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Home , Hartmut Michel, Johann Deisenhofer, Robert Huber

EUGENE GARFIELD INSTITUTE FOR SCIENTIFIC lNFORMATION~ 3501 MARK ETST PHILADELPHIA PA 19104 The 1988 in Goes to Joham Deisenhofer, , and Hartnmt Michel for Elucidating Photosynthetic Processes

Number 22 Mav,. 29, 1989

The 1988 was awarded to , Robert Huber, and , for revealing the atomic structure of a membrane-bound that drives in a purple bacterium. After briefly recounting the work that led to the prize, this essay examines the laureates’ most-cited papers and the IS1° research fronts in which they appear.

Photosynthesis, as most schoolchildren As Douglas C. Youvan, Massachusetts In- learn, is the process by which sunlight is stitute of Technology, Cambridge, and converted into chemical energy—a process Barry L. Marrs, E.I. du Pent de Nemours essentird to most life on earth. During the & Company, Inc., Wihnington, Delaware, 1980s researchers made significant progress point out, recent studies on photosynthesis in elucidating the structural and chemical have employed bacteria belonging to the features of photosynthesis. The 1988 Nobel genus Rhodopseubrwtas. Although rhodo- Prize in chemistry recognizes three West pseudomonad photosynthesis differs in cer- German scientists for their contributions to tain respects from that carried out by higher the understanding of photosynthetic pro- plants, it is generally similar; and these bac- cesses and protein structure at the atomic teria are well suited to the techniques of mo- level. The three are Joham Deiserthofer, lecular genetics. The interiors of the bacteria Howard Hughes Medical Institute, and De- are filled with photosynthetic membranes, partment of , University of which are small, hollow spheres (or stacks Texas southwestern Medical Center, Dallas, of membranes, depending on the species) Texas; Robert Huber, Max Pkrnck Institute made of lipid bilayers. The photosynthetic for Bicsehemistry, Martinsried, Federal reaction centers, which are composed pri- Republic of (FRG); and Hartmut marily of , are embedded in the pho- Michel, Max Planck Institute for Biophys- tosynthetic membranes. z ics, am Main, FRG, who were The challenge for researchers, as noted honored for determining the three-dimen- by Richard Henderson, Laboratory of Mo- sional structure of the photosynthetic reac- lecular Biology, Medical Research Council, tion center in the purple bacterium R/rodo- Cambridge, UK, lay in releasing the pro- pseadomonas viridis. As noted in the an- teins in undamaged and stable form from the nouncement from the Royal Swedish Acad- lipid bilayers that constitute the mem- emy of Sciences, Stockholm, “They are the 1 branes.s The next step would be to crystal- first to succeed in unraveling the full de- lize the proteins, so that their structures tails of how a membrane-bound protein is could be analyzed by means of X-ray crys- built up, revealing the structure of the mol- tallography. For decades, however, attempts ecule atom by atom. The protein is taken at crystallization had failed. The proteins from a bacterium which, like green plants have components that are not soluble in wa- and algae, uses light energy from the sun ter, since they must interact with lipids to build organic substances. ” 1 (within the membrane) and water (at the

148 membrane surface). As Roger Lewin, depu- wash them out of their membranes using a ty news editor, Science, notes, the proteins variety of detergents.5 Working with the are “partly hydrophilic and partly hydro- bacterial membrane protein bacteriorhodop- phobic, and will not line up in neat crystal- sin, Michel and Dieter Oesterhelt, collabo- line arrays when exposed to water.”4 rating first at the University of Wi,irzburg, In the late 1970s and early 1980s, Michel FRG, and later at the Max Planck Institute was one of many researchers attempting to for Biochemistry, began to experiment with crystallize membrane-bound proteins and a novel combination of detergents and small

Table 1: Papers authored by J. Defaerrbofer,H. Mkhel, and R. Huber, cited in the SCI” from 1945 to 19S8. The papers am arranged in deacenefing order, according to number of citations. A =total citations, B= bibliographic data.

A B

361 DeiaenftoferJ, Epp O, Miki K, HrrberR & Michel H. X-ray structure analysis of a membrane protein complex. Electron density map at 3 A resolution and a model of the chromophores of the photosynthetic reaction center from Rhodopseudomonas viridis. J. Mol. BioL 180:385-98, 1984, 344 Deisenhofer j & Steigem.mrrW. Crystallographic refinement of the structure of bovine pancreatic tIYPSIII inhibitor at ].5 A resolution. A.cm CrystaUogr. 11-,hrcr, .Sci, 31:238.50, 1975, 303 Huber R, Krrkh+D, Bode W, !lchwager P, Bartels K, Deiaenhofer J & .WeigemannW. Structure of the compIex forme+ by bovine trypsin and bovine pancreatic trypsin inhibitor. II. Crystallographic refinement at 1.9 A resolution. J, kfof. Biof. 89:73-101, 1974. 282 Deisenhofer J, Epp O, Mild K, HrrberR & Michel H. Structure ,of the protein subunits in the photosynthetic reaction center of Rhodopseudornonus viridis at 3 A resolution. Na[ure 318:618-24, 1985. 264 Ruehhrmmr A, Kukla D, Sebwager P, Bartels K & Huber R. Structure of the complex formed by bovine trypsin and hovine pancreatic trypsin inhibhor. Crystal structure determination and stereochemistry of the contact region. J. IUof. Biof. 77:417-36, 1973. 221 Huher R, Defaenhofer J, Cuhnan P M, Marsushhna M & PabrrW. Crystallographic structure smdies of an lgG molecule and an Fc fragment. 264:415-20, 1976. 194 Deiaeahofer J. Crystallographicrefinement and atomic mndels of a human Fc fragme~t and its complex with fragment B of protein A from Staphylococcus aureus at 2.9- and 2.8-A resolution. Biochemistry 20:2361-70, 1981. 194 Huber R & Bode W. Structural basis of the activation and action of trypsin. Account. Chem. Res. 11:114-22, 1978, 1s1 Epp O, Cobmm P, Fehlhammer H, Bade W, ScbifFer M, Huber R & Pabn W. Crystaland molecular structure of a diner comprised of the variable portions of tbe Bence-lones protein REI. Eur. J. Biochem. 45:513-24, 1974, 159 Huber R, Kukfa D, Ruehlnram A, Epp O & Formanek H. Basic trypsin inhibitor of bovine pancreas. 1, Structure analysis and conformation of the pnlypeptide chain. Naturwimenschajlen 57:389-92, 1970. 153 Huher R, Epp O, Steigemmm W & Formanek H. Atomic structure of erythrcmuorin in light of the chemical sequence and its comparison with . Eur. J. Biochem. 19:42-50, 1971. 152 Huber R & Hoppe W. Zur Chemie des Ecdyanns, VII. Die Kristafl- und Molekulstmkturanalyse des Insektenverpuppungshormons Ecdyson mit der Autnmatisierten Faltmolekalmethmie (Chemistry of the ecdyamres. 7. Crystal and molecular strucNre analysis of the insect pupation hormone ecdysone with the automatic molecular methnd). Chem. Ber. 98:2403-24, 1965. 140 Michel H. Three-dimensional crystals of a membrane protein complex. The photosynthetic reaction center from Rhodopseuriomonas viridis. J. Mol. Biol. 158:567-72, 1982. 128 HrrberR, Epp O & Forroanek H. Structures of denxy - and carbonmonoxy-erythrncruorin. J. hfo/. BioL 52:349-54, 1970. 122 Huber R, Kukla D, Ruehbnamr A & Steigemarm W. Pancreatic trypsin inhibitor (Kunitz). I. Stmcturc and fimction, CoId Spring Harbor Syrnp, 36:141-8, 1971. 118 Cobnmr P M, Defsenftofer J, HrrberR & Palm W. ~trucNre of the human molecule Koll (inumrnoglobrrlin G]): an electron density map at 5 A resolution. J. Mol. Biol 100:257-78, 1976. 99 DeiaerrhoferJ, Cobnan P M, Epp O & Huber R. Crystallograp~ic SIfUcNral stales of a human Fc fragment. II. A complete mmfel based on a Fourier map at 3.5 A resolution. Hoppe Sey/ers Z. Physiol. Chem. 357:1421-34, 1976. 58 Michel H & Ckaterhelt D. Light-induced changes of the pH grad[ent and the membrane potential in H. halobium. FEBS Lat. 65:175-8, 1976, 85 Deiaenhofer J, Jones T A, Huber R, Sjndabf J & Sjnqrrist J. Crystallization, crystal structure anrdysis and atomic model of the complex formed by a human Fc fragment and fragment B of protein A from Staphylococcus aureus. Hoppe .%ylers Z. Physiol. Chem. 359:975-85, 1978.

149 ‘‘amphiphilic” molecules, which have both The next step was to analyze the structure hydrophilic and hydrophobic elements. As through X-ray . At this Youvan and Marrs point out, the hydro- point, Michel began his collaboration with phobic ends of these molecules bind to the Deisenhofer, who worked in the pro- hydrophobic parts of the membrane pro- tein-structure laboratory in Martinsried (kr- teins, exposing the hydrophilic ends of the cated outside ) headed by Huber. small molecules. The resulting combination Within two years, in 1984, the team (includ- of small molecules and proteins can then be ing colleagues Otto Epp and Kunio Miki) dissolved in a water-based solution and solved the stmcture.7 One year later, in Na- thence crystallized.z ture, they gave a complete description of the Using these techniques, Michel and Rh. viridis reaction center—making it the Oesterhelt managed to get true three-dimens- first membrane protein whose stmcture was ional crystafs of bacteriorhodopsin; how- solved at the atomic levei. g ever, the crystals were not sufficiently well The structure elucidated by Michel, ordered for a high-resolution X-ray crystal- Deisenhofer, and Huber confirmed much of lographic analysis. Michel then turned his what had been learned about Rh. viridis in efforts to crystallizing the photosynthetic re- previous studies. There are four protein sub action center of Wt. viridis. In 1981 he suc- units, denoted L, M, H, and C, in the reac- ceeded in producing large, well-ordered tion center, The