLE ISSUE OF BLOOD COAGULATION IS A VERY IMPORTANT CLINICAL PROBLEM. AND THERE HAVE BEEN A LOT OF WORK ON TISSUE FACTOR PATHWAY INHIBITOR, PROTEIN C PATHWAY, AND ANTITHROMBIN 3 PATHWAY, ALL OF WHICH ARE IMPORTANT, BUT THEY ALL OCCUR AFTER CLOTTING TAKES PLACE. TEAR A WAY OF TURNING IT OFF. THEY'RE NOT A WAY OF PREVENTING CLOTTING. WE DON'T WANT TO SIT HERE AND CLOT. SO I PROPOSE THE MASTER SWITCH HYPOTHESIS. THAT WE WANT TO BE PROTECTED FROM ACCIDENTAL THROMBOSIS, SO IN THE ABSENCE OF PDI, THROMBUS FORMATION CANNOT BE INITIATED. PDI LIVES INSIDE THE CELL. AND A NEEDS TO BE RELEASED WITH TISSUE INJURY AND PDI ACTIVATES SUBSTRATES BY MODIFICATION DISULFIDE BONSDZ, THE SO CALLED ALLOSTERIC DISULFIDES. SO THAT'S THE -- RELEASES SUPPRESSION OF THROMBUS FORMATION. I LIKE THIS ANALOGY. THIS IS THE ELECTRICAL SYSTEM IN OUR HOUSES. WE HAVE A MASTER SWITCH IN THE ELECTRICAL BOX, WHICH YOU CAN THROW AND NONE OF THE LIGHTS WILL WORK. BUT WHEN IT GOES ON, THEN THE INDIVIDUAL LIGHTS WILL WORK. JUST IN FINISHING, LET ME JUST MENTION THAT WE'VE USE ANOTHER TRAPPING MUTANT, FIRST COUSIN OF PDI, TO SHOW THAT THIS TARGETS THE LEK TIN PATHWAY OF COMPLEMENT ACTIVATION, THAT THESE PROTEINS ALL LEAD TO THE ACTIVATION OF COMPLEMENT. AND THIS IS THE PATHWAY, THE SO-CALLED LECTIN PATHWAY. THIOISOMERASES TARGET THESE PROTEINS, MODULATE THEM AND TURN THEM OFF. SO IN FINISHING, LET ME JUST SAY A COUPLE COMMENTS ABOUT CHRIS ANFINSEN. WHEN I CAME TO THE LAB, I WAS VERY GREEN AS A BIOCHEMIST, BUT VERY ACCOMPLISHED ACTUALLY AS A SAILOR. CHRIS TOLD ME, HE USED TO LOOK TO RECRUIT PEOPLE WHO COULD FULFILL THE NEEDS OF HIS STRING QAWR TE. HE PLAYED THE VIOLA. BUT TWO YEARS BEFORE I ARRIVED, HE BOUGHT HIMSELF A 31-FOOT BOAT, AND I'M GOING TO LET OTHERS TELL THE STORY, BUT I USED TO SAIL WITH HIM. AND THIS IS ON ONE OF THE CRUISES WHERE I WAS SAILING WITH HIM FROM ANNAPOLIS PART OF THE WAY TO FLORIDA. AND THIS IS CHRIS ON HIS BOAT. IT WAS VERY INTERESTING TO SPEND SO MUCH TIME WITH CHRIS. YOU'RE WITHIN 30 FEET OF HIM FOR TWO, THREE, FOUR DAYS, AND THIS WAS NOT A SCIENTIFIC EXPEDITION. HE TALKED ABOUT ALL KIND OF THINGS. AS ALAN MENTIONED, HE STARTED THE RESEARCH PROGRAM FOR PHYSICIANS, AND I STILL REMEMBER HIS STATEMENT TO ME. HE SAID, YOU KNOW, PHYSICIANS DON'T KNOW THE DIFFERENCE BETWEEN SERUM ASPARAGINE AND SERUM ASPARAGUS. SO HE WAS A VERY WARM PERSON. HE WAS EXTREMELY THOUGHTFUL, INTERESTING, AND THAT'S WHY THIS WAS FOR ME THE TURNING POINT IN MY CAREER AND DEVELOPMENT. THANK YOU VERY MUCH. [APPLAUSE] >> THANK YOU. QUESTIONS? >> DO YOU THINK PDI MIGHT BE IN GRANULES THEN, IN ENDOTHELIAL CELLS? >> I'M DEAF. >> OKAY. IS IT IN SECRETORY GRANULES AS OPPOSED TO -- >> GOOD QUESTION. IT'S BEEN SHOWN IN PLATELETS THAT IT'S IN ITS OWN GRANULES, A NEW GRANULE CALLED THE DENSE GRANULE -- THE T GRANULE. AND IN ENDOTHELIAL CELLS, IT APPEARS TO BE YET ANOTHER GRANULE. BUT IT'S NOT IN WHAT WE EXPECT EXPECTED. >> CONGRATULATIONS FOR SHOWING US SOME OF THE DYNAMICS OF THE PLATELET THROMBOSIS ON THE -- WALL OF THE BLOOD VESSEL. SO WE -- THE PLATELETS AND -- WITH IODINE AND I FOUND OUT THAT THERE ARE MORE THAN 10 TO 50 TIMES MORE -- MAN PLATELET. SO I WAS WONDERING, AS YOU WITH STUDYING THE DYNAMICS, HOW DOES IT RELATE TO THIS? >> THE QUESTION, AS YOU'RE STUDYING THE DIE IN THE DYNAMICS? >> THE ISSUE OF PLATELET TO FIBRO KNOW SIN ON THE -- WALL OF THE BLOOD VEST WILL. >> DR. FURIE HAS TROUBLE HEARING YOU. MAYBE YOU SHOULD DISCUSS THIS AFTERWARDS. >> SURE, YES. VERY WELL. THANK YOU. >> THANK YOU. >> WE ARE AMAZINGLY A FEW MINUTES AHEAD OF OUR SCHEDULE SO MAYBE AFTER THE NEXT LECTURE, WE CAN TAKE A FIVE OR 10 MINUTE BREAK BEFORE WE HAVE THE HALF A DOZEN OR SO COMMENTS BY ALUMNI OF THE LABORATORY. HOPEFULLY NOT LOSING MANY OF YOU AT THAT BREAK. WE'RE VERY PLEASED THAT OUR NEXT SPEAKER IS DR. ARTHUR HORWICH OF YALE UNIVERSITY. DR. HORWICH WAS IN THE FIRST CLASS OF UNDERGRADUATE MEDICAL STUDENTS AT BROWN UNIVERSITY AND FINISHED THAT, DID AN INTERNSHIP IN PEDIATRICS AFTERWARDS, AND ACTUALLY WE HAVE ANOTHER PERSON WHO HAS A SIMILAR BACKGROUND WHO WILL SPEAK LATER TODAY. BUT THEN WENT TO THE SAULK INSTITUTE TO CONTINUE HIS TRAINING AND WAS LURED FROM CLINICAL MEDICINE TO BASIC RESEARCH IN THAT EXPERIENCE, AND AFTERWARDS WENT BACK TO YALE, FIRST JOINING A GOOD COLLEAGUELY ON COLLEAGUE LEON ROSENBERG, AND DEVELOPING HIS OWN LABORATORY WHERE HE HAS THRIVED FOR THE LAST 30 YEARS OR SO, BEING ONE OF THE FIRST DISCOVERERS OF THE PROTEINS WE NOW CALL CHAPERONINS, AND VERY PLEASED THAT DR. HORWICH AS WITH OUR OTHER SPEAKERS HAS RELATED HIS OWN WORK TO THE WORK THAT WAS DONE HERE 60 YEARS AGO, AND WE'LL HEAR THE STORY OF CHAPERONIN-ASSISTED PROTEIN FOLDING AS A MODERN WAY OF LOOKING AT SOME OF THE REFOLDING STUDIES THAT WERE DONE HERE IN EARLIER ERA. DR. HORWICH HAS RECEIVED MANY HONORS FOR THESE STUDIES, WHICH I WILL NOT ENUMERATE IN THE INTEREST OF TIME. ARTHUR? >> THANK YOU, ALAN. I'M REALLY HUMBLED AND HONORED TO BE HERE. I ALSO UNFORTUNATELY DID NOT MEET CHRIS DURING HIS LIFETIME, BUT HE HAS HAD A STRONG INFLUENCE ON MY WORK AND ON THE WORK OF HUNDREDS OF OTHER PEOPLE WHO STUDY MOLECULAR CHAPERONES IN A FIELD THAT SORT OF I'VE WATCHED GROW UP, AND SO IT'S REALLY AN HONOR TO GET TO TALK TO YOU A LITTLE BIT TODAY ABOUT WHAT I VIEW TO BE A SORT OF KINETIC ADD-ON TO WHAT CHRIS SO BEAUTIFULLY DESCRIBED IN THERMODYNAMIC TERMS. SO THIS IS JUST TO REITERATE THAT WE'RE TALKING ABOUT THE LAST STEP OF INFORMATION TRANSFER IN THE CELL. NAMELY PROTEIN FOLDING. AND A LOT OF PEOPLE WERE RECOGNIZING THESE THINGS IN THE LATE 50s. AND HOWEVER, CHRIS' WORK MADE A BIG RECOGNITION OF THIS STEP THAT OTHER MOLECULAR BIOLOGISTS WERE NOT REALLY PAYING SUCH ACUTE ATTENTION TO AT THAT POINT THE. AND I THINK THAT THE DISCOVERY THAT HE MADE TRULY DOES RING DOWN 60 YEARS AS ONE OF THE GREATEST EXPERIMENTS OF ALL TIME. SO I WAS AN UNDERGRADUATE AT BROWN IN OCTOBER OF 1972, AND I'D NEVER HEARD ABOUT THIS EXPERIMENT THAT I WAS READING ABOUT, I WAS IN A BIOCHEMISTRY COURSE AND READING ABOUT OTHER WORK. AND THIS EXPERIMENT WAS JUST THE MOST SPECTACULAR THING I'D EVER HEARD. I WAS WORKING IN THE LAB OF JOHN FANE WHICH STUDIED FAT CELLS UNDER THE TUTELAGE OF MIKE CHECK CHECK, U. MASS WORCESTER. IN FACT, HE TAKES CLAIM TO RECRUITING CRAIG MELLOW THERE, BUT HE'S DONE A LOT OF NICE EXPERIMENTS HIMSELF ON THE INSULIN AND GLUCOSE PATHWAYS. MIKE AND I WERE SO STUNNED LOOKING AT THE "NEW YORK TIMES" ARTICLE ON CHRIS' EXPERIMENT THAT WE LITERALLY ROUSED ABOUT THE LAB TO FIND PROTEINS THAT WE COULD ACTUALLY TEST REFOLDING WITH. BUT THERE WERE A LOT OF OTHER EXPERIMENTS THAT WE DID HAVE TO DO AT THAT POINT. BUT THIS REALLY STUCK IN MY MEMORY. AND SO REALLY, I NEVER THOUGHT I WOULD COME IN THIS DIRECTION PARTICULARLY, AND 15 YEARS WENT BY DURING WHICH I RECEIVED MY MEDICAL TRAINING AND BASIC SCIENCE TRAINING, SOME OF IT ON RETURN TO YALE. AND SO THAT BROUGHT US TO FALL 1987, WHERE WE SUDDENLY WERE IN A POSITION TO ASK AN INTERESTING QUESTION ABOUT FOLDING IN THE CELL. MOST MITOCHONDRIAL PROTEINS, MITOCHONDRIA AS THESE ORGANELLES THAT SUPPLY ENERGY TO OUR CELLS, MOST OF THE PROTEINS INSIDE ARE ENCODED IN THE NUCLEAR GENOME, THEN THEY'RE TRANSLATED IN THE ALL BEAR A SORT OF END TERMINAL AMINO ACID SEQUENCE, A LITTLE PEPTIDE TICKET THAT CONNECTS THEM TO MITOCHONDRIA AND DIRECTS THEM INTO THE ORGANELLES, BUT IMPORTANTLY, AN EXPERIMENT IN 1986 FROM A VERY FAMOUS MY MY COTOE -- HAD SHOWED TO CROSS THE MITOCHONDRIAL MEMBRANES, THEY HAVE TO BE UNFOLDED. SO WE WERE STUD STUDYING THE CYST IT TEM WE WERE USING AS A REPORTER ON PROTEIN IMPORT IN HAPLOID YEAST WHERE WE WERE USING A HUMAN MITOCHONDRIAL PROTEIN, IT PROGRAMMED ITS INDUCIBLE EXPRESSION IN THE YEAST CELLS AND SURE ENOUGH, THE PROTEIN WENT IN TO MITOCHONDRIA AND IT HAD ITS SIGNAL PEPTIDE CHOPPED OFF AND ASSUMED AN ENSIGHENZYMATIC FORM. IN THE YEAST WE WERE ABLE TO SUDDEN SUDDENLY ASK A QUESTION, NAMELY, WHEN PROTEINS CROSS THE MITOCHONDRIAL MEMBRANES TO REACH THIS INNERMOST MATRIX COMPARTMENT, ARE THEY SPONTANEOUSLY FOLDED, AS MANY OF CHRIS' EXPERIMENTS CERTAINLY SUGGESTED THAT MANY PROTEINS WERE ABLE TO DO, OR DID THEY REQUIRE SOME KINETIC ASSISTANCE? IN OTHER WORDS, SURE, THEIR PRIMARY STRUCTURE WAS NECESSARY TO DIRECT THEM TO THE NATIVE STATE, BUT MAYBE INSIDE CELLS, PROTEINS DO NEED SOME KINETIC ASSISTANCE SO THEY DON'T GO THE WRONG WAY WHEN THEY'RE USING THEIR AMINO ACID SEQUENCE TO DIRECT FOLDING. SO COULD THERE BE A MACHINE IN THE MITOCHONDRIAL MATRIX THAT ACTUALLY ASSISTS PROTEIN FOLDING IN THIS COMPARTMENT? IF THAT WERE SO, THEN IN OUR PARTICULAR MUTANT, THE PRO TEEPS WOULD BE UNFOLDED, THEY'D CROSS THE MEMBRANES, THEY'D FULLY ENTER THE MATRIX COMPARTMENT, THEY'D HAVE THEIR SIGNAL PEPTIDES CLEAVED SO THEY'D GET SLIDELY SMALLER AS A RESULT OF THAT, IF YOU LOOK AT THEM, FOR EXAMPLE, IN AN SDS -- BUT THEY WOULD HAVE NO ENZYMATIC ACTIVITY. SO WITHIN A WEEK OR TWO OF HAVING THIS IDEA THAT MAYBE THERE COULD BE A MACHINE THAT FOLDS PROTEINS IN MITOCHONDRIA, WITH HE ACTUALLY FOUND A MUTANT WITHIN OUR COLLECTION OF MITOCHONDRIAL IMPORT MUTANTS WHERE EXACTLY THAT HAPPENED. A REPORTER PROTEIN WENT FULLY INTO THE MITOCHONDRIAL MATRIX, ITS SIGNAL PEPTIDE WAS CLEAVED, THERE WAS A TON OF THE MATURE FORM OF THE PROTEIN, BUT IT WAS ABSOLUTELY INACTIVE BIOLOGICALLY BIOLOGICALLY. SO THIS SEEMED LIKE REAL HERESY AT THE TIME, ALTHOUGH I NEVER THOUGHT IT WAS PARTICULARLY HERESY, I ALMOST ALWAYS BELIEVED IN THE THERMODYNAMIC HYPOTHESIS, BUT WE DECIDED TO LOOK AT ANOTHER PROTEIN, AND THEN WE STARTED TO LOOK AT A NUMBER OF OTHER PROTEINS, SO FIRST WE LOOKED AT THE BETA SUBUNIT OF THE F1ATPA, SO THIS IS THE ENERGY PROVIDING GIZMO THAT POINTS INTO THE MITOCHONDRIAL MATRIX, THE BETA SUBUNIT AS PART OF THIS STRUCTURE, JOHN WALKER AND HIS CO-WORKERS SOME YEARS LATER DETERMINED ITS STRUCTURE IN THE WHOLE ROTARY MECHANISM OF ATP SYNTHESIS. AND SURE ENOUGH THE BETA SUBUNIT WAS ALSO PRETURNED, IT NEVER MADE IT INTO THIS STALK. SO NOW WE WERE FACING SOME REAL DOUBTS. WE FELT LIKE WE'RE SEEING SOMETHING THAT IS EXACTLY WHAT WE WERE LOOKING FOR, BUT NOBODY WILL EVER BELIEVE US. AND SO AT THAT POINT, THE PHONE RANG AND IT WAS A CALL FROM ONE OF THE MAJOR MITOCHONDRIAL INVESTIGATIVE LABORATORIES IN EUROPE FROM WALTER NOYBERT'S LAB. HE ASKED WHETHER WE NEEDED SOME HELP STUDYING OUR MUTANTS, AND I SAID WE ABSOLUTELY NEED HELP. THIS IS A LEVEL OF BIOCHEMISTRY THAT'S A LITTLE BIT BEYOND US, AND I DIDN'T TELL HIM ABOUT THIS MUTANT AT THAT POINT, BUT HE SAID YOU SHOULD COME OVER AND HAVE A CHAT WITH US. SO I GO OVER AND IT PRENTSZ SOME OF THE MUTANTS WE HAVE THAT AFFECT, FOR EXAMPLE, THE CLEAVAGE ENZYME THAT TAKES OFF THE SIGNAL PEPTIDE AND EVERYBODY IS NODDING YES, YES, YES, AND THEN I TELL THEM ABOUT THIS MUTANT THAT WE HAVE WHERE PROTEINS ENTERED THE MITOCHONDRIA AND THEY DON'T SEEM TO BE ABLE TO FOLD AN ACTIVE STATE THE AT THAT POINT. AND I WATCHED THEIR JAWS DROP BECAUSE THEY ALSO FELT LIKE THIS WAS REALLY A HERETICAL IDEA, AND SO THEY THOUGHT, WELL, YOU KNOW, MAYBE THE PROTEINS ARE COMING IN THROUGH THESE SITES -- THESE MEMBRANE IMPORT SITES AND THEY'RE JAMMING IN THE IMPORT SITE. SO IMAGINE THAT YOU'RE COMING IN END TERMINUS FIRST, THE SIGNAL PEPTIDE COULD BE CLEAVED BUT THE REST OF THE PROTEIN WOULD BE JAMMED IN THE IMPORT SITE. SO THAT WOULD LOOK LIKE A FOLDING DEFECTIVE MUTANT BECAUSE IT CAN'T REALLY FOLD AND IT'S NOT REACHING THE MATRIX, IT'S JUST THAT ITS END TERMINUS IS BEING CLEAVED. I SAID, WELL, I HAVE SOME DOUBTS ABOUT THAT, BUT WE'LL SEND YOU THE STRAIN, YOU CAN MAKE MITOCHONDRIA FROM IT AND SEE WHAT YOU SEE. SO TWO WEEKS LATER, A SENIOR POSTDOC IN THE GROUP CALLED BACK AND SAID, YOU KNOW, YOU'RE RIGHT, THE PROTEIN IS FULL, WE'VE MADE MITOCHONDRIA FROM YOUR MUTANT STRAIN, THE PROTEIN, WHEN IT'S IMPORTED, GOES FULLY INTO THE MATRIX COMPARTMENT, AND IT'S NOT SUSCEPTIBLE TO EX-OJ EXOGENOUS PROTEASES -- THROUGH WHICH IT ENTERS THE ORGANELLES, AND YOUR IDEA THAT MAYBE THIS IS A FOLDING DEFICIENCY SEEMS REALISTIC. SO SDLY WE HAD SUDDENLY WE HAD THE ATTENTI ON OF A REALLY SERIOUS GROUP OF MY TOE MITOCHONDRIAL BIOCHEMISTS,. YOU'LL NOTICE MOST OF THESE PROTEINS ARE OLIGOAMERICA ASSEMBLIES BUT THIS ONE IS NOT. THIS IS THE IRON SUL FAWR PROTEIN, A MEMBER OF THE BC1 COMPLEX OR COMPLEX 3 THAT HAS TO DO WITH THE RESPIRATORY CHAIN IN THE INNER MITOCHONDRIAL MEMBRANE. FROM STUDIES THAT THEY AND OTHERS HAD CONDUCTED, WE KNEW THAT THE RISCHE PROTEIN COMES IN AND IS CLEAVED TWICE, AND IT'S A MONOMER DURING ITS ENTIRE LIFETIME IN THE MITOCHONDRIAL MATRIX BEFORE IT'S INSERTED INTO THE INNER MEMBRANE, WE KNOW NOW THE INSERTASE IDENTITY IN ALL OF WHAT I'VE TOLD YOU IS, IN FACT, CONFIRMED. AND SO THE FACT IS THAT WHEN WE LOOKED AT THIS PROTEIN, IT REACHED THE MATRIX COMPARTMENT BUT IT ONLY UNDERWENT ONE CLEAVAGE. THE SECOND CLEAVAGE IS DEPENDENT ON GETTING AN IRON SULFUR CLUSTER WHICH REQUIRES FOLDING OF A DOMAIN OF THIS PROTEIN. WE KNOW THIS FOR A FACT NOW. AND SO WHEN WE SAW THAT, WE SAID, WOW, IT LOOKS LIKE A MONO AMERICA PROTEIN IS AFFECTED SO WE'RE NOT LOOKING AT SOMETHING THAT HAS TO DO WITH ASSEMBLING SUBUNITS, IT HAS TO DO WITH FOLDING MONO AMERICA PROTEINS ENTERING THE MITOCHONDRIAL MATRIX. SO WE WENT AHEAD TO RESCUE THIS PARTICULAR MUTANT AND IT WAS RESCUED BY A YEAST GENE ENCODING WHAT HAD BEEN IDENTIFIED A YEAR OR TWO BEFOREHAND AS A HEAT SHOCK PROTEIN TEEN THAT RESIDES IN MITOCHONDRIA. SO WHEN YOU TAKE CELLS TO A HIGH TEMPERATURE, THEY INDUCE A LOT OF PROTECTIVE PROTEINS AND THIS IS ONE THAT RICHARD HALL BERG IN HIGH WA HAD IDENTIFIED, AS A THERMALLY INDUCED PROTEIN, ONLY TWO OR THREE FOLD INDUCED BUT NONETHELESS PRESENT IN MITOCHONDRIA AS A LARGE ASSEMBLY. SO HE'D BEEN STUDYING THIS PROTEIN. WE CALLED HIM UP AND SAID, WELL, THE YEAST SEQUENCE? AND HE SAID YES, I AM. I IDENTIFIED THE HOMOLOGUE IN YEAST. AND WE MATCHED OUR DNA SEQUENCE DIRECTLY WITH HIS IN CODING THIS ASSEMBLY. SO SUDDENLY WE KNEW THIS THIS LARGE DOUBLE RING ASSEMBLY THAT HALL BERG HAD BEEN LOOKING AT AS A HEAT SHOCK PROTEIN WAS, IN FACT, SOME SORT OF A FOLDING MACHINE INSIDE THE MITOCHONDRIAL MATRIX. AND SO THIS DOUBLE RING ASSEMBLY IS TWO STACKED RINGS, EACH WITH 7 RADIALLY SMIMENT TRICK IDENTICAL MEMBERS, SO IT'S A TETRA DECK MER OF IDENTICAL SUBUNITS AND DECIDED TO DUB IT HEAT SHOCK PROTEIN 60. NOW THAT'S A LITTLE BIT OF A MISNOMER BECAUSE WHEN WE MADE THE DELETIONS IN YEAST, YOU NEED HSP60 UNDER ALL CONDITIONS TO HAVE A VIABLE CELL. THE MINUTE YOU SHIFT -- THE MINUTE YOU REMOVE THE NORMAL COPY OF HSP60, YOU HAVE A DEAD CELL AND OUR ORIGINAL MUTANT WAS A TEMPERATURE SENSITIVE LETHAL MUTANT. WITHIN 15 MINUTES OF THE TIME YOU SHIFTED FROM 23 TO 37 DEGREES, THE MUTANT COMPLETELY STOPPED GROWING IN WHATEVER PHASE OF THE CELL CYCLE IT WAS IN AND ARRESTED COMPLETELY. SO IN A SUBSEQUENT EXPERIMENT, TO MAIL DOWN THIS FOLDING REACTION A LITTLE BETTER, YOAKAM OWES TERMAN AND ULLRICH CARRIED OUT SOME EXPERIMENTS WHERE THEY COULD SHOW THAT A MONOMERIC PROTEIN, WHEN IT HAS AN END TERMINAL SIGNAL PEPTIDE ATTACHED TO IT BY MOLECULAR MANIPULATION, IT NOW EB TERES THE MITOCHONDRIAL MATRIX AND AT LOW LEVELS OF ATP, IT BECOMES STABLY ASSOCIATED WITH THE HSP60 COMPLEX IN A NON-NATIVE FORM THAT'S VERY PROTEASE SUSCEPTIBLE F YOU ADD -- BACK TO THAT SYSTEM, IT NOW ASSUMED A LEVEL HE PRO TEE -- FURTHER SUPPORT FOR THE IDEA THAT FOLDING INVOLVES COMPLEX FORMATION WITH HSP60 AND THEN SOME FORM OF ATP MEDIATED RELEASE. SO THE REST OF EVERYTHING I HAVE TO SAY NOW WILL JUST BE RELATED TO THIS BINDING, HOW DOES THIS GIZMO ACTUALLY WORK. SO I JUST WANT TO SAY FIRST THAT DOUBLE RING GIZMOS ARE NOT FOREIGN TO THE VARIOUS REDUCING COMPARTMENTS OF THE CELL. ARY NOT FOUND IN THE OXIDIZING COMPONENTS FOR EXAMPLE WHERE PDI AND REDUCTIONASE DO A MAJOR JOB ALONG WITH OTHER MACHINERIES. SO THESE DOUBLE RING ASSEMBLIES THAT ASSIST DE NOVO FOLDING TO THE NATIVE STATE ARE FOUND, FOR EXAMPLE, AND WE REALIZED THIS IMMEDIATELY WHEN WE SAW THE HSP60 SEQUENCE, THEY'RE FOUND IN MITOCHONDRIA, THEY'D BEEN STUDIED BY JOHN ELLISON, CO-WORKERS -- WHERE THEY HAD IMPLICATED THEM IN THE ASSEMBLY OF RUBISCO, COMPOSED OF LARGE SUBUNITS AND SMALL SUBUNITS, EIGHT OF EACH, AND HE'D SHOWN THAT THE LARGE SUBUNIT WHICH IS CHLOROPAST ENCODED IS ASSOCIATED WITH THIS BINDING PROTEIN, A DOUBLE RING ASSEMBLY IN THE CHLOROPAST STROMA, AND WHEN THE SMALL SUBUNITS IMPORT, NOW IT'S RELEASED FROM THE BINDING PROTEIN AND ASSEMBLES INTO THE HEXADECAMER. OBVIOUSLY THE SHOTS EXPERIMENTS AND OUR OWN EXPERIMENTS SUGGESTED THAT NO, THIS HAS TO DO WITH FOLDING OF THE RUBISCAL LARGE SUBUNIT. THE GROWING PROTEIN THAT HAD BEEN IDENTIFIED AS EARLY AS 1972 IN JAPAN AS A HEAT INDUCIBLE PROTEIN REQUIRED FOR LAMDAPHAGE ASSEMBLY WHEN IT INFECTS BACTERIA. SO FOR EXAMPLE, A MUTANT THAT IS AFFECTING THE E. COLI GROW EL GENE WILL NOT ALLOW LAMBDA TO MAKE NEW PHAGE PARTICLES AND BURST THE CELL SO YOU ESSENTIALLY ARE RESISTANT TO LAMBDA PHAGE DESTRUCTION AND THEY IMPLICATED THIS IN PHAGE ASSEMBLY BUT THEY ALSO RECOGNIZED THAT THIS THING WAS ESSENTIAL FOR VIABILITY OF E. COLI, FOR EXAMPLE, AND THAT THERE MUBL POST MUST BE HOST FUNCTIONS INVOLVED BUT AGAIN THESE EXPERIMENTS IN MITOCHONDRIA WERE SHIFTING IT TO POLYPEPTIDE CHAIN FOLDING. SO THEN WE LOOKED IN THE EUKARYOTIC CYTOSOL AND FOUND BY VIRTUE -- TO A THERMALLY INDUCIBLE CHAPERONE, THE EU CARE OH TICK CYTOSOL TCP1 OR CCT AS IT'S CALLED THAT HAS EIGHT DISTINCT SUBUNITS PER RING THAT'S CRITICAL FOR FOLDING ACTIN AND TUBE EU LIN TUBULIN, IT HAS VERY SPECIALIZED BINDING CAPACITIES THAT I WON'T TALK ABOUT HERE. SO ALL OF THIS IS HAPPENING IN 1989. INCLUDING PUBLISHED IN THE LAST ISSUE OF NATURE OF THE YEAR, A PAPER FROM GEORGE LAWYER MER'S GROUP FROM DU PONT IN WILMINGTON, RECONSTITUTING GROW EL GROW ES MEDIATED FOLDING IN VITRO. SO WHAT GEORGE AND HIS CO-WORKERS DID WAS TO DILUTE A SIMPLIFIED FORM OF ROUX RUBISCO, AND WHEN IT'S DILUTED FROM DENATURE -- THE PROTEIN JUST CRASHES, IT FORMS A WHITE MESS AT THE BOTTOM OF THE TUBE. IF YOU ADD A STOIK METRIC AMOUNT, YOU NOW FORM A STOIK STOIKMETRIC COMPLEX WITH NON-NATIVE RUBISCO AND IT'S LATENT, IT HAS NO CO2 FIXING ACTIVITY, IT'S A DEADEN SIEM, BUT IT'S NOT AGGREGATING. SO THE TETRADEC AMER APPARENTLY COMBINED A NON-NATIVE -- IN 91, 92 WE ALL DID ELECTRON MICROSCOPY AND SHOWED THAT NON-NATIVE PROTEINS BIND IN THE CENTRAL HOLE OF THIS DOUBLE RING CYLINDER, AND SO THIS IS A WAY OF PREVENTING AGGREGATION. THEN IN A MAGICAL STEP, IF YOU ADDED THE COCHAPERONIN WHICH IS A SINGLE RING COMPOSED OF 10,000 MOLECULAR MASS SUBUNITS AND MAGNESIUM A IT TP, HYDROLYZABLE ATP, OVER A PERIOD OF A FEW MINUTES, YOU STARTED TO SEE R.D UBISCO DIMERS FORMED THAT WERE ACTIVE. SO LAWYERMER LOOKED AT THE KINETICS AND DEDUCED THAT IT WAS FOLDING MON MERES OF RUBISCO AND THEY WERE SPONTANEOUSLY DIMERIZING, AGREEING VERY MUCH WITH OUR MITOCHONDRIAL EXPERIMENTS. SO HERE YOU HAD TO HAVE STOI COMETRIC OESs TO MAKE ASYMMETRIC COMPLEXES WITH GROW ES BINDING AT ONE OF THE ENDS COAXIALLY BINDING ONE OF THE ENDS OF THE CYLINDER AND WE REALLY DIDN'T UNDERSTAND WHAT WAS GOING ON IN THIS BLACK BOX. WE ALSO DIDN'T UNDERSTAND BINDING VERY WELL BUT THOSE WERE REALLY EARLY DAYS. SO I'M GOING TO REALLY FAST FORWARD, I THINK THE THING THAT REALLY HELPED US ALL WAS ACTUALLY THIS GUY, PAUL SIGLER, WHO MOVED FROM THE UNIVERSITY OF CHICAGO AWAY FROM STEVE'S GANG IN THAT DEPARTMENT, I THINK HE WAS MOLECULAR BIOLOGY, TO YALE. THIS IS ONE REASON WHY YALE RELU SOUGHT HIM, HERE'S A TRIPPER PRESSER OPERATOR STRUCTURE THAT HE'S LOOKING AT WITH WHOM HE COLLABORATED ON SOME BIOCHEMISTRY, AND ONE OF THE MASTER CRYSTAL OG FERS OF OUR TIME, HE'S OF THE DENZO PROGRAM USED TO REDUCE VIRTUALLY ALL X-RAY DATA EVERYWHERE, AND HE WAS THE PERSON WHO -- I HAD TO GO SEE PAUL FOR ADMINISTRATIVE MANNER, I GO ACROSS TOWN, PAUL SAYS I DON'T WANT TO TALK ABOUT ADMINISTRATIVE STUFF, LET'S JUST TALK ABOUT THESE PAPERS YOU PUBLISHED ON THE CHAPERONINS, YOU'RE GOING TO NEED A STRUCTURE IF YOU REALLY WANT TO UNDERSTAND WHAT'S GOING ON. I SAID GREAT, LET'S WORK ON THIS. AND HE COMES IN THE ROOM AND PAUL TELLS HIM ABOUT THIS DOUBLE RING THING THAT IS 850 -- HE JUST SAYS, THERE'S NO COMPUTING THAT CAN HANDLE THIS THING AT THIS POINT IN HISTORY. I BET IT'S GOING TO TAKE YOU TWO OR THREE YEARS TO SOLVE THIS STRUCTURE AND WHEN YOU DO, BY THAT POINT, WE'LL BE ABLE TO HANDLE THIS. HE TURNED OUT TO BEING EXACTLY RIGHT. IT TOOK US THREE YEARS TO GENERATE DECENT CRYSTALS AND HERE IS MY STUDENT WHO GENERATED THESE WONDERFUL CRYSTALS THAT DEFRACTURED TO BETTER THAN 3 ANG 3 ANGSTROMS, AND I CERTAINLY REMEMBER THE DAY SHE SNATCHED ONE OF THESE CRYSTALS AND PUT THE IT IN THE AREA DETECTOR IN OUR SIDE ROOM, WE'RE NOW SIDE BY SIDE IN A NEW BUILDING, AND PAUL JUST COMES RUNNING IN AND JUMPING OFF THE CEILING BECAUSE THIS THING IS DEFRACTURING TO 3 1/2 ANGST 3 1/2 ANGST 3 1/2 3 1/2 ANGST STROMS. WE HAVE WOON DER FULL NATIVE DATA SE. PAUL SITS US DOWN FOR A SIX-HOUR REVIEW OF HOW TO MAKE HEAVY ATOM DERIVATIVES FROM STEM TO STERN, AND A MONTH LATER, WE HAD A DECENT HEAVY ATOM DERIVATIVE, COLLECTED THE DATA ON THAT AND SPISHAK LITERALLY SOLVED THE STRUCTURE IN 24 HOURS. HE'D BEEN THINKING ABOUT THIS PROBLEM AND HOW HE WAS GOING TO DEAL WITH ALL THESE SYMMETRIES AND SOLVE A PATTERSON IN NO TIME. SO I WON'T GO THROUGH THE DETAILS OF STRUCTURAL SOLUTION BUT HERE IS THE THING IN LIVING COLOR. SO AT ROUGHLY 2.8 ANGSTROMS RESOLUTION, SO WE WERE ABLE TO RESORT TO NON-CHRIS TOLL GRAPHIC SYMMETRY AVERAGING AROUND THE RINGS TO HELP WITH EXTENDING PHASES AND RESOLVING THIS LARGE ASSEMBLY. SO THE RINGS ARE POSITIONED BACK TO BACK, AND THEY'RE ESSENTIALLY SEVEN TWOFOLD SYMMETRY AXE GOING AROUND THE RING. SO WHAT ONE OBSERVES IS THAT THE BASE OF THIS ASSEMBLY IS THESE EQUATORIAL DOMAINS, WHICH MAKE VERY TIGHT CONTACTS GOING AROUND THE RINGS AND WITH EACH OTHER ACROSS THE RINGS. SO THEY'RE HOLDING THE MACHINE TOGETHER AND HAVE AN ATP BINDING SITE IN EACH OF THEIR DOUGH DOMAINS. THEY'RE CONNECTED COVALENTLY THROUGH THESE INTERMEDIATE DOMAINS THROUGH THE TERMINAL APICAL DOMAINS AT EACH END OF THE CYLINDER. AND THEY ARE REALLY SORT OF FLYING IN THE BREEZE IF YOU LOOK, THEY JUST HAVE ONE OR TWO CONTACTS WITH EACH OTHER, AND SO YOU CAN MANL WHEN ATP BINDS, THIS THING CAN SORT OF OPEN LIKE A FLOWER, THE APICAL DOMAINS COULD OPEN. BUT WE HAD ALREADY COLLECTED ALL THIS EM DATA THAT INDICATED THAT -- POLYPEPTIDES BIND IN THIS HOLE. SO WHAT ARE THEY BINDING TO? WE THOUGHT THERE MIGHT BE SOME SURFACES THAT THEY WOULD DEFINITELY BEING ASSOCIATING WITH, AND THAT WAS SHOWN BY MEU TA GENESIS EXPERIMENTS. SO NOW IMAGINE YOUR STANDING INSIDE THE TOP RING OF A GROWING OEL BUT IMAGINE THIS IS THE TOP RING AND YOU'RE JUST LOOKING AT THE BACK WALL. ON THE APICAL DOMAINS, WE SAW ALL THESE RESIDUES POINTING INTO THE CAVITY. SO WE MUTATED INDIVIDUALLY, ALL THESM AT ONCE AND TESTED WHETHER E. COLI WAS VIABLE IN THE SETTING OF MUTATIONS THAT TURN EACH HYDROPHOBIC RESIDUE INTO A HYDROFI LIQUOR ELECTROSTATIC RES RESIDUE. HE WITH CAME INWE CAME IN THE NEXT MORNING AND ALL THEM WERE DEAD AS A DOOR NAIL. SO THEY'RE CRUCIAL FOR THE VIABILITY OF GROW EL AND WHAT THEY MUST BE DOING IS OBVIOUSLY BINDING OTHER HYDROPHOBIC SURFACES, SO THINK ABOUT OUR PROTEIN. NORMALLY IT HAS A -- BUT WHEN IT'S UNFOLDED THAT INTERIOR BECOMES EXPOSED TO SOLVENT, AND SO HERE THIS THING IS SPECIFICALLY BONDING NON-NATIVE PROTEINS THROUGH ITS OWN PRO IF HE EVERRED HYDRO -- IT'S SURROUNDING THE POLYPEPTIDE WITH SEVEN BINDING SURFACES SO IT'S ESSENTIALLY ENGULFING IT. SO IT HAS NO CHANCE TO AGGREGATE WITH OTHER PROTEINS. IT'S TRAPPED IN A BINARY COMPLEX IN THIS ASSEMBLY. AND SO THIS WAS ACTUALLY SHOWN BOTH AT A GENETIC LEVEL WHERE MY LONG TIME RIGHT HAND TECHNICAL SUPPORT PERSON AND I MADE COVALENT RINGS IN E. COLI SO AT THE CODING SEQUENCE LEVEL, WE MADE THE RINGS A SINGLE COVALENT MOLECULES, AND THEY FORMED DOUBLE RINGS IN E OWE LIE, SO NOW YOU COULD MAKE MUTATIONS IN ANY ONE OF THE APICAL DOMAINS, IN ANY COMBINATION OR PERMUTATION. IN FACT, WHAT WE OBSERVED WAS THAT YOU NEEDED THREE OR FOUR CONSECUTIVE NORMAL WILD TYPE HYDROPHOBIC APICAL DOMAINS. AND THAT WAS U.S. PISHT TO FIND AND FOLD VIRTUALLY ANYTHING WE RECOGNIZED AS A SUBSTRATE OF THE MACHINE FOR OUR VARIOUS IN VITRO EXPERIMENTS AND IN VITRO, THEY ALSO SUPPORTED GROW EL DEFICIENT MUTANTS. IF YOU MADE LARGER NUMBERS OF DEFECTIVE SUBUNITS, NOW YOU HAD AN INVALUABLE CELL. THEN MORE RECENTLY IN CRYO-EM STU DISIS CARRIED OUT COLLABORATIVELY OVER MANY YEARS, WE'VE OBSERVED, FOR EXAMPLE, THAT -- IN FACT, CAN BECOME ABUTTED AGAINST THREE OR FOUR OF THESE APICAL DOMAINS IN A STABLE COMPLEX. AND I SHOULD POINT OUT THAT THIS IS A NON-NATIVE FORM OF MDH SO IT'S AN ENSEMBLE OF A ZILLION DIFFERENT CONFORMATIONS AND IT REPRESENTS ONLY A PART OF THE DENSITY OF WHAT WOULD BE THE NATIVE PROTEIN OF AN MDH SUBUNIT. SO THEN IN COLLABORATION WITH CURT FUTRICH, WE CARRIED OUT AB AN EXPERIMENT LOOKING AT A SELECTIVELY LABELED SUBSTRATE PROTEIN WHILE IT WAS BOUND IN A SINGLE RING VERSION OF GROW EL AND WHAT WE SAW WAS JUST RANDOM COIL RESONANCES, SO IN THIS EXPERIMENT YOU HAVE AN N15 THAT'S BECOME BOUND TO A SINGLE RING VERSION OF GROW EO AND IT'S ACTING LIKE IT HAS NO ORDERED SECONDARY STRUCTURE, NO ORDERED TERTIARY STRUCTURE. WHEN YOU ADD, HOWEVER, TO THAT SAME COMPLEX GROW ES AND ATP AND ALLOW -- TO REFOLD IN IT AS I'LL COME TO A IN EKD IS, YOU'LL NOW SEE A NATIVE SPECTRAL PROPERTY, SO HERE COME THE 180 RESIDENCES BACK TO DISCRETE -- DISBURSED RESIDENCES AND HERE ARE ESSENTIALLY 19 LUCINES THAT ARE PRESENT IN DHFR. THE TRICK TO POLYPEPTIDE BINDING IS AGAIN WHEN YOU START EXPOSING THESE HYDROPHOBIC INTERNAL SEQUENCES EXTERNALLY DUE TO STRESS OR DURING THE PROCESS OF FOLDING OR FOR SOME OTHER REASON CAUSING MISFOLDING, NOW A PLEAK ALREADY CHAPERONE CAN PREVENT THIS FROM OCCURRING, CAN PREVENT HYDROPHOBIC SURFACES FROM ASSOCIATING WITH EACH OTHER, AND STABILIZING NON-NATIVE PROTEIN IN THIS CASE IN A CENTRAL CAVITY. THERE'S ALSO A LITTLE BIT OF A STRETCHING ACTION THAT'S BEEN OBSERVED ON THESE NON-NATIVE PROTEINS. ONE OF MY POSTDOCS CARRIED OUT EXPERIMENTS IN 2004 THAT SHOW THE PROTEINS ARE BEING ESSENTIALLY STRETCHED ON THE RACK BY THE SEVEN BINDING SURFACES WHEN THEY'RE BOUND IN THE CENTRAL HOLE OF GROW EO AND ADDITIONAL EPR EXPERIMENTS -- SO HOW DO PROTEINS FOLD? SO ONE OF THE FIRST QUESTIONS ABOUT HOW THIS FOLDING MACHINE WORKS IS THE PROTEINS HAVE TO BE RELEASED INTO SOLUTION WHERE THEY FOLD, OR ARE THEY ACTUALLY FOLDED INSIDE OF THE MACHINE? WELL, WHEN WE LOOKED AT ONE OF OUR FAVORITE SUBSTRATE PROTEINS, THE MON MORE, 33,000 MOLECULAR MASS SULFUR TRANSFERASE, WHEN WE IMPOSE JUST THE NATIVE STRUCTURE, WHICH IS A MORE COLLAPSED STRUCTURE THAN WOULD OCCUR IN THE NON-NATIVE STATE, IT BANGED INTO THE APICAL DOMAINS. WE JUST IMPOSED THIS -- HERE'S THE NATIVE STRUCTURE AND WE THOUGHT, HOW CAN A PROTEIN POSSIBLY FOLD IN THERE? IT CAN ONLY BE BOUND THEN IT HAS TO BE RELEASED INTO SOLUTION WHERE IT FOLDS? THEN HELEN SAY BELL CARRYING OUT EMs COMPLETELY CHANGED OUR MIND WITHIN A FEW WEEKS OF THE TIME WE HAD THAT PARTICULAR IMAGE ON THE SCREEN. IN THE LAB. SHE SHOWED THAT WHEN ATP AND GROW ES BIND TO ONE SIDE OF GROW EO TO FORM THIS I SYMMETRIC COMPLEX, THERE IS A BIG CAVITY OPENING SO HERE'S THE ORIGINAL UNLIGANDED TYPE OF RING WHICH IS CALLED THE TRANSRING OF THIS ASYMMETRIC COMPLEX, YOU CAN SEE IT'S THE USUAL DIAMETER HOLE THAT'S OPEN TO THE GREAT WORLD, BUT IT'S NOT VERY BIG AND AGAIN, WE COULDN'T SEE HOW ANYTHING COULD FOLD IN THERE, BUT LOOK WHAT HAPPENS WHEN GROW ES, THIS LITTLE TOP HAT, BINDS IN THE PRESENCE OF NUCLEOTIDE TO GROEL. THE APICAL DOMAINS OPEN UP HERE, REMEMBER THEY WERE FACING THE CAVITY, HYDROPHOBIC SURFACE FACING THE CAVITY, NOW THEY'RE OPENED UP AND ABUTTING GROES, AND ALL THIS IS A CHAMBER WHERE FOLDING COULD POTENTIALLY TAKE PLACE. SO JONATHAN WEISSMAN, POSTDOC IN THE LAB AT THE TIME, CARRIED OUT AN ORDER OF ADDITION PROTEOLYSIS EXPERIMENT. HE SAID OKAY, I'M GOING TO BIND A PROTEIN, I'M GOING TO ASK WHETHER IT CAN BE PROTECTED FROM PROTEASE UNDERNEATH A BOUND GROES. SO ONE ORDER WAS TO ADD GRO OH ES FIRST AND THEN POLYPEPTIDE. OKAY, YOU CAN'T -- YOU HAVE TO FIND IN TRANS. NOW WHEN YOU ADD PROAT NAIS K, THE PROTEIN IS COMPLEEPTLY DIGESTED, SO NOW REVERSE THE OR THE OF ADDITION, POLYPEPTIDE FIRST, THEN GROES. NOW POLYPEPTIDE -- OR IT'S A TRANSARRANGEMENT WITH POLYPEPTIDE HERE AND GROES ON THE OPPOSITE RING, IN WHICH CASE YOU SEE DIGESTION. SO THE POINT IS WE DID SEE A 50/50 DISTRIBUTION OF PROTECTION, AND IN FACT, WE COULD DO HIT-AND-RUN CROSS LINKING EXPERIMENTS THAT SHOWED THAT THIS PROTEIN IN HERE, IF IT HAD A HIT-AND-RUN CROSS LINGER ON IT WOULD CROSS THIS RING AND NOT THIS RING WHOSE TAILS ARE CLIPPED, SO THEY DANGLE INTO THE CENTRAL CALFITY AT THE LEVEL OF THE EQUATORIAL DOMAINS. I HAVEN'T REALLY TALKED ABOUT THEM, BUT THE POINT IS THAT WE COULD SEE THAT YOU COULD BE ENCAPSULATED. SO THE QUESTION THEN WAS, OKAY, CAN THIS ENCAPSULATED COMPLEX BE PRODUCTIVE? OR IS THIS THE PRODUCTIVE COMPLEX? I'VE GOT TO SAY THAT WE HAD 50/50 BETTS IN THE LAB, SOME PEOPLE SAY ONLY TRANSCAN REALLY BE FOLDING ACTIVE, YOU HAVE TO EJECT FROM THERE. JONATHAN AND I BOTH THOUGHT IT WOULD BE WHAT WE CALL CYSTS, IN THIS RING THAT WOULD BE FOLDING ACTIVE AND WE TURNED OUT TO BE RIGHT. SO IN SINGLE TURNOVER EXPERIMENTS THAT I WON'T DETAIL, WE COULD SHOW THAT IF YOU START WITH THIS TOPOLOGY, THE PROTEIN IS FULLY ACTIVATED IN ONE ROUND. THIS WAS WITH OUR OLD FAVORITE SUBSTRATE. SO NOW THE QUESTION WAS, IF YOU START FOLDING UNDER GROES IN A RING, CAN YOU COMPLETE IT IN THAT RING. SO WE MADE SAYING EL RING VERSION OF GROEL. THE REASON FOR THIS WAS THAT GEORGE LORE MER HAD SHOWN THAT YOU NEED ATM ACTION IN THE TRANSRING, HE THOUGHT IT WAS HYDROLYSIS BUT IT TURNS OUT IT'S JUST BINDING. IF YOU BIND ATP IN THE OPPOSITE RING, THAT EJECTS ALL THE LIGANDS FROM THIS RING. SO GROES LEAVES, POLYPEPTIDE IF IT'S IN THIS CAP TI LEADS, SO DOES -- LEAVE FROM THE EQUATORIAL DOMAINS. AND SO WHAT WE DECIDED TO DO WAS JUST REMOVE THIS RING ALL TOGETHER, SO WE MADE A MUTANT VERSION OF GROEL THAT'S AN OBLIGATE SAYING EL RING VERSION. BY MUTATING FOUR ELECTROSTATICS THAT LIE AT THE END OF EACH SUBUNIT THAT INTERACT WITH THE -- SUBUNITS IN THE OPPOSITE RING. SO NOW YOU COULD MAKE A SINGLE RING VERSION OF GROEL, YOU'D ADD TO IT AND IT WOULD ENCAPSULATE IT AND THE GROES WOULD JUST STAY ON INDEFINITELY. NOW WE PURIFYIED -- TO FORM A BINARY COMPLEX AND THEN ADDED AT P&G ROES TO SEE WHAT WOULD HAPPEN. COULD YOU REACH THE NATIVE STATE INSIDE OF THAT RING, AND HERE'S THE KINETICS OF THAT EXPERIMENT. SO INDEED SR1 IS FOLDING ACTIVE, IN THE SAME WAY THAT WILD TYPE GROEL, GROES ATP IS ACTIVE, GROWING AT EACH ROUND OF THE CHAPERONIN CYCLE AS I'LL OUTLINE IN THE NEXT COUPLE SLIDES, LEAVING AT EACH ROUND OF THE CYCLE WHETHER IT'S FOLDED OR NOT, AND SO THIS IS A NON-CYCLING STABLE COMPLEX ON TOP, AND WHAT WE COULD SHOW IS THAT YOU CAN ACTUALLY -- FILTER THESE TIME POINTS FROM THE SR1 REACTION AND CARRY OUT A -- ASSAY ON THIS COMPLEX OF SINGLE RING BOUND WITH GROES BOUND AND THE POLLLY PEPTIDE ENCAPSULATED UNDERNEATH AND OBSERVE THE SAME RECOVERY OF ACTIVITY OFF THE GEL FILL TRAIG TRAITION COLUMN OF THIS COMPLEX. SO IT COULD GO ALL THE WAY TO THE NATIVE STATE INSIDE THIS CHAMBER. SO THIS IS A SORT OF LIKE MINI COMPARTMENT WHERE FOLDING CAN OCCUR IN AN UNDISTURBED WAY. SO IT'S TRIGGERED BY ESSENTIALLY HAVING ATP IN THE RING, AND ACTUALLY WE COULD BREAK THAT DOWN. WE COULD PERFORM A -- WHERE THE POLYPEPTIDE IS STUCK ON THE CAVITY WALL AND DOES NOT FOLD. NOW WE SIMULATE ADDITION OF THE GAMMA PHOSPHATE AS AN ALUMINUM FLUORIDE METAL COMPLEX AND NOW YOU SEE ALL HELL BREAKS LOOSE AND THE POLYPEPTIDE FOLDS INSIDE THIS CAVITY. SO IT'S RELEASED FROM THE WALL BY THE GAMMA FOS FAI. YOU CAN SAY THIS IS A TRANSITION STATE ANALOG, WE CAN ALSO ADD THE GROUND STATE AND THAT WILL DO THE SAME THING, SO TRIGGERS -- SIMILARLY HAYS CARRIED OUT AN -- IT'S A STABLE FOLDING ACTIVE COMPLEX AND IT COULD FOLD AN EVEN LARGER PROTEIN, RUBISCO. SO WHAT DOES THE FOLDING CHAMBER LOOK LIKE? SHO NOW WE ARE L. TO MAKE GROEL GROES COMPLEXES. WHAT WAS REALLY DIFFICULT TO DO WAS TO HAVE THEM FREEZE THEM AND STAY STABLE AND DEFRACTURED AND BE ABLE TO DEFRACTURE REASONABLY WELL. SO A POSTDOC IN PAUL'S CREW SPENT ABOUT A YEAR TRYING TO FIGURE OUT HOW TO FREEZE THESE CRYSTALS AND EVENTUALLY WITH A EXTINGUISH TECHNIQUE WAS ABLE TO SOLVE THE PROBLEM. SO WHAT YOU SEE NOW, SO THIS WAS A MOLECULAR REPLACEMENT SOLUTION BUT ULTIMATELY ALL OF THE PHASES FROM THAT DETERMINATION WERE THROWN OUT AND WE DID RANDOM AVERAGING FROM RANDOM PHASES TO GET AN UNBIASED MODEL. WHAT YOU SAW WAS THAT THE TRANSRING LOOKS JUST LIKE AN UNLIGANDED OEL RING. DID TOAND INTERACT WITH THE POLY PEPTIDE BINDING HYDROPHOBIC SURFACE AND ACTUALLY IN THESE LOOPS IS THE HYDROPHOBIC SEQUENCE AND AN IVL EDGE TO A LOOP THAT INTERACTS WITH THE GROEL APICAL DOMAINS. SO THIS JUST SUMMARIZES THE MAJOR RIGID BODY MOVEMENTS OF ATP GROES BINDING SO THE APICAL DOMAINS ELEVATE 60 GREESES AND MAKE A HARD RIGHT TURN 90 DEGREES, SO HERE'S THE INITIAL UNLIE LYE GANDED OEL, NOW IT'S LOOKING OUT OF THE BOARD AT US AND IT'S BEEN REPLACED BY A COMPLETELY HYDROPHILLIC SURFACE, SO HERE'S GROES ON TOP OF THIS NOW ELEVATED STRUCTURE, ACTUALLY THE INTERMEDIATE DOMAINS CLUMP DOWN ON TO THE NUCLEOTIDE, LOCKING IT INTO PLACE AND ASSURING COMMITMENT TO HYDROLYSIS, AND THIS IS THE FOLDING CHAMBER. SO THIS HAS HYDROFI LICK WALLS, SO PROBABLY A PROTEIN OF UP TO 50 OR 60,000 DALTONS COULD FOLD INSIDE THE CAVITY. SO THIS COULD INITIALLY RECRUIT A NON-NATIVE PROTEIN INTO THE RING, AND HERE'S THE ENCAPSULATED CHAMBER IN WHICH SO ALL THE OF THIS SURFACE IS BLUE AND MOST OF THE YELLOW IS GONE, AND YOU COULD MUTATE THESE YELLOWS AND IT HAS NO EFFECT ON THE FOALT FOLDING REACTION. SO WHAT IS IT ABOUT THAT CHAMBER? WELL, IT ABSOLUTELY PREVENTS AGGREGATION. SO THERE ARE PERMISSIVE CONDITIONS WHERE YOU CAN STUDY THIS CHAPERONIN REACTION. A PROTEIN CAN REACH THE NATIVE STATE FREE IN SOLUTION IF YOU DILUTE IT UNDER PARTICULAR CONDITIONS, IT CAN REACH THE NATIVE STATE. AND UNDER THOSE SAME CONDITIONS, IT SEEMS LIKE IT FOLDS FASTER IF YOU HAVE GROEL/GROES, BUT THAT'S ACTUALLY NOT THE CASE. THE CASE IS THAT WHEN YOU TRY TO FOLD THIS THING WITHOUT THE BENEFIT OF THIS CAVITY, IT'S AGGREGATING, IT'S MAKING LOW ORDER -- VIRTUALLY ALL OF THE PROTEINS WE'VE STUDIED UNDER THIS CONDITION WITH MAYBE ONE EXCEPTION PERHAPS ARE SUBJECT TO THIS. IF YOU RELIEVE THIS BY ADJUSTING THE CONDITIONS OF SPONTANEOUS -- NOW YOU ACHIEVE THE SAME RATE AS OCCURS HERE. BUT UNDER CONDITIONS WHERE YOU'RE STILL MAKING THIS GARBAGE THAT GOES OFF PATHWAY AND HAS TO REVERSIBLY ULTIMATELY POPULATE THIS STATE AND REACH THE NATIVE STATE, WHEN YOU HAVE THE STRAIGHT TO THE NATIVE STATE. THERE IS NONE OF THIS STUFF BEING POPULATED BECAUSE THE PROTEIN IS IN SOLITARY CONFINEMENT. SO THIS TERM HAS BEEN USED FOR THIS CHAMBER. THE ANFINSEN FOLDING CHAMBER. IT WOULD BE LIKE CHRIS' EXPERIMENT CARRIED OUT THE IN INFINITE DILUTION, IN AN INFINITE SEA OF SOLVENT WHERE THE MOLECULE HAS NOTHING TO AGGREGATE WITH, SO IT'S JUST GOING TO EXPERIMENT IN THE IT FINDS ITS WAY DOWN THE ENERGY LANDSCAPE INTO THE NATIVE STATE. NEGATIVELY CHARGED RESIDUES, BLUE IS BASIC RESIDUES, SO IT'S BEING DIRECTED BY THE PRIMARY STRUCTURE IN THIS CHAMBER WITH NO CHANCE OF AGGREGATION. SO I'M JUST GOING TO CLOSE BY JUST A COMMENT OR TWO ON THE NUCLEOTIDE CYCLE. THIS IS THE FOLDING ACTIVE STATE IN ATP, THIS HAS HALF LIE LIFE OF ABOUT 10 SECONDS AT ROOM TEMPERATURE. THE END OF THAT TIME THERE'S A COMMITMENT TO HIGH DROALIZE ATP TO THE ADP FORM IN IS HAVE SHOAP SHOWN THERE'S A NEGATIVE COUPTIVITY BETWEEN RINGS. WHEN THIS GUY IS FULLY OCCUPIED WITH ATP, THIS GUY CANNOT HAVE ATP BOUND. THERE'S A NEGATIVE COUP COOPER COOPERATIVITY, AND WHEN THIS HYDROLYZES, NOW IT GATES THE ENTRY OF ATP INTO THE OPPOSITE RING AND THAT ARRIVAL AS I MENTIONED BEFORE TRIGGERS ALLOSTERIC REJECTION OF EVERYTHING THAT HAD BEEN IN THE CHAMBER. GROES LEAVES, POLYPEPTIDE LEAVES, IT WON'T REACH THE NATIVE STATE IN SOLUTION AS OTHERS HAVE SHOWN IN SOME REALLY ELEGANT EXPERIMENTS, IT HAS TO GO MAKE ANOTHER ROUND AT THE MACHINE. OKAY. SO I JUST WANT TO MAKE THIS COMMENT THAT IT COST HAVE TO DOES HAVE TO LEAVE AT EACH ROUND. IT CAN ESSENTIALLY BIND NON-NATIVE POLYPEPTIDES IF THEY WERE IN SOLUTION AND NEVER RELEASE THEM. SO IF THE REACTION IS COMMITTED IN SUCH A WAY THAT A POLYPEPTIDE HAS TO STAY AT THE MACHINE UNTIL IT REACHES THE NATIVE STATE, A TRAP WOULDN'T BOTHER THE REACTION. BUT IF IT'S NOT COMMITTED AND NON-NATIVE PROTEINS ARE LEAVING AT EACH ROUND AS WELL AS NATIVE FOLDED ONES, THEN THIS TRAP WOULD SLOW THE REACTION BECAUSE IT WOULD BIND THESE NON-NATIVE THINGS IRREVERSIBLY AND HALT FOLDING. THIS IS, IN FACT, WHAT WE OBSERVED. MUTANTS OF GROW EL THAT WE CALL TRAPS COMPLETELY STOPPED THE REACTION ON A DIME WHEN YOU ADD THEM, AND YOU CAN -- BY CARRYING OUT VARIOUS CHROMATOGRAPHY SEPARATION. SO I'M GOING TO END BY SHOWING YOU THIS WHOLE THING AS A CONNECT THE DOTS IN SUCH A WAY THAT WE'RE CONNECTING THE X-RAY AND EM STRUCTURES THAT WE HAVE TO GET AN IDEA OF WHAT THE REACTION CYCLE LOOKS LIKE. SO THIS IS STILL A SLIGHTLY UNRESOLVED AREA AFTER ALL THESE YEARS OF WHETHER YOU FORM SYMMETRIC COMPLEXES FOLDING IN BOTH RINGS OR WHETHER IT REALLY IS AN ASYMMETRIC MACHINE WHICH I HAVE FAVORED HISTORICALLY BUT I'M OPEN TO WHATEVER THE DATA IS GOING TO TELL US. SO CAN YOU RUN THE MOVIE FROM UP THERE, PLEASE? I DON'T HAVE A WAY TO INITIATE IT. THANK YOU. JUST CLICK THE BAR. YOU HAVE TO CLICK ON THE SCREEN. THEN CLICK THE BAR AND THE MOVIE WILL PLAY. SO NOW YOU'RE LOOKING AT A SUBUNIT OF GROW EO AND NOW YOU'RE LOOKING DOWN THE ASSEMBLY, HERE'S ALL THAT HYDROPHOBIC BINDING SURFACE, AND REMEMBER, THERE'S THOSE C TERMINAL TALES BLOCK THINGS OFF AT THE EQUATORIAL DOMAIN, SO IT'S REALLY TWO CALFITIES, HERE'S A NON-NATIVE PROTEIN COMPUTATIONALLY PULLED APART DHFR BINDING IN A RING. HERE COMES ATP THIS CAUSES A COUNTERCLOCKWISE TWIST OF THE APICAL DOMAINS BUT THEY'RE STILL MAKING CONTACT WITH POLYPEPTIDE. THERE IS HYDROPHOBIC SURFACE AND THERE'S A HUGE RIGID -- 120 DEGREE TWIST IN ELEVATION AND FOLDING IS REPRESENTED BY THE BLEUL BLINKING DOT. AND A NON-NATIVE PROTEIN CAN BIND IN THE OPPOSITE OPEN RING. THEN HYDROLYSIS OCCURS, PHOSPHATE LEAVES, ATP ENTERED HERE IS A GREEN. AND NOW YOU SEE THE APICAL DOUGH MAIPS HERE MOVING AND NOW GROW ES IS GOING TO BIND AND NOW EVERYTHING IS LEAVING THIS RING POST HYDROLYSIS, HERE THE'S THE -- WHICH HAS NOW BECOME THE FOLDING AK ITTIVE RING. SO THIS MACHINE CAN DEDICATE MULTIPLE CYCLES AND SOME ATP, ALTHOUGH IT'S NOT A HUGE ENERGY COST BUT IT COSTS SOMETHING, MUCH LESS THAN PROTEIN SYNTHESIS, TO MEDIATE CORRECT FOLDING OF POLYPEPTIDES. SO THAT IS OUR ADD-ON TO WHAT REALLY CHRIS SHOWED AT A THERMODYNAMIC LEVEL THAT IS REALLY THE GROUND ZERO OF PROTEIN FOLDING AS WE KNOW IT. SO THANKS VERY MUCH FOR YOUR ATTENTION. I'LL TAKE ANY QUESTIONS. [APPLAUSE] >> FIRST OF ALL, CONGRATULATING YOU, THIS IS FANTASTIC. I'VE BEEN FOLLOWING THIS WORK FOR -- >> THIS IS MOTHER NATURE, NOT US. >> I'VE BEEN FOLLOWING THIS FOR MANY YEARS. COULD YOU PLEASE COMMENT A LITTLE BIT ON THE ROLE OF THE P.I.? YOU SAID THE ATP SPLIT, THEN THE P.I. IS GOING IN AND COULDING SOME SIGNALING? >> WELL, WHEN ATP HIDE LOSES, PHOSPHATE IS REALLY FREE TO LEAVE AT THAT POINT. IT IS TO THE REALLY SEQUESTERED AT ALL. SO IT JUST DIFFUSES AWAY IMMEDIATELY. BUT ADP IS REALLY TRAPPED UNTIL ATP ARRIVES IN THE OPPOSITE RING AND CAUSES ESSENTIALLY A LITTLE BIT OF AN OPENING OF THE NUCLEOTIDE POCKET TO ALLOW ADP TO LEAVE. >> SO WE'RE REALLY TALKING ABOUT BINDING ENERGY CHANGE, RIGHT? IS THAT WHAT YOU ARE SAYING, THAT -- >> I MEAN, YOU COULD THINK OF IT THAT WAY. I MEAN, IT'S AN ORDERED SORT OF FORWARD PROGRESSION OF THIS MACHINE FROM CYCLE TO CYCLE. YEAH. I THINK THAT'S FAIR. I THINK PAUL -- HE WOULD DEFINITELY AGREE. >> WHAT PART OF THE LORIMAR MECHANISM DO YOU AGREE WITH AND DISAGREE WITH? >> NOW YOU'RE TRYING TO DWELL ON MY UNPUBLISHED DATA. I'M NOT GOING TO GO THERE. >> WE'LL TAKE A BREAK NOW AND REASSEMBLE IN 10 MINUTES THAT WOULD MAKE IT, I THINK, QUITE WHAT I WOULD SAY INTERESTING FOR CHRIS BECAUSE IT WAS A REAL CHALLENGE, WHAT WE KNEW IS THAT ISAAC AND LINDEMAN HAD DISCOVERED INTERFERON IN 1957, AND OF COURSE FLASH GORDON IMMEDIATELY PICKED UP ON THIS AND DECIDED THAT IT WAS POTENTIALLY MEDICALLY IMPORTANT AND CURED US FROM AN EXTRATERRESTRIAL VIRUS, AND I THINK WHEN LOOKING AT THIS, MANY PEOPLE REALIZE THAT THERE WAS A POTENTIAL MEDICAL BENEFIT FROM THIS MOLECULE, AND AT THAT TIME, CAREY CANTELE, WORKING IN FINLAND WITH A NUMBER OF COLLEAGUES, HAD PURIFIED PARTIALLY INTERFERON ABOUT 1% PURE AND WAS WORKING WITH CLINICIANS TO SEE IF INTERFERON IT ACTUALLY HAD ANY BIOLOGICAL ACTIVITY AGAINST VIRUSES AND POTENTIALLY AGAINST CANCER. BUT THERE WAS THIS ALWAYS UNDERLYING THOUGHT THAT MAYBE IT WASN'T INTERFERON BECAUSE THIS WAS ONLY 1% PURE, SO CHRIS AND HIS LAB EMBARKED ON THIS PARTICULAR PURIFICATION, AND IT WAS QUITE A DIFFICULT PROJECT, AND MANY, MANY POSTDOCS AND INDIVIDUALS PARTICIPATED IN THIS& AND I WOULD SAY THE TEAM THAT WORKED ON THIS FROM THE 70s TO THE 80s INCLUDEED MANY OF CHRIS' COLLEAGUES FROM THE WEISSMAN INSTITUTE, HERE IN THE UNITED STATES, LILA CORLEY, HIS TRUSTED TECHNICIAN WHO WORKED WITH HIM MANY YEARS, THEN PAM, MARK SMITH AND MYSELF. THIS WAS A PROJECT THAT WENT ON FOR SEVERAL YEARS. UNTIL FINALLY, WE MANAGED TO PURIFY INTERFERON TO HOE KNOW HOMO JE NATE AND WORKED ON THE SEQUENCE, BUT IT HAD AN INTERESTING PATH AND WHEN THEY SAY THESE PROJECTS SOMETIMES TAKE A VILLAGE, AND THEN SOME, THIS IS REALLY TRUE FOR THIS INTERFERON PROJECT. WE STARTED ON THE NINTH FLOOR AND BUILDING 10, WHERE A LOT OF VERY EXCITING AND INTERESTING WORK STARTS. AND STARTED TO MAKE A HUMAN LYMPHOBLASTOID CULTURES IN SMALL QUANTITIES AND FOUND THAT THEY MADE A FAIRLY LARGE AMOUNT OF INTERFERON PER CELL. BUT WE RECOGNIZED THAT JUST GROWING CELLS IN SOME ROLLER BOTTLES OR TEA FLASKS WEREN'T GOING TO BE SUFFICIENT TO DO THE PRODUCTION THAT WE NEEDED TO DO, SO WORKING IN THE BASEMENT OF BUILDING 5, WE MOVED OVER, GOT OUR LITTLE -- WENT OVER TO BUILDING 5 AND WORKED WITH LEVY'S GROUP, AND AT THAT TIME CHUCK WAS THERE WITH HILTON AND SAM BARON AND WE STARTED PRODUCING INTERFERON WITH HUMAN LYMPHOBLASTOID CELLS USING THE PARAMIXO VIRUS MDV. WELL, EVEN IN CARBOYS, THAT WASN'T ENOUGH, SO WE MARCHED OVER TO THE BUILDING 6 PILOT PLANT WHERE SHYLOCK WAS AND WE WORKED ON A SCHEME TO DO 200-LITER CULTURES WHICH WERE THEN DILUTED TO 800-LITERS IN THESE HUGE FERMENTERS AND INDUCE THEM WITH A VIRUS TO PRODUCE INTERFERON AND USING LARGE SCALE EQUIPMENT COULD THEN GET FROM 800- LITER 1 MILLIGRAM OF INTERFERON THAT WAS PURIFIED. AND SO THIS WAS QUITE A FEAT, AND AFTER WORKING ON THE PURIFICATION FOR TWO YEARS, WE FINALLY GOT ENOUGH MATERIAL THAT WE COULD ACTUALLY WILL DO CHARACTERIZATION AND SEQUENCING ON. SO CHRIS AT THAT TIME KNEW LEROY HOOD AND MIKE HUNKAPILLAR AT CAL TECH AND THEY HAD JUST DEVELOPED A NEW MICRO SPINNING CUB SEQUENCER AT THAT TIME, SO CHRIS SAID WHY DON'T YOU GO TO CALIFORNIA AND WORK WITH LEE AND MIKE AND SO I DID, AND THAT'S WHERE THE NIH VILLAGE AND THEN SOME COMES FROM, BECAUSE WE MARCHED OUT TO CALIFORNIA AND WORKED WITH THEM TO GET THE AMINO ACID SEQUENCE OF THE FIRST 20 AMINO ACID RESIDUES. THIS WAS QUITE A FEET AT THIS TIME BECAUSE OF ITS HIGH SPECIFIC ACTIVITY IN THE VERY SMALL QUANTITY WE HAD, IT WAS SUCH PRECIOUS MATERIAL. BUT WE HAD DONE THIS, AND AT THE SAME TIME, ERNEST NIGHT AT DU PONT WAS WORKING ON THE PURR FIX OF FIBROBLASTS FROM HUMAN CELLS AND AT YALE, PETER LINYELL WAS WORKING AND WHAT WE DECIDED TO DO WAS ALL PUBLISH AT THE SAME TIME THE SEQUENCE OF THESE THREE DIFFERENT INTERFERONS IN "SCIENCE" MAGAZINE. THIS WAS QUITE A TIME BECAUSE THE IMPORTANCE OF CYTOKINES IN INTERFERON AT THAT TIME REALLY BECAME MORE PARAMOUNT AND MORE INTERESTING, ESPECIALLY AS IT'S USED IN THE FIELD OF CANCER HAD DEVELOPED. SO WITH THE SEQUENCE, THIS ALLOWED A NUMBER OF LABS TO ACTUALLY CLONE INTERFERON INTO E. COLI AND GENENTECH WAS ONE OF THOSE LABS AND CHARLIE WEISMAN'S LAB OUT IN ZURICH WAS ANOTHER ONE OF THE LABS, AND THAT ENDED UP BEING DEVELOPED INTO A PRODUCT FROM HOFFMAN LA ROCHE, THEY HAD INTERFERON ALPHA 2A AND SHARON HAD ALPHA INTERFERON 2B, AND BOTH OF THESE WERE FIRST LICENSED BY THE FDA BACK IN 1986, AND I HAD GONE FROM CHRIS' LAB TO SET UP THE CYTOKINE LAB AT FDA WITH PHIL AND OTHERS AND SO I THOUGHT THIS WAS A REALLY NICE TRANSITION AND AGAIN, THE FIRST LICENSING OF INTERFERON UNIQUELY WAS NOT FOR -- ACTIVITY BUT -- TUMOR ACTIVITY IN HAIRY CELL LEUKEMIA. SO AGAIN, INTERFERON GOT LICENSE FOR A NUMBER OF DIFFERENT INDICATIONS IN THE UNITED STATES, IT INCLUDED EVERYTHING FROM HEPATITIS B, HEPATITIS C, NOW NEW DRUGS HAVE BEEN APPROVED TO TAKE OVER FROM THESE, BUT AT THE TIME, THAT WAS THE ONLY MEDICINE AVAILABLE, AND WAS QUITE IMPORTANT. SO I WANT TO SHARE THINGS WE'VE HEARD REPEATEDLY BY A NUMBER OF MEMBERS OF THE AUDIENCE BUT TO ADD A FEW OF MY OWN. ONE IS TO BE KIND AND RESPECTFUL OF ALL MEMBERS OF THE TEAM, EVEN THE FOLKS THAT PICK UP OUR TRASH. ALWAYS BE PREPARED TO GIVE A TALK ON YOUR RESEARCH ON SHORT NOTICE, AND CHRIS ALWAYS ASSUMED YOU WERE GOING TO HAVE YOUR SLIDES WITH YOU, SO THAT IF SOMEBODY ASKED YOU TO TALK ON YOUR SCIENCE, YOU WERE ALWAYS PREPARED. WHEN YOUR EXPERIMENT DIDN'T WORK, ALWAYS CHECK THE PH. NINE TIMES OUT OF 10, THAT WAS THE CORRECT PROBLEM. NEVER GIVE UP, EVEN WHEN FACED WITH FAILURE. WHICH SOUNDS LIKE HE GOT THAT FROM WINSTON CHURCHILL. AND THE LAST ONE WAS BE HUMBLE IN YOUR SUCCESSES. I THINK BESIDE A NUMBER OF PEOPLE OF MENTION, CHRIS WAS A REAL REN VANS INDIVIDUAL. HIS LOVE OF MUSIC, HIS LOVE OF SCIENCE, YOU KNOW, HE WAS GREAT. BUT HE ALSO SHARED HIS PASSION OF SAILING WITH THE LAB TOO. HE GAVE US AN OPPORTUNITY TO GO DOWN AND SAIL ON HIS BOAT AND JUST KIND OF SIT AROUND AND TALK AND JUST REALLY RELAX AND BE IN AN ENVIRONMENT WHERE ONE COULD LEARN NOT ONLY ABOUT SCIENCE BUT ALSO ABOUT BEING A HUMAN BEING. SO I WANT TO THANK THE ORGANIZERS AND CHRIS' FAMILY AND FRIENDS ALLOWING ME TO SHARE CHRIS' JOURNEY AND MANY THANKS TO CHRIS FOR SHARING HIS WISDOM AND HIS ENTHUSIASM FOR SCIENCE AND BEING A WONDERFUL MENTOR. THANK YOU. [APPLAUSE] >> THANK YOU. YOUR SLIDES REMIND ME THAT IT WAS CHRIS WHO WHEELED THE CARBOYS CARRYING THE LYMPHOBLASTOID -- THESE GUY CAN TICK CARBOYS FROM BUILDING 10 TO 6 OR 5 AND PEOPLE WOULD ASK ME, IS THAT CHRIS ANFINSEN, THE NOBEL PRIZE WINNER, PUSHING THE CARTS FROM ONE BUILDING TO THE OTHER? AND IT WAS. I THINK NOW CONTINUING THE SAILING THEME, DAVID SACHS WILL TALK A LITTLE. >> AS I SAID, THIS WILL BE ALSO A PERSONAL ACCOUNT MORE THAN A SCIENTIFIC LECTURE. VOYAGES WITH CHRIS. YOU GOT THE WRONG ONE HERE, I'M AFRAID, ON MY SCREEN SO I GUESS I'LL LOOK AT IT UP HERE. SO THESE ARE SOME OF THE COMMENTS I MADE AT THE MEMORIAL FOR CHRIS THE YEAR AFTER HE DIED. CHRIS WAS MY MENTOR, TEACHER, CO-WORKER, BOSS, AND MOST OF ALL, MY FRIEND. HE WAS SO EASY TO TALK TO, AND IT WAS SUCH FUN TO GET HIS OPINION, SO VALUABLE TO GET HIS ADVICE. HIS GUIDANCE AND FRIENDSHIP WERE AN IMPORTANT PART OF MY LIFE, NOT ONLY DURING THE YEARS I SPENT IN HIS LAB, BUT ALSO FOR THE ENTIRE 21 YEARS I SPENT AT NIH. AFTER I MOVED TO BOSTON, WE CORRESPONDED AND TALKED SEVERAL TIMES BY PHONE, AND I ALWAYS THOUGHT I'D SEE HIM AGAIN SOON. HE DIED MUCH TOO SOON. SO MY VOYAGE AT THE NIH WAS A TWO-YEAR VOYAGE, TWO YEARS FROM MY MILITARY SERVICE, THAT LASTED 21 YEARS. THAT'S QUITE A LOT DUE TO MY EXPERIENCE DURING THOSE TWO YEARS WORKING WITH ALAN SCHECHTER AND CHRIS ANFINSEN. THIS IS A PICTURE OF CHRIS. HE MADE DAILY ROUNDS IN THE LABORATORY. WE ALL WOULD WAIT UNTIL HE CAME AROUND, WE KNEW AT SOME POINT, HE'D BE COMING AROUND, WE'D BE WORKING AWAY, HE WAS ALWAYS INTERESTED IN WHAT YOU WERE DOING, AND HE ALSO WAS VERY ENCOURAGING TO ANY IDEA THAT SOUNDED LIKE A REASONABLE IDEA TO HIM. WHEN THE FIRST ARRIVED, ALAN LEARNED I HAD SOME EXPERIENCE PREVIOUSLY IN ORGANIC CHEMISTRY, HE PUT ME TO WORK ON A PROJECT USING THE NUCLEAR MAGNETIC RESONANCE TO STUDY HISTIDINE RING PROTONS IN PROTEINS, AND IN A VERY SHORT TIME WE TURNED OUT QUITE A FEW PAPERS ON THIS, IT WAS A REAL INTRODUCTION ON HOW TO DO RESEARCH AT NIH. ANOTHER EXAMPLE, THERE WAS SOME OTHER WORK GN ON IN THE LAB OF PROTEIN SYNTHESIS BY THE MERRIFIELD METHOD, AND IT WAS TAKING A VERY LONG TIME, IT TOOK -- MUCH OF THE TIME WAS TAKEN UP IN THE WASHES. I HAPPENED TO NOTICE THAT THE RESIN SWELLED AND SHRANK WHEN YOU ADDED DIFFERENT SOLVENTS AND FROM MY PREVIOUS WORK, I REALIZED THAT WOULD BE A VERY QUICK WAY OF WASHING. SO I MENTIONED THAT TO CHRIS AND HE QUITE QUICKLY ASKED LILA CORLEY, HIS TECHNICIAN, TO WORK WITH ME AND IN THE MATTER OF A FEW WEEKS, WE DEVISED AN INSTRUMENT WHICH WOULD ELIMINATE THOSE LENGTHY WASHES AND WE SYNTHESIZEED BRAD KIE NEN IN LESS THAN FIVE HOURS, A SYNTHESIS WHICH AT THAT TIME TOOK A WEEK. SO IT WAS, AGAIN, ANOTHER EXAMPLE OF HOW MUCH FUN IT WAS TO TALK OVER IDEAS WITH CHRIS AND THEN PUT THEM INTO ACTION. BUT MOST EXCITING FOR ME WAS WHEN I GOT INTO THE USE OF MY OWN AREA OF IMMUNOLOGY TO STUDY A MOLECULE THAT WAS OF GREAT INTEREST TO CHRIS, STAPHYLOCOCCAL NUCLEASE. I STARTED MAKING ANTIBODIES TO NUCLEASE AND THEN FRACTIONATING THEM ON COLUMNS BEARING PEPTIDES THAT WE KNEW HOW TO MAKE FROM STAPHYLOCOCCAL NUCLEASE, AND THAT DEMONSTRATED THAT THE WHILE PREPARED AGAINST THE INTACT NATIVE MOLECULE, THESE ANTIBODIES COULD BIND TO THE DISORDERED POLYPEPTIDE FRAGMENTS, THE RANDOM STRUCTURE THAT WE'VE HEARD SO MUCH ABOUT ALREADY THIS MORNING. OR THIS AFTERNOON. IT IS POSTULATED -- WE POSTULATED IN THAT ARTICLE THAT THE NATIVE ANTIGENIC DETERMINE NAPT MIGHT BE GENERATED IN SUCH FRAGMENTS BY SPONTANEOUS FOLDING OF THE POLLLY PEPTIDE CHANGE HE CHAIN WITH WAY THEN PULLED IN THE EQUILIBRIUM IN THE ANTIBODY MADE AGAINST THE NATIVE STRUCTURE WAS PULLED IN TO THE NATIVE FORMAT. THOSE FINDINGS WERE OF GREAT INTEREST TO A COMMON VISITOR TO MICHAEL SELA. THAT ENDED UP IN AN INVITATION FOR MY FIRST VISIT, MY WIFE AND MY FIRST VISIT TO ISRAEL. I VISITED THE WEIZMANN INSTITUTE, MY FIRST OF MANY VISITS TO THE INSTITUTE, AND CHRIS GAVE ME A PERSONAL TOUR OF OLD JERUSALEM, YOU SEE HERE IN THE BOTTOM RIGHT. DURING WHICH TIME HE DELIGHTED ME AND EMBARRASSED CHRISTINA BY ARGUING VIGOROUSLY WITH EVERY MERCHANT AND ENDING UP BUYING THE RUGS AND THE VARIOUS OTHER THINGS WE TOOK HOME AT MUCH LESS THAN HALF THE PRICE THAT CHRISTINA WAS ALREADY WILLING TO PAY. THE WORK WITH THE ANTIBODIES TO STAPHYLOCOCCAL NUCLEASE LED TO THIS PAPER IN PNAS, AND I THANK AND IMMUNOLOGIC APPROACH TO THE CONFORMATIONAL EQUILIBRIA OF POLYPEPTIDES. I WAS ABSOLUTELY DELIGHTED WHEN I NOTED THAT IN CHRIS' NOBEL LECTURE IN 1972, HE HAD SEVERAL FIGURES FROM THOSE PAPERS THAT WE WROTE ON THE IMMUNOLOGIC APPROACH TO THE CONFORMATIONAL EQUILIB BREE A OF POLYPEPTIDES. ONE OF THEM RIGHT HERE SHOWS YOU THE INACTIVATION OF STAPHYLOCOCCAL NUCLEASE ACTIVITY BY THE ANTIBODY AGAINST THE RANDOM CONFORMATION, THE -- I MEAN AGAINST THE NATIVE CONFORMATION BUT -- AGAINST THE SAME POLYPEPTIDE FRAGMENT AND OF A SIMILAR AFFINITY, BUT WITH RECOGNIZING THE RANDOM CONFORMATION OF THAT LARGE FRAG ENT OF STAPHYLOCOCCAL NUCLEASE. AND WE PUBLISHED IN "NATURE" A PAPER ON THE IMMUNOLOGIC DISTINCTION BETWEEN POSSIBLE ORIGINS OF ENZYMATIC ACTIVITY IN THIS LARGE POLYPEPTIDE FRAGMENT OF STAPHYLOCOCCAL NUCLEASE AND OUR CONCLUSION IS THE RESULTS WERE CONSISTENT WITH THE PRESENT CONCEPTS OF FOLDING POLYPEPTIDE FRAGMENTS FORMING STRUCTURES SIMILAR TO THE NATIVE PROTEIN AND MAY OFFER A METHOD FOR INVESTIGATION OF OTHER MOLECULAR SYSTEMS AND IT DID SO. THERE ARE MANY STUDIES THEREAFTER IN WHICH ANTIBODIES HAVE BEEN USED BY OTHER INVESTIGATORS TO REGAIN ENZYMATIC AND OTHER ACTIVITIES IN PROTEINS THAT HAVE OTHERWISE DENATURED. SO AS OTHER SPEAKERS HAVE MENTIONED, CHRISTEN JOYED VERY MUCH CHRIS ENJOYED VERY MUCH SAILING. BEING THE SON OF TWO IMMIGRANTS, HE WAS QUITE A MARINER, NATURAL MARINER, AND DIDN'T NEED A LOT OF FANCY EQUIPMENT THAT WAS AVAILABLE, AND SO US A SEE HERE ON THE LEFT, USING A SEXTON FOR FINDING OUT WHERE WE WERE. HE CONTINUED THAT UNTIL AS I RECALL ONE PERIOD WHERE WE GOT LOST, AND SUBSEQUENTLY MORE MODERATE EQUIPMENT, HERE YOU SEE HIM ON THE RIGHT. THESE ARE TWO OF OUR TRIPS BETWEEN ANNAPOLIS AND ST. AUGUSTINE. HE LEFT THE BOAT ALWAYS IN ST. AUGUSTINE OVER THE WINTER. HERE WE SEE HIM AT A BIRTHDAY CELEBRATION, AND FROM THE YEAR, I GATHER IT WAS MY 45TH. AND HERE AT ONE OF OUR NEW YEAR'S EVE PARTIES, AT WHICH HE WAS A GUEST MANY TIMES. SO I GIVE THANKS TO CHRIS FOR SOME OF THE MOST IMPORTANT AND MOST ENJOYABLE VOYAGES OF MY LIFE. THANK YOU. [APPLAUSE] FOLLOWING ALAN SCHECHTER'S CALL IN LATE JULY ANNOUNCING TODAY'S EVENT, I FOUND MYSELF SIFTING THROUGH A LOT OF MEMORIES OF FAMILY LIFE WITH CHRISTIAN B. ANFINSEN, DADDY TO ME, SOME FUNNY, OCCASIONALLY EXASPERATING AND I WILL SHARE SOME OF THOSE WITH YOU. WHILE THERE WAS ONLY ONE OF HIM, HE MANAGED TO DISTRIBUTE HIS MANY TALENTS, THOUGH NOT AS FINELY HONED TO HIS OFFSPRING. DESPITE MY EARLY RESISTANCE IN STUDYING LANGUAGES IN POLITICAL SCIENCE I WAS NOT SURPRISE LIG DRAWN NO -- ULTIMATELY PLANT PHYSICIAN YOL AND BIOCHEMISTRY. DADDY'S PASSION FOR THE NATURAL WORLD AND SCIENTIFIC DISCOVERY WAS PAST ALONG TO ME. SOMETHING I HAVE ENJOYED SHARING WITH MY OWN STUDENTS FOR 41 YEARS AT PROVIDENCE COLLEGE. HIS SCIENCE GEEK GENE, IF I CAN CALL IT THAT, SEEMS TO HAVE PERMEATED THE ENTIRE FAMILY. MARGO WORKED AS A SALES REP FOR GLAX COSMITH KLINE AND KRIFT SHIFTED HIS BUSINESS MANAGEMENT FOCUS TO TEACHING FOR TERM NEXT WITH BOARD CERTIFICATION IN ENTOMOLOGY. FURTHER UP THE FAMILY TREE, HIS GRANDSON ROGER IS A MATH AND SCIENCE TEACHER. HIS GRANDDAUGHTER KATE WHO'S UP HERE IN THE AUDIENCE AN EQUINE VETERINARIAN, AND HIS GREAT GRANDCHILDREN, LAURA AND CHRISTIAN, ALREADY SHOW AN INTEREST IN AND TALENT IN SCIENCE AND MATH AS WELL. MY SISTER MARGO AND CHRIS III, OR AS I SOMETIMES TEASE HIM AND HE'S IN THE AUDIENCE TOO, CHRISYY, HE'LL KILL ME FOR THAT, INHERITED DADDY'S TALENT FOR MUSIC. DAD WAS AN ACCOMPLISH THE PIANIST AND PLAYED VIOLA FOR THE SYMPHONY. MARGO MAJORED IN MUSIC AND LATER PLAYED VIOLIN WITH THE ALEXANDRIA ORCHESTRA, AND CHRIS IS A TEANLTED TALENTED CELLOIST AND GUITARIST AND YO-YO MA FAN. MARGO AND MOM PLAYED FIRST AND SECOND VIOLINS, DADDY, THE VIOLA, AND CHRIS, THE CELLO. WHILE I PLAYED PIANO, DADDY'S FIRST INSTRUMENT, I PLAYED BADLY. SO I GENERALLY SERVED AS THE APPRECIATIVE AUDIENCE. GROWING UP, DAD SPENT SUMMERS WITH HIS FAMILY IN STONE HARBOR, NEW JERSEY. HE LOVED THE WATER -- SLIDES THAT I DID BRING, THERE'S A WONDERFUL PHOTOGRAPH OF HIM AS A STUDENT LIFEGUARDING AT STONE HARBOR BEACH. BOY, WAS HE A HUNK. HE LOVED THE WATER. HE WAS A GREAT SWIMMER AND HE SERVED AS A LIFEGUARD IN NEW JERSEY WHERE HE LIVED. IN THE SUMMERTIME WITH HIS PARENTS. AS MOST OF YOU KNOW, HE WAS AN AVID AND ADVENTUROUS SAILOR. SOMETIMES ASSUMING BAD THINGS WOULD NEVER HAPPEN TO HIM. HE SURVIVED A BROKEN MAST OFF CAPE MAY WITH HIS SHIP TO SHORE RADIO ON THE FRITZ, A VIOLENT STORM WHILE SAILING FROM THE BAHAMAS TO THE COAST OF GEORGIA WITH ME AND TWO OTHERS, CONCERNED ABOUT OUR SAFETY, MOM CALLED THE COAST GUARD WHOSE RESPONSE WAS, IF THEY'RE OUT THERE, LADY, THERE'S NOTHING WE CAN DO. CHRIS III HAS INHERITED THAT PASSION AND SKILL. OVER THE YEARS, HE'S CRUISED THE WATERS OF THE CHESAPEAKE BAY. FORTUNATELY WITH CONSIDERABLY MORE CAUTION AND DARE I SAY MORE COMMON SENSE THAN DADDY SOMETIMES SHOWED. DAD'S LOVE OF LANGUAGES AND HIS TRAVEL GENE WAS ONE I INHERITED AS HAS HIS GREAT GRANDDAUGHTER, HARA, WHO AT 14 IS ALREADY FLUENT IN THREE LANGUAGES. WHICH IS AMAZING. DADDY AND I ENJOYED CHALLENGING EACH OTHER IN SWEDISH, DANISH, FRENCH, HEBREW, AND OCCASIONALLY GERMAN. HIS FACILITY WITH LANGUAGE PERHAPS COMBINED WITH HIS STUBBORNNESS GENE WAS OCCASIONALLY HIS DOWNFALL. WHEN I GRADUATED FROM COLLEGE, I WORKED AT THE GENETICS INSTITUTE IN COPENHAGEN. DAD CAME FOR A VISIT. AND ONE EVENING WHEN WE WENT OUT TO DINNER, HE CONFIDENTLY PLACED OUR ORDER IN DANISH. MUCH TO HIS CHAGRIN, THE WAITER LISTENED PATIENTLY WHILE THE ORDER -- UNTIL THE ORDER WAS COMPLETE, AND THEN REPLIED IN PERFECT ENGLISH, "WILL THAT BE ALL, SIR?" I'M NOT SURE WHO WAS SHOWING OFF HIS LANGUAGE SKILLS MO BUT I HAD SKILLS MO RE BUT I HAD A GREAT LAUGH KNOWING HE SPOKE DANISH WITH A THICK NORWEGIAN ACCENT. DADDY WAS ALSO SOMETHING OF A TEASE. HE HAD A WONDERFUL SENSE OF HUMOR, AND THOUGHT HE COULD PRETTY MUCH GET AWAY WITH ANYTHING. A TREAT THAT MY SISTER MARGO HAS INHERITED. ONE AFTERNOON AT NIH, DAD MADE A KWIK TRIP TO PICK UP A BOOK AT THE LIBRARY. MY HUSBAND TELLS ME I HAVE A PATIENCE OF A GNAT ON SPEED. EVIDENTLY I INHERITED THAT TRAIT FROM DADDY. WHEN HE REALIZED THAT HE WOULD NEED TO STAND IN LINE TO CHECK OUT THE BOOK, HIS IMPATIENCE KICKED IN. HE TUCKED THE BOOK DOWN THE BACK OF HIS PANTS, HEADED FOR THE EXIT, ONLY TO TRIGGER THE LIBRARY ALARM. SOMEWHAT SHEEPISHLY, ACCOMPANIED BY THE ALARM HORN, HE RETURNED TO CHECK OUT THE BOOK PROPERLY. MY SISTER MARGO THOUGHT HER FATHER COULD DO NO WRONG AND FOLLOWING HIS EXAMPLE, ONE NIGHT CLIMBED THROUGH A WINDOW IN HIS ANNAPOLIS COTTAGE TO RETRIEVE SOME OF HER SON CHAD'S FAVORITE BOOKS. FORTUNATELY, NO ALARM SOUNDED. A FEW DAYS LATER, HOWEVER, SHE RECEIVED A CALL FROM LIBBY, WHO NOTED THAT SHE COULD HAVE JUST ASKED. NOW WHERE'S THE FUN IN THAT? RECENTLY, REMINISCING WITH MY HUSBAND, I RECOUNTED THE MOMENT I HEARD ABOUT THE NOBEL AWARD. MANY OF MY SUBSEQUENT WRECK LENGS LEXES RECOLLECTIONS ARE SOMETHING OF A BLUR FROM THE EXCITEMENT. PACKING TO FLY HOME TO BE WITH THE FAMILY, SHOPPING FOR A DRESS FOR THE NOBEL DINNER, ORDERING AT THE HOT DOG STAND IN FRONT OF THE GRAND HOTEL IN STOCKHOLM, HEARING DADDY SPEAK AND RECEIVE THE PRIZE FROM THE KING OF SWEDEN. NONE OF THESE MEMORIES IS QUITE AS VIVID AS MY MEMORY OF THE DAY I HEARD OF THE AWARD. I WAS SITTING IN THE LAB, PLANT PHYSIOLOGY GRADUATE STUDENT AT INDIANA UNIVERSITY. BEMOANING THE PREVIOUS DAY'S EVENTS. I HAD CULTURED CARBOYS OF FUNGI FOR A MONTH AND SPENT HOURS IN THE COLD ROOM RUNNING THE EXTRACTS THROUGH CROW CHROMATOGRAPHY. AS I EX-ECTED THE COLD ROOM AND WALKED TOWARD THE LAB WITH MY COLUMN I FLUENT TO MY HOR HORROR, THE BOTTOM DROPPED OUT OF THE FLASK, LEAVING A MONTH'S WORTH OF WORK IN A PUDDLE ON THE FLOOR. PREDICTABLY THE FOLLOWING MORNING I SAT SULGING OVER MY MISFORTUNE. THERE WAS A KNOCK ON THE LAB DOOR AND GENETICIST MARCUS RHODES POKED HIS HEAD IN. WITH A BIG GRIN ON HIS FACE, HE SAID, GREAT NEWS, CONGRATULATIONS! OF COURSE AS AN EGO CENTRIC GRADUATE STUDENT, I THOUGHT HE WAS SARCASTICALLY REFERRING TO MY EARLIER DISASTER. THEN HE SAID, REALIZING I DIDN'T KNOW, "YOUR FATHER WAS JUST AWARDED THE NOBEL PRIZE IN CHEMISTRY." NEEDLESS TO SAY, MY DAY BRIGHTENED. AND I RACED DOWN THE HALL TO THE PAYPHONE TO CALL HOME. YES, THOSE WERE THE DAYS OF WIRES. SO WHILE DADDY IS NO LONGER WITH US, HE IS NOT GONE. THERE ARE BITS OF HIM EVIDENT IN HIS OFFSPRING, AND ALL OF YOU HERE CARRY ON HIS WORK, HIS TALENTS, HIS PASSIONS, HIS PHILOSOPHY, HIS OPTIMISM, HIS SENSE OF JUSTICE, AND FOR ALL OF THAT, HE WILL BE REMEMBERED THROUGH YOU, AND I AM VERY THANKFUL FOR THAT AND THANKFUL FOR YOUR EARS. [APPLAUSE] >> I THINK EVERYTHING AFTER THAT IS ANTICLIMAX, BUT LET'S CONTINUE BEFORE WE HAVE OUR RECEPTION. DR. STEINER, WOULD YOU GIVE YOUR PERSONAL VIGNETTES, PLEASE? >> I'D LIKE TO START WITH A THANK YOU, THANKS TO ALAN SCHECHTER FOR INVITING ME TO COME AND HONORING ME BY INVITING ME TO THE SYMPOSIUM. AND I'D ALSO LIKE TO THANK THE NIH, WHICH SUPPORTED MY RESEARCH IN IMMUNOLOGY FOR A GOOD MANY YEARS. I HAVEN'T ACTUALLY COUNTED HOW MANY, BUT IT MUST BE PUSHING 40 -- MORE THAN 40 PROBABLY. AND I ALSO MADE MANY FRIENDS HERE OVER THE YEARS AND VARIOUS PEOPLE AT THE NIH HELPED ME BY PROVIDING ME WITH MATERIAL PROMINENT AMONG THOSE ARE MICHAEL POTTER, WHO SENT ME MICE ACTUALLY WHILE I WAS FINDING SOME INTERESTING THINGS IN ANTIBODIES, AND I WANTED TO LOOK AT THE GENETICS SO HE SEPTEMBER ME SOME VARIOUS MICE FROM, YOU KNOW, ALL PARTS OF THE WORLD, CZECHOSLOVAKIA AND OTHER PLACES, AND THAT WAS QUITE INTERESTING. AND THE OTHER PERSON I'D LIKE TO SINGLE OUT AT THE NIH WITH WHOM I INTERACTED WAS DAVID DAVIES. I GUESS HE WAS AT THIS INSTITUTE, AT THE ALLERGY -- THE ARTHRITIS, ET CETERA. HE'S A CRYSTALLOGRAPHER, AND ONE OF THE FIRST THINGS I DID WHEN I CAME TO M.I.T. ON THE FACULTY WAS TO TRY TO PARTLY INFLUENCED BY THE WORK I HAD DONE EARLIER WITH RIBONUCLEASE TO MAKE DERIVATIVES OF ANTIBODIES THAT COULD BE CRYSTALLIZED AND THAT -- WHICH -- AND TO MAKE MERCURY DERIVATIVE SO YOU COULD HAVE A HEAVY METAL WHICH WOULD BE HELPFUL IN INTERPRETING THE CRYSTALLOGRAPHY, AND DAVID DAVIES SENT ME -- AT THAT TIME HAD IDENTIFIED AND ISOLATED A PROTEIN, PURE ANTIBODY IN A SENSE, THAT CRYSTALLIZED, AND SO I WAS QUITE INTERESTED IN THAT, AND HE SENT IT TO ME AND I WORKED AT IT, AND I HAD -- IT WAS RATHER EARLY IN MY TIME, I ASKED HIM TO TAKE THE PROTEIN AND RUN IT ON AN SDS DRILL TO SEE WHAT WE HAD, AND HE SAID -- HE CAME TO ME AFTER DOING IT TWICE, HE SAID I MUST HAVE DONE SOMETHING WRONG BECAUSE THE PROTEIN INSTEAD OF GIVING ONE BAND FOR THE -- GAVE TWO BANDS, AND I WAS -- WE WERE BOTH SURPRISED, AND EVENTUALLY MANY YEARS LATER, IT TURNED OUT THAT THIS PROTEIN HAD, UNBEKNOWNST TO THE PEOPLE DOING THE CRYSTALLOGRAPHY HAD A DELETION OF THE HINGE REGION OF THE HEAVY CHAIN, IT THEREFORE LOST THE CYSTINE RESIDUES THAT BOND TO THE LIGHT CHAIN, SO IT WAS AN ABNORMAL IMMUNOGLOBULIN. THAT WAS A SHOCK ALL AROUND, BUT I REALLY ENJOYED INTERACTING WITH DAVID DAVIES. HE WAS A VERY NICE PERSON, AND MICHAEL POTTER WAS FANTASTIC. THE OTHER PERSON I THOUGHT I'D MENTION, I DON'T THINK HE ACTUALLY IS EMPLOYED BY THE NIH BUT HE CERTAINLY SPENT A LOT OF TIME HERE, WAS ILLFAN CABOT, WHICH WHOM I INTERACTED QUITE A BIT BECAUSE I BECAME INTERESTED IN THE -- COMPARING DIFFERENT ANTIBODIES AND HE WAS BEFORE THE AGE OF COMPUTERS, HE HAD COMPILED THESE INCREDIBLE EXEND CHAIN, THE LIGHT CHAINS, ET CETERA, WHICH WERE VERY USEFUL FOR PEOPLE AT THAT TIME. SO THAT'S THE THANKS PART. THE HOW AM I HERE PART IS I WANT TO JUST GET TO RATHER BRIEFLY BECAUSE I DON'T BELIEVE -- I MAY HAVE MET CHRIS ANFINSEN. I CERTAINLY KNEW ABOUT HIM, AND I KNEW A LOT ABOUT HIM ALSO BECAUSE I WORKED VERY VERY CLOSELY AT YALE MEDICAL SCHOOL BECAUSE I WORKED WITH FRED RUCHARDS WHO WAS A CONTEMPORARY, THEY HAD FOLLOWED SIMILAR PATHS. HOW DID I GET TO FRED'S LAB? THAT'S A MINOR STORY. I WENT TO COLLEGE -- ANOTHER CONNECTION WITH CHRIS ANFINSEN, AND IN A RECENT SWARTMORE ALUMNI BULL, THERE WAS A RATHER INTERESTING STORY ABOUT A WOMAN IN JERUSALEM WHO MET -- IT WAS ANOTHER ALUM, NOT CHRIS' CLASS, BUT SOMEONE WHO WAS WALKING WITH HIS DAUGHTER IN JERUSALEM, AND ONE OF THEM WAS WEARING A SWARTHMORE T-SHIRT AND THEY MET A WHOM WOULD SAID, DID YOU GO TO SWARTHMORE, STOP THEM, AND IT TURNED OUT TO BE THE WIDOW OF CHRIS, WHO WAS LIVING IN JERUSALEM, I GUESS. AND THAT WAS ACTUALLY QUITE AMAZING. I HAD CLIPPED IT OUT SO IF ANYONE WANTS IT, IN FACT, IF ANYONE WANTS ME TO EXPAND ON ANYTHING THAT I SAY TODAY, I'D BE HAPPY TO GET EMAIL AND TRY TO DO IT. MY EMAIL ADDRESS IS MY FIRST INITIAL L., MY CLAST NAME STEINER WITH NO PUNCTUATION,@M MIT.EDU. ANYWAY, HOW DID I GET TO WORK ON RIBONUCLEASE? I WAS A MATH MAJOR, I REALLY LOVED MATHEMATICS, I LOVED IT IN HIGH SCHOOL, I HAD NO QUESTION WHAT I WOULD MAJOR IN WHEN I TWOANT SWARTHMORE. THEY HAD A VERY GOOD MATH DEPARTMENT. IN FACT, THE CHAIR OF THE MATH DEPARTMENT I'M SURE ALSO WHEN CHRIS WENT THERE WAS A MAN NAMED ARNOLD DRESDEN, A VERY RESPECTED MATHEMATICIAN ESPECIALLY OUT OF A SMALL COLLEGE, AND HE ALSO WAS A MEURS MOUSSAOUI A MUSICAL FANATIC. HE LOVED CHAMBER MUSIC, AND HIS HOME WHICH WAS RIGHT OFF THE CAMPUS, HE HAD OPEN HOUSE FOR DOING CHAMBER MUSIC FOR ANYONE FROM THE COMMUNITY, STUDENTS, FACULTY, ANYBODY ELSE WHO WAS INTERESTED, WOULD JUST SHOW UP WITH THE INSTRUMENT AND THAT WAS WHERE I AND MANY OTHER PEOPLE FIRST PLAYED CHAMBER MUSIC. HE HAD THE WHOLE COLLEGE -- HE HAD THE COLLEGE'S COLLECTION AT HIS HOME. HE HAD -- HE LIVED IN A COLLEGE HOUSE THAT HAD A SPECIALLY BUILT LIVING ROOM SO THAT IT COULD ACCOMMODATE TWO PIANOS WITHOUT TAKING UP THE ENTIRE LIVING ROOM, AND THAT WAS -- MANY PEOPLE WHO WENT TO SWARTHMORE WERE INTERESTED IN MUSIC TOOK -- I WOULD BE INTERESTED TO KNOW IF CHRIS DID THAT BECAUSE I WOULD THINK WITH AN INTEREST IN A STRINGED INSTRUMENT, HE WOULD HAVE, BUT I DON'T KNOW THAT. BUT THAT WAS A VERY IMPORTANT PART OF MY COLLEGE EXPERIENCE ACTUALLY. AND THEN I NEVER HAD ANY QUESTION THAT I WOULD MAJOR IN MATH BECAUSE I HAD ALWAYS LOVED MATH, AND I STILL DID WHEN I ENTERED SWARTHMORE AND ALL THE WAY THROUGH. I WAS SOMEONE LOOKING DOCTOR THIS IS, OF COURSE, THE PRECOMPUTER ERA. I WAS LOOKING FOR SOMETHING THAT I COULD APPLY MATH TO THAT WAS SORT OF MORE REAL WORLD, BUT IT WAS NOT EASY FIND. THERE WERE PEOPLE AT THAT TIME WHO WERE APPLYING -- TRYING TO APPLY MATHEMATICS TO A VARIETY OF OTHER THINGS BUT WHEN I LOOKED INTO IT A LITTLE BIT, NONE OF IT SEEMED VERY SOLID TO ME, AND I DON'T THINK MUCH EVER CAME OF IT, MOST OF THOSE I'VE HEARD. HOWEVER, AND THEN SOMEWHERE, I TOOK BIOLOGY AND I WAS QUITE FASCINATED WITH BIOLOGY BECAUSE OF -- YOU KNOW, IT'S LIFE AND HOW DOES IT HAPPEN AND HOW DOES IT EVOLVE. THERE WERE SO MANY INTERESTING QUESTIONS, AND THE OTHER THING THAT MADE A BIG IMPRESSION ON ME AT SWARTHMORE WAS THE QUAKER PHILOSOPHY. I REALLY KNEW NOTHING ABOUT QUAKERS -- I GUESS I KNEW SOMETHING ABOUT THEM AS SOMEWHAT EDUCATED AS A FRESHMAN IN COLLEGE BUT I DIDN'T KNOW ANY QUAKERS WITH ONE EXCEPTION ACTUALLY. MY FAMILY, MYSELF WAS BORN IN VIENNA IN A JEWISH FAMILY, AND WE FLED FORTUNATELY, AND WITH SOME DIFFICULTY, CAME TO THE UNITED STATES. AT M.I.T., I'M TEACHING A FRESHMAN SEMINAR ON REFUGEES, WHICH IS A VERY TOPIC IN OUR COUNTRY AT THE PRESENT TIME. AND I DID THIS BECAUSE I THOUGHT IT WAS AN INTERESTING SUBJECT AND I THOUGHT THE STUDENTS WOULD GET SOMETHING OUT OF IT, AND I HAD PERSONAL EXPERIENCE WITH BEING A REFUGEE. ALTHOUGH I DIDN'T REALLY SUFFER BUT I WAS VERY FORTUNATE THAT WE WERE ABLE TO GET FIRST OUT OF YEMEN AND THEN OUT OF FRANCE, JUST AS THE WAR WAS BEGINNING, AND CAME TO THE UNITED STATES. CERTAINLY THE MAJOR GOOD FORTUNE OF MY LIFE WAS THAT DEPARTURE FROM EUROPE. SO THAT BRINGS ME TO SWARTHMORE, AND I MAJORED IN MATH. I MY MORED MINORED IN ECONOMICS AND BIOLOGY, IN SWARTHMORE YOU COULD DO A MINORS PROGRAM, IT WAS BROUGHT OVER BY A PRESIDENT WHO HAD BEEN TO OXFORD AND IT WAS MODELED ON THE OXFORD TUTORIAL SYSTEM. I THRIVED ON THAT SYSTEM. THERE WERE NO LECTURES, AND IT WAS VERY BENEFICIAL FOR ANYONE WHO HAD AN INCLINATION TO BE A SCHOLAR BECAUSE THE EMPHASIS WAS ON LEARNING YOURSELF. JUST HAD TO GO AND DIG THE INFORMATION OUT OF THE LIBRARY, OUT OF A TEXTBOOK OR OUT OF SOMEWHERE. AND I LIKED THAT. AND SO I BEGAN TO THINK AS I WAS NEARING GRADUATION, I ALWAYS ASSUMED I'D GO TO GRADUATE SCHOOL IN MATH BECAUSE THAT WAS MY PATH, BUT I BEGAN TO THINK, WELL, MAYBE I WANTED TO DO SOMETHING THAT WAS MORE RELATED TO HUMAN BEINGS, YES, I'VE GOT TWO MINUTES MORE? OKAY. I'M GOING TO -- I'M SORRY, I GET CARRIED AWAY WITH THIS. SO I WENT TO MEDICAL SCHOOL IN THE END AT YALE. YALE ALSO WAS A LITTLE BIT LIKE SWARTHMORE, THEY HAD NO EXAMS, WHICH UPSET MANY OF THESE STUDENTS, HOW DO WE KNOW IF WE KNOW ANYTHING, BUT HAVING BEEN THROUGH THE SWARTHMORE HONORS PROGRAM, I HAD NO PROBLEM WITH THAT. AT YALE, I MET A SART SWARTHMORE GRADUATE, MAXINE SINGER, I SAID I NEEDED A SUMMER JOB THAT FIRST YEAR, DID SHE KNOW ANYBODY WHO MIGHT HAVE SUCH A JOB. SO SHE REFERRED ME TO A NEWLY ARRIVED ASSISTANT PROFESSOR NAMED FRED RICHARDS, AND SAID SHE THOUGHT HE WOULD HAVE -- HE HAD A GRANT, HE HADN'T HIRED ANYONE, SHE THOUGHT HE'D HAVE ROOM FOR ME, SO I DID THAT, AND MET FRED RICHARDS, HE DISCUSSED A PROJECT WITH ME WHICH WAS TO REDUCE RIBONUCLEASE AND SEE WHAT HAPPENED TO IT, TO THE ACTIVITY. SO THAT WAS MY PROJECT, AND I'M GOING TO SUMMARIZE THIS IN ABOUT TWO SENTENCES, I WORKED VERY HARD AT IT THE ENTIRE REST OF MY TIME THROUGH MEDICAL SCHOOL, WROTE A THESIS, BUT PRED DID NOT FRED DID NOT MENTION PUBLICATION. I THEN HAD DECIDED TO GO TO MEDICAL SCHOOL ANYWAY, I WAS IN MEDICAL SCHOOL, I WAS AT YALE, I DECIDED TO CONTINUE AND THE NEXT THING AFTER YOU GRADUATE FROM MED SCAL KAL MEDICAL SCHOOL, I WROTE THE THESIS, SUBMITTED IT, AND THEN ENTERED -- BECAME AN INTERNAL AND WENT THROUGH A RESIDENCY PROGRAM AT YALE. IN THE END, DECIDED THAT CLINICAL MEDICINE WAS PROBABLY TO NOT FOR ME. I GOT TOO UPSET WHEN MY PATIENTS DIED, AND THEY DID DIE A LOT WHEN YOU'RE AT A BIG HOSPITAL IN A CITY AS AN INTERN AND RESIDENT. SO I THOUGHT MAYBE I COULD COULD ACADEMIC MEDICINE AND IT WOULD BE A LITTLE GENTLER IN TERMS OF THE HOURS AND SO SON, SO I THOUGHT I'D TRY THAT, BUT I KNEW THAT I NEEDED A SPECIALTY, SO I DECIDED TO GO INTO IMMUNOLOGY, AND I THEN DID TWO POSTDOCS, I HAD AN M.D. DEGREE, WHICH WERE VERY FORTUNATE, I MEAN, I WAS VERY LUCKY AND POSSIBLY SANGUINE IN CHOOSING TWO MARVELOUS MENTORS. THE FIRST ONE IN ST. LOUIS WAS HER MANNIZE MAN AND THE SECOND IN LONDON WAS ROD PORTER, VERY EMINENT, ROD PORTER WAS REALLY THE BASIC PERSON FIGURING OUT WHAT THE STRUCTURE OF THE ANTIBODY MOLECULE AND GOT A NOBEL PRIZE FOR THAT SHORTLY AFTER I LEFT HIS LAB. IN HIS LAB, I HAD OPPORTUNITY TO DO A LITTLE BIT OF THE WORK I LEARNED AT YALE ABOUT DISULFIDE BRIDGES BECAUSE ONE OF THE THINGS ROD PORTER HAD MADE A MISTAKE ABOUT IN HIS BASIC ANALYSIS OF THE ANTIBODY MOLECULE FAB AND FC AND ALL THAT WAS CORRECT, HE HAD MISTAKEN THE DISULFIDE BRIDGING AND HE ASKED ME TO TRY TO STRAIGHTEN THAT OUT. I'M NOT EVEN SURE HE KNEW I'D WORKED ON DISULFIDE BRIDGES. SO THAT WAS -- WELL, ANYWAY, EVENTUALLY I DID THAT, WITH THE DISULFIDE MOLECULE AND I THINK I'VE GOT TO STOP NOW. SORRY. >> ALL RIGHT. DR. STEINER IS BEING MODEST. THE REASON WE INVITED HER TO TALK A TODAY IS THAT SHE HAD ACTUALLY DONE VERY SIMILAR EXPERIMENTS TO WHAT CHRIS WAS DOING AT NIH AT YALE WITH FRED RICHARDS DURING THE 50s AND GOT VERY SIMILAR RESULTS. BUT FOR REASONS WE CAN DISCUSS LATER, THEY WERE NEVER PUBLISHED, AND SO DAVID EISENBERG, THE CRYSTALLOGRAPHER WROTE A PAPER IN "CELL" IN JUNE OF THIS YEAR COMMENTING ON THE SIMILARITIES AND DIFFERENCES. BUT IT'S INTERESTING -- >> IT WAS THE SAME WORK. >> IT WAS THE SAME WORK AND IT'S BEING DONE HERE, AND THAT CRISPR SUED AND EVENTUALLY PUSHED TO GET THE RESULTS AND THE NOBEL PRIZE. THANK YOU VERY MUCH. [APPLAUSE] >> I WOULD JUST SAY THAT WHEN I LEARNED THAT CHRIS GOT THE NOBEL PRIZE FOR THESE EXPERIMENTS WHERE I'D DONE EXACTLY THE SAME THING, I DIDN'T FEEL -- I DIDN'T -- I FELT ACTUALLY HAPPY BECAUSE HE REPLICATED WHAT I'D FOUND. HE GOT THE SAME RESULTS. I FEEL BAD FOR FRED RICHARD, BECAUSE I THOUGHT -- I DIDN'T -- YOU KNOW, I WAS REALLY FOLLOWING HIS DIRECTIONS AND RATHER BADLY. I HAD VERY LITTLE EXPERIENCE IN THE LAB, I CAUSED FIRES, I BROKE U VET, I DID ALL SORTS OF DREADFUL THINGS, AND HE WAS VERY PATIENT WITH ME. AND I THINK HE -- YOU KNOW, HE WAS A GOOD FRIEND OF CHRIS. THEY SAILED TOGETHER. I SAILED WITH FRED SOME AND HE WAS A REMARKABLE PERSON. THAT'S LIFE. >> THANK YOU VERY MUCH. [APPLAUSE] >> JUST TWO MORE TALKS AND THEN WE'LL LET YOU FOR THE REFRESHMENTS. THE NEXT ONE IS BUHM SOON PARK, IN THE HISTORY OFFICE FOR ATE FOR EIGHT OR NINE YEARS WHO WILL TALK A LITTLE ABOUT HIS ANALYSIS OF CHRIS' ROLE IN EDUCATION. AND I SHOULD MENTION THAT I WAS VERY INTERESTED TO HEAR DR. STEINER TALK ABOUT HER EXPERIENCE IN SWARTHMORE BECAUSE I'VE INFERRED THAT SOME OF THE THINGS THAT MADE CHRIS A GREAT SCIENTIST WERE FROM THE YEARS THAT HE WAS AT SWARTHMORE EXPERIENCING THE SAME STIMULI THAT DR. STEINER AND OTHERS HAVE EXPERIENCED OVER THE YEARS. BUHM SOON PARK. >> THANKS, ALAN, FOR INVITING ME HERE. I'M HONORED AND GLAD TO COME BACK HERE TO THIS CAM CAMPUS WHERE I SPENT SEVERAL YEARS AS A RESEARCH FELLOW AT THE OFFICE OF NIH HISTORY, NIDDK AND NHLBI. I'M ALSO DELIGHTED TO TALK ABOUT CHRIS ANFINSEN AND THE DEVELOPMENT OF HIS TEACHING ASPIRATION WITHIN THE NIH INTRAMURAL PROGRAM. AND MY TALK WILL BE A BIT DIFFERENT BECAUSE I'M NOT DIGGING UP MY OWN MEMORIES OF CHRIS ANFINSEN BECAUSE I HAVE NO MEMORY, AND I CAME TO NIH IN 1999 AND ALREADY HE PASSED AWAY, BUT I AS A HISTORIAN AM DIGGING UP MATERIALS AND DIGGING UP DOCUMENTS AND PAPERS ABOUT HIM, AND SO THIS IS SORT OF A RECONSTRUCTION OF MY UNDERSTANDING OF CHRIS ANFINSEN AND HIS LIFE. MY TALK IS CENTERED ON TWO EVENTS. ONE, THE CREATION OF THE RESEARCH ASSOCIATE PROGRAM, AND THE OTHER, THE FOUNDATION FOR ADVANCED EDUCATION IN THE SCIENCES. THE IDEA OF THE RESEARCH ASSOCIATE PROGRAM WAS PROPOSED AND APPROVED IN 1956, AND A TOTAL OF 14 WAS SELECTED IN THE FOLLOWING YEAR. ANFINSEN WAS DEEPLY INVOLVED FROM THE BEGINNING. FAES WAS INCORPORATED YEARS LATER, 1959, AND ANFINSEN WAS, AS YOU CAN SEE HERE, ONE OF THOSE 11 PEOPLE WHO SIGNED FOR ITS CREATION. ONE MAY WONDER WHY ANFINSEN WAS SO MUCH INTERESTED IN TEACHING AT NIH. ONE CLUE CAN BE FOUND IN HIS OWN EXPERIENCE AS A VISITING RESEARCHER AT KARLS BERG LABORATORY IN 1939. THIS PICTURE WAS TAKEN AROUND THAT TIME. ACTUALLY I FOUND THIS PHOTOFROM NLM'S PROFILES IN SCIENCE FOR ANFINSEN. ANFINSEN LATER RECORDED HIS EXPERIENCE THERE AS PERHAPS THE MOST FORMATIVE AND EXCITING YEAR OF MY LIFE, AND THE ENDURING INFLUENCE OF THE SCIENTIFIC AND SOCIAL ATTITUDES THAT HE LEARNED THERE. DURING HIS STAY, HE ALSO MET SOME OTHER VISITORS FROM HARVARD UNIVERSITY, I THINK THE PERSON IS HATTIE BECHER. WHO MIGHT INFLUENCE HIS DECISION TO GO TO HARVARD FOR HIS DOCTORAL STUDY. AFTER RECEIVING HIS PH.D. IN 1943, HE BEGAN TO TEACH WHOM HE CALLED THOSE BRIGHT HARVARD MEDICAL STOOL STUDENTS AS HARVARD MEDICAL SCHOOL STUDENTS AS AN INSTRUCTOR. AND HE STAYED AT HARVARD FOR SEVERAL MORE YEARS UNTIL IN 1950, WHEN HE LURED AWAY TO NIH BY JAMES SHANNON, WHO WAS AT THE TIME SCIENTIFIC DIRECTOR OF THE HEART HEART INSTITUTE. ANFINSEN VISITED KARLS BURG LABORATORY FOR HIS SABBATICAL IN 1954 TO WORK WITH LINDERSTROM-LANGE ON THE BIOCHEMISTRY OF RNAs. NO DOUBT THIS TOPIC BECAME THE FOCAL POINT OF HIS RESEARCH FOR MANY YEARS TO COME. HE ADMIREED LINDERSTROM-LANGE AS SOMEONE WHO COULD TURN A GOOD IDEA INTO A TERRIFIC IDEA WITH HIS CHARISMA. HE RANKED CARLSBERG LAB AS ONE OF THE SCIENTIFIC ME CAN SCIENTIFIC MECCAS , AND PA TOUR INSTITUTE. IT IS INTERESTING AND IMPORTANT TO NOTE THAT HE ALSO THOUGHT THAT NIH WAS ONE OF THOSE MECCAS BENEFITING FROM ITS FOREIGN VISITORS AND THE YOUNG MEDICAL DOCTORS WHO CAME DURING THE YEARS. THE DR. DREFT BEGAN WITH OUTBREAK OF THE KOREAN WAR IN 1950 AND WAS ENFORCED UNTIL THE END OF THE VIETNAM WAR IN 1973. FROM EARLY ON, THE SERVICE WAS DEEPLY INVOLVEED IN ITS ADMINISTRATION EITHER BY THE LOCAL SELECTIVE BOARD OR ASSIGNING -- DOCTORS TO VARIOUS POSTS OF ITS OWN FACILITIES INCLUDING NIH. INDEED, THERE WAS AN URGENT NEED TO FILL HOUSE STAFF POSITIONS OF THE CLINICAL CENTER A NEW TERM, "CLINICAL ASSOCIATES," WAS USED FOR NEW DOCTORS IN ORDER TO DISTINGUISH THEM FROM ORDINARY RESIDENTS CALLED" CLINICAL FELLOWS." AS YOU CAN SEE, IN THE PRESENT TRUMAN SPEECH DURING HIS VISIT TO THE NIH IN 1951 FOR LAYING THE CORNERSTONE CEREMONY, THERE WAS A HIGH EXPECTATION TO THE CLINICAL CENTER THAT SHOULD TRANSLATE SCIENTIFIC KNOWLEDGE INTO MEDICAL THERAPIES. SOME SCIENTISTS INCLUDING ARTHUR CORNBERG -- SUCH FREEDOM. ANFINSEN SAW IT DIFFERENTLY. TO HIM, IT WAS A GREAT OPPORTUNITY TO PURSUE SOMETHING FUNDAMENTALLY IMPORTANT BECAUSE OF ALL THE VISITING FELLOWS PASSING THROUGH NIH. HE WAS CONVINCED THAT IF TRANSLATION WERE TO TAKE PLACE, IT WOULD BE DONE BY PEOPLE WHO WERE CONVERSANT BOTH IN MEDICAL PRACTICES AND ADVANCED SCIENCES. FOR THIS REASON, HE BEGAN TO EX-PROPER WAYS TO TRAIN MEDICAL DOCTORS IN BASIC SCIENCES BY UTILIZING A LARGE RESERVOIR OF POTENTIAL TEACHERS WITHIN THE INTRAMURAL PROGRAM. BUT FINDING A SUITABLE WAY OF INSTITUTIONALIZING A TEACHING PROGRAM AT ANYWAS NOTNIH WAS NOT EASY, SO ANFINSEN AND OTHERS SOUGHT A PARTNERSHIP WITH THE U.S. DEPARTMENT OF AGRICULTURE GRADUATE SCHOOL BY OFFERING COURSES TO BE TAUGHT AT NIH AND BY THE NIH PEOPLE, AND FOR THE NIH PEOPLE. BUT HE ADMINISTERED REMOTELY BY USDA. SO THE YEARS NIH, USDA, EVENING SCHOOL, STUDIED THIS WAY IN 1954. FROM THE OUTSET, THE EVENING COURSE WAS VERY POP LARK BUT SEVERAL MEMBERS OF THE SCIENTIFIC ADVISORY COMMITTEE WHICH WAS IN CHARGE OF COURSES FOR BASIC SCIENCE REALIZED THAT THIS FORMULA OF BEING OPEN TO THE GENERAL PUBLIC HAD THE LIMITATIONS OFFERING A SERIES OF INTENSIVE COURSES FOR MEDICAL DOCTORS. SO IN 1956, THEY BEGAN TO DISCUSS THE IDEA OF TWO-YEAR PROGRAM FOR PEOPLE WHO HAVE ALREADY HAD THEIR M.D. DEGREE AND INTEND TO GO INTO MID CALORIE SECH AS A CAREER CAL MEDICAL RESEARCH AS A CAREER. THE IDEA WAS THESE PEOPLE WOULD SPEND A QUARTER OF THEIR TIME TAKING FORMAL LECTURES AND IN THE REST OF THE TIME IN THE LABORATORY WITH A PRECEPTOR. THEY WOULD BE DIFFERENT FROM VR ORIENTED -- HENCE IN THIS REGARD, THEY WERE CALLED RESEARCH ASSOCIATES. THIS IDEA DREW A CONSIDERABLE INTEREST FROM JIM SHANNON AT THE TIME NIH DIRECTOR. SHANNON REQUESTED A MORE DETAIL DETAILED -- AFTER INTENSIVE REVIEW OF SCIENTIFIC PROGRAMS IN OTHER SCHOOLS SUCH AS HARVARD MEDICAL SCHOOL AND THE ROCKEFELLER INSTITUTE THE, THE -- MEMBERS PREPARED THE PROPOSAL AND DISCUSSED IT AT THE SCIENTIFIC DIRECTOR'S MEETING. TWO POINTS WERE EMPHASIZED. THE IMPORTANCE OF HAVING THE RESEARCH ASSOCIATE WALK ON WORK ON PROBLEMS OF HIS OWN CHOICE RATHER THAN BE SERVANTS OF THE RESEARCH PROBLEMS OF THE PRECEPTOR. AND THE IMPORTANCE OF PROVIDING THE STUDENTS WITH SOME INTEGRATED ORGANIZED BASIC RESEARCH, BASE A IK KNOWLEDGE AS FOUNDATION THAT WOULD PERMIT THEM TO DO THEIR OWN INTEGRATING OF KNOWLEDGE LATER. THIS PROPOSAL WAS GENERALLY WELL RECEIVED AMONG THE SCIENTIFIC DIRECTORS. THERE WAS EVEN A SUGGESTION THAT NIH SHOULD BECOME PROGRESSIVELY MORE IMPORTANT AS A TRAINING AND EDUCATIONAL INSTITUTION, AND THAT IN THE FUTURE IT MIGHT BE DESIRABLE TO ESTABLISH A FULL GRADUATE SCHOOL. A FURTHER DISCUSSION CONTINUED AT A NOVEMBER MEETING. OF COURSE THERE WERE SOME OBJECTIONS TO THE PROGRAM BECAUSE OF THE LARGE AMOUNT OF TIME IT MIGHT REQUIRE OF THE NIH MANPOWER -- FINALLY, A COMPROMISE WAS REACHED. THE RESEARCH ASSOCIATE PROGRAM SHOULD BE AN NIH FUNCTION, BUT AN INSTITUTE, EACH INSTITUTE THE MAY TAKE PART OR NOT AS ITS DISCRETION DEPENDING ON WHETHER THEY WISH TO ALLOCATE PROVISIONS AND MONEYS FOR THIS PURPOSE. IT SHOULD BE BORNE IN MIND THAT THIS PROGRAM WAS NOT THE IMPOSED FROM TOP NIH DIRECTOR, IT WAS RATHER CONCEIVED BY BOTTOM, BY MEMBERS OF THE SCIENTIFIC ADVISORY COMMITTEE. PARTICIPATION WAS VOLUNTARY, AND ABSOLUTELY UP TO EACH INSTITUTE. BUT SHANNON'S CONTRIBUTION WAS NOT THE SMALL. IN THE REPORT OF THE EDUCATION PROGRAM, ACKNOWLEDGED HIS MORAL SUPPORT. DR. SHANNON MEETING WITH THE SCIENTIFIC ADVISORY COMMUNITY POINTED OUT THE INCREASING EXTENT TO WHICH BASIC RESEARCH IN THE MEDICAL SCIENCE WAS BEING CONTRIBUTED BY MEDICALLY TRAINED INVESTIGATORS AND THE IMPORTANCE, THEREFORE, OF MAKING AVAILABLE THE BEST IN TRAINING OPPORTUNITIES FOR THIS THESE MEN. IN FACT, NIH'S MD HAD BEEN PH.D. PROGRAM KNOWN AS MEDICAL SCIENCE TRAINING PROGRAM, SORT OF EXTRAMURAL VERSION OF RESEARCHERS' PROGRAM, WAS BEING PREPARED AT THE TIME AND MSTP PROGRAM OFFICIALLY STARTED IN 1964. THE ESTABLISHMENT OF FAES ALSO COULD BE SEEN AS THE OUTCOME OF FURTHER DEVELOPMENT OF RESEARCHER ASSOCIATE PROGRAM. THE SCIENTIFIC ADVISORY COMMITTEE WANTED TO AWARD A FORMAL RECOGNITION, MEANING DEGREE, BUT IT WOULD BE IMPOSSIBLE UNLESS NIH ESTABLISHED THE -- OF ITS OWN. THE -- MEMBERS APPROACHED SHANNON WITH THE IDEA OF NIH -- SCHOOL, WHICH AT THE TIME WAS ORIGINAL PROPOSAL WAS CALLED THE ACADEMY OF ADVANCEMENT OF CONTINUING EDUCATION IN THE CENSUS. AND THEY SAID WE ARE PROUD OF THE SUCCESS OF THE PROGRAM AND HOPE THAT NIH CAN NOW RECOGNIZE IT AS ITS OWN PROGRAM IN A MORE POSITIVE WAY AND SUPPORT IT IN A MANNER THAT WILL ENCOURAGE ITS MATURATION IN THE RIGHT DIRECTIONS. SHANNON WAS SUPPORTIVE, BUT HIS STAFF MEMBERS WERE GENERALLY SKEPTICAL ABOUT THE IDEA. THEY WONDER, HOW DOES THE PROPOSED -- SCHOOL FIT INTO THE PRIMARY MISSION OF THE HIV? OTHERS SAID WHAT REAL AND TANGIBLE BENEFITS ARE OFFERED TO THE NIH IN THE FURTHERANCE OF OUR PRIMARY MISSION? HERE CAME THE IDEA OF CREATING A THIRD PARTY FOUNDATION. SHANNON APPROVED IT IN 1959 AND IT TOOK TWO MORE YEARS TO GET SURGEON GENERAL'S APPROVAL BECAUSE OF THE CONCERNS ABOUT THE RUMOR THAT ULTIMATE AIM OF THE FOUNDATION IS TO DEVELOP A CLASSICAL UNIVERSITY STRUCTURE AT THE NIH, WHETHER UNIVERSITY OF MEDICAL SCIENCES. FAES WAS FULLY FUNCTIONAL SINCE 1961 BUT DID NOT -- THE ISSUE OF -- AUTHORITY. THE BOARD OF DIRECTORS OF THE FAES ARE -- THE PROPOSAL AT AN INFORMAL MEETING IN THE SUMMER OF 1962. DAN STEINBERG TU TIME THE PRESIDENT OF FA ES SAID, IT WOULD BE MOST -- FOR US TO BE SET UP TO GRANT A DIFFERENT KIND OF DEGREE, POSSIBLY IN MEDICAL RESEARCH, DOCTORAL -- CHRIS ANFINSEN ADDED, WE ARE BEST EQUIPPED HERE, I THINK, TO -- THOSE WHO ARE REASONABLY WELL TRAINED. WE'VE DONE BEST WITH THE RESEARCH TYPE OF PERSONS. WHY DON'T WE EXPEND WHAT WE HAVE NOW AND FORMALIZE THE PROGRAM TOWARD THE GRANTING OF DEGREES. A SECOND DOCTORAL DEGREE TO THOSE WHO ALREADY HAD AN M.D. AND ALL P.H.D. WAS PROPOSED. SHANNON AT THE TIME WAS NOT ENTIRELY ON THE NEGATIVE ABOUT THIS IDEA BUT HE WAS NOT ENTHUSIASTIC ABOUT IT EITHER BECAUSE OF ALL THE LEGAL PROBLEMS AND SO ON. SO NO FURTHER PROGRESS WAS MADE THEREAFTER. HANS STETTEN AND ANFINSEN IN THE MEANTIME LEFT NIH, ST. STETTEN TO RUTGERS, ANFINSEN TO HARVARD AS A PROFESSOR, BOTH OF THEM EVENTUALLY CAME BACK. SO WHAT DOES EMPHASIS TEACHING INSPIRATION TELL US TODAY? I'M SHOWING BACK AT ALL WILL YOUR SLIDE. I THINK THAT TO HIM, THE SCIENTIFIC AND THE SOCIAL WERE NOT TWO SEPARATE THINGS IN ONE'S LIFE. AS HE SAID ABOUT HIS EXPERIENCE AT THE CLARKSBURG LABORATORY. OF COURSE HE DID NOT COME TO THE NIH TO TEACH TO BE SURE, BUT AS SOON AS HE SAW AN INFLUX OF MEDICAL DOCTORS, HE DID NOT HESITATE TO CREATE A SOCIAL ENVIRONMENT FOR THEM TO MAXIMIZE THEIR SCIENTIFIC POTENTIAL. HE TAUGHT WHILE LEARNING. HE LEARNED WHILE TEACHING. WE CAN SEE THIS FROM THE FACT THAT THE MOST CREATIVE YEAR -- HIS MOST CREATIVE YEAR AS A RESEARCHER COINCIDES WITH HIS MOST ENERGETIC TIME AS A TEACHER. THANK YOU VERY MUCH. >> SINCE MANY OF THE POINTS I WAS GOING TO MAKE HAVE BEEN COVERED, I'LL GIVE MY TALK IN ABBREVIATED FORM, BUT I'D LIKE TO COMMENT ON WHAT DR. PARK JUST SAID THAT THE LAST TWO SUMMERS, CHRIS FROM THE HISTORY OFFICE AND I HAVE BEEN THE HOST OF A WOMAN SENT BY THE CHINESE ACADEMY OF SCIENCES TO NIH TO FIND OUT HOW THIS PROGRAM HAS BEEN SO SUCCESSFUL AND WHAT THINGS THE CHINESE ACADEMY OF SCIENCES CAN LEARN ABOUT TRAINING THEIR OWN SCIENTISTS, AND ALTHOUGH THIS WOMAN IS VERY SHARP AND A VERY QUICK LEARNER AND ENGLISH IS QUITE GOOD, I THINK IT WILL TAKE HER ANOTHER 10 OR 15 SUMMER VISITS TO NIH TO UNDERSTAND ANYTHING ABOUT THE COMPLEX HISTORY OF THE INTRAMURAL PROGRAM AND WHY IT WAS SUCCESSFUL AND WHAT HAS NOT BEEN SUCCESSFUL, BUT YOU CAN SEE SOME OF THE COMPLEXITY WITH RESPECT TO EDUCATION IN DR. PARK'S PRESENTATION. I WANT TO JUST BRIEFLY CLOSE BY AMPLIFYING A LITTLE ABOUT WHAT I'VE TERMED CHRIS ANFINSEN AS A FULLY ENGAGED SCIENTIST. THIS IS AN OUTLINE OF HIS CAREER, YOU'VE HEARD ALL THE DETAILS SO FAR SO THERE'S NO REASON FOR ME TO GO OVER IT FURTHER THE. I JUST WANT TO ADD ONE POINT ABOUT WHEN I'D GONE THROUGH HIS PUBLICATION RECORD IN DETAIL, ONE SEES THAT THERE ARE EIGHT OR 10 DIFFERENT AREAS OF RESEARCH THAT HE WORKED ON DURING HIS CAREER, WHAT I CALL 55 YEAR SIGN TIIVE JOURNEY FROM THE MICRO METHODS IN RETINAL HISTOCHEMISTRY IN CARLSBERG AND AS A GRADUATE STUDENT AT HARVARD TO THE LAST WORK ON ON HIGHLY THERMOSTABLE -- AFTER HE RETIRED FROM THE NIH WHICH UNFORTUNATELY WE'VE NOT HAD TIME TO GO INTO IN MORE DETAIL, ALTHOUGH -- AND OTHERS FROM HOPKINS WHO INTERACTED CLOSELY WITH CHRIS DURING THOSE YEARS ARE HERE TODAY FORTUNATELY. BUT UNLIKE MANY SCIENTISTS, CHRIS ALWAYS CHANGED HIS FOCUS OF INTEREST EVERY FIVE OR SEVEN YEARS, AND I THINK THAT WAS A GREAT STRENGTH, AND I THINK THE FACT THAT IT DOESN'T HAPPEN SO OFTEN IN THESE DAYS IS A WEAKNESS OF OUR SCIENTIFIC SYSTEM, NOT A STRENGTH. I WANT TO HIGHLIGHT WHAT I'VE CALLED SOME OTHER ASPECTS OF THE ANFINSEN'S CAREER. ONE, SOMETHING THAT HASN'T BEEN MENTIONED UP TO NOW WAS HI HIT OR MISS PUBLICATION OF A SMALL BOOK CALLED THE MOLECULAR BASE OF EVOLUTION, PUBLISHED BY JOHN WIRILY AND SONS IN 1959. HE WROTE THIS SPENDING AN HOUR OR TWO EACH MORNING WORKING ON IT BEFORE HE GOT TO HIS RESEARCH, AND THIS WAS THE RESEARCH THAT LED TO HIS NOBEL PRIZE AND SO THIS WAS NOT A INSIGNIFICANT CONTRIBUTE OF HIS TIME, AND IN THE PREFACE OF THE OF THAT BOOK, HE SAYS THAT EVERY BIOLOGIST WOULD REALIZE THAT EVOLUTION IS CENTRAL TO ANY STUDIES OF BIOLOGY, AND THIS IS A THEME THAT WAS LATER ARTICULATED IN 1971 OR '72 AND USUALLY CREDITED TO JOHN BUT IT'S CLEARLY STATED IN ANFINSEN'S BOOK WRIT WRITTEN IN 58 AND 59, DOESN'T GET NUT ENOUGH CREDIT FOR THIS REALIZATION, VERY UNUSUAL FOR A BIOCHEMIST TO HAVE SUCH A BROAD PERSPECTIVE. AS MINGED, HE CHANGED HIS RESEARCH FOCUS EVERY FIVE TO 10 YEARS. HE ALSO EMPHASIZED THE THERMODYNAMIC OR EEK EQUILIBRIUM APPROACHES TO BIOCHEMICAL PROBLEMS AS SEVERAL OF THE SPEAKERS HAVE SAID TODAY, HE WAS NOT INTERESTED IN THE KINETICS IN THE PROTEIN FOLDING FIELD, IT'S REALLY SPLIT INTO TWO, THE EQUALLY BREEM PEOPLE AND THE KINETIC PEOPLE, AND THEY DON'T INTERACT, THEY DON'T SPEAK TO EACH OTHER, BUT I THINK THAT THE THERMODYNAMIC APPROACH AS YOU'VE HEARD FROM SEVERAL LECTURES TODAY HAS BEEN QUITE ROBUST IN ITS CONTRIBUTIONS. AND AS IMPLIED A MOMENT AGO, HE WORKED AS WELL WITH M.D.s OR PERHAPS BETTER WITH M.D./PH.D.s, AND RELATED TO THIS, HE HAD AN EQUAL INTEREST IN BASIC AND APPLIED RESEARCH. HIS FIRST WORK AT HARVARD AFTER GETTING HIS DEGREE ON MALARIA, THEN WITH A.K. SOLOMON AND OTHERS IN THE ANESTHESIOLOGY DEPARTMENT, AND HE NEVER FELT THAT APPLIED PROBLEMS WERE BELOW HIS INTEREST. SOME OTHER ASPECTS OF HIS CAREER, HIS APPRECIATION OF THE IMPORTANCE OF EDUCATION OF COURSES AT FAES, SEMINARS WHICH HE ACTIVELY PARTICIPATED IN, TEACHING IN THE SEMINAR SERIES, NOT ONLY HELPING ORGANIZE IT, AND LASTLY AND I'LL COME TO IT IN THE LAST SLIDE, HE WAS ACTIVE THROUGHOUT HIS CAREER IN POLITICS, ESPECIALLY ABOUT NUCLEAR TESTING AND HUMAN RIGHTS, AND THIS IS A TIME WHEN THE GOVERNMENT WAS NOT NECESSARILY ANY MORE COOPERATIVE IN THIS ACTIVITY THAN IT IS NOW BUT HE SOMEHOW MANAGED TO DO IT. THIS IS A PICTURE MIGHT HAVE COULD COPY WHICH LED ME TO CHOOSE HIS LAB AND MANY OTHER PEOPLE THAT I MEET OVER THE YEARS WERE STIMULATED TO COME WORK WITH HIM BECAUSE OF THE BOOK WHICH WAS FOR A DECADE OR MORE A MAJOR CONTRIBUTION TO THE FIELD. DR. STEINER DIDN'T SHOW THIS SLIDE, BUT THIS IS FROM THE CELL PAPER IN JUNE OF THIS YEAR BY DAVID EISENBERG, POINTING OUT EISENBERG'S DISCOVERY VIA JOHN EDSEL THAT DR. STEINER AND FRED RICHARD HAD BEEN DOING VERY SIMILAR EXPERIMENTS TO WHAT ANFINSEN HAD BEEN DOING HERE, AND EISENBERG ENTITLES HIS PAPER "HOW HARD IT IS SEEING WHAT'S IN FRONT OF YOUR EYES." THIS ONE QUOTE FROM HIS PAPER IN "CELL," WE SCIENTISTS MAKE SIGNIFICANT DISCOVERIES ONLY WHEN OUR OBSERVATIONS CAN BE LINKED BY SCIENTIFIC CONTEXT TO SIGNIFICANT QUESTIONS. WHEN LISA STEINER WAS RENATURING RIBOKNEE CLEE ACE A, THE SCIENTIFIC CONTEXT HAD NOT YET POSED THE QUESTION OF HOW THE WIGGLE EE PROTEIN CHAIN ASSUMES ITS THREE DIMENSIONAL STRUCTURE. THAT WE WAS BEFORE ITS TIME AND THAT'S A CON -- I THINK THERE WERE OTHER FACTORS FOR ANFINSEN IN HIS DISCOVERY THAN THE QUESTION BEING AT THE RIGHT TIME, ONE, I THINK WAS EQUAL INTEREST IN BASIC AND APPLIED RESEARCH, IN PARTICULAR, HE HAD FORMULATED BY 1960 OR '61 THE GOAL OF TOTAL CHEMICAL SYNTHESIS OF PROTEINS AND FOR THAT, SHOWING THAT THE PROTEINS COULD REFOLD TO AN ACTIVE FORM IN VITRO WAS ESSENTIAL IF DR. KENT'S LECTURE INDICATED. SO I THINK HAVING AN APPLIED GOAL AS WELL AS A BASIC GOAL WAS ONE OF THE REASONS THAT THE YALE LAB DID NOT COME TO THE SAME CONCLUSIONS ABOUT THE IMPORTANCE OF THE WORK. SECONDLY, ANFINSEN'S KNOWLEDGE OF INTEREST RANGING FROM ORGANIC CHEMISTRY THAT HE HAD LEARNED AS A GRADUATE STUDENT FOR TWO YEARS AT PENN TO THE ENTOMOLOGY OF HIS LATER EXPERIENCES GAVE HIM A BREADTH OF KNOWLEDGE AND APPROACHES THAT WERE RELATIVELY UNIQUE IN THE BIOCHEMICAL FIELD, AND THEN ALSO THE OVERVIEW OF THE IMPORTANCE OF EVOLUTION IN BIOLOGY ALSO, I THINK, IF YOU'LL READ HIS PAPERS, FOR EXAMPLE, THE CLASSIC PAPER, YOU'LL SEE THE REFERENCES TO EVOLUTION AND SELECTION WHICH WERE VERY IMPORTANT TO HIM ALL THROUGH HIS WORK. I COME BACK TO THIS SLIDE, THE TWO BOTTOM ASPECTS, NOW TALK FURTHER ABOUT THE SCIENCE. ONE IS THE INTEREST IN CONTINUING EDUCATION WHICH DR. PARK TALKED ABOUT, BOTH THE FAS AND THE WEEKLY EDUCATIONAL SEMINARS FOR PHYSICIANS, HIS DEEP INTEREST IN TRAINING PHYSICIANS BASED UPON HIS SWARTHMORE EXPERIENCES AND ALSO AT OXFORD. ONE THING THAT MAY NOT HAVE BEEN CLEAR, DR. PARK'S PRESENTATION WAS THAT JAMES SHANNON WAS FOR THE MOST PART AGAINST SUCH ACTIVITIES AND IT WAS ONLY RELUCTANTLY DRAGGED HIM INTO BELIEVING THE IMPORTANCE OF EDUCATIONAL PROGRAMS, AND I THINK THAT WAS ESSENTIAL FOR THE SUCCESSES OF THE INTRAMURAL PROGRAM. AND LASTLY, THE POLITICAL ACTIVISM, HE BECAME CHAIR AT THE NATIONAL ACADEMY OF SCIENCES FOR HUMAN RIGHTS, A POSITION HE HELD UNTIL 1989, HE SPENT MANY HOURS AFTER WRITING LETTERS TO LEADERS AROUND THE WORLD WITH HIS NOBEL PRIZE WHERE GOVERNMENTS WERE REPRESSING SCIENTISTS, IN '81, HE AND OTHER MEMBERS TRAVELED TO SOUTHERN AMERICAN COUNTRIES ON A MISSION TO LIBERATE 12 SIGN IT TISES WHO WERE BEING COERCED IF NOT ARRESTED BY THE MILITARY GOVERNMENT. IN THE NATIONAL LIBRARY OF MEDICINE PAPERS, I RECENTLY DISCOVERED THIS SENTENCE FROM A NOTE THAT HE DID SOME YEARS LATER. AT THAT POINT IN MY LIFE, I HAD STOPPED SMOKING FOR 10 YEARS, BUT TWO WEEKS IN BENES ARIES GOT ME BACK ON THE WEED AS AN ANECDOTE AFTER NERVE-WRACKING INTERVIEWS WITH RELATIVES AND OFFICIALS AT ALL HOURS OF THE DAY AND NIGHT, THE TENSION THAT EXISTED. BUT SADLY, I THINK, IT WAS THE RETURN OF HIS SMOKING THAT HE HAD STOPPED FOR MANY -- I BELIEVE FOR MANY YEARS, ALTHOUGH LIBBY CAN TELL ME MORE PRECISELY, THAT THAT CONTRIBUTED TO HIS DEATH IN 1995. THIS IS MY FERRET PICTURE FAVORITE PICTURE OF HIM, JUST BEFORE HE RETIRED, THE SHELF ABOVE HIS DESK IS EMPTY BECAUSE THE BOOKS, THE ADVANCEMENT OF PROTEIN CHEMISTRY HAVE BEEN SHIPPED OFF TO ISRAEL, SO I CAN REASONABLY DATE THIS PICTURE AS WELL AS HIS WORLD PORTABLE TYPEWRITER, WHICH IS IN EXHIBIT IN THE MAIN LOBBY. AND FOR THOSE OF YOU INTERESTED IN MORE INFORMATION, ALTHOUGH YOU'VE HAD A LOT OF IT THIS AFTERNOON, FOUR OR FIVE YEARS AGO, I PUBLISHED A BIOGRAPHICAL MEMOIR BY THE NATIONAL ACADEMY OF SCIENCES, WE HAVE SOME COPIES UP ABOVE WHERE YOU CAN ACCESS IT THROUGH THE BIOGRAPHICAL MEMOIRS PAGE ON THE NATIONAL ACADEMY OF SCIENCES WEB, ALSO THE NATIONAL LIBRARY OF MEDICINE AND THE OFFICE OF NIH HISTORY HAVE PAGES DEVOTED TO CHRIS' LIFE, THE NATIONAL LIBRARY OF MEDICINE IN PARTICULAR HAS MANY OF THE IMPORTANT DOCUMENTS RELATED TO HIS CAREER, BUT LET ME END NOW AND THANK YOU ALL FOR PARTICIPATING AND INVITE ALL OF YOU TO JOIN US ON THE TERRACE FOR SOME REFRESHMENTS. THANK YOU.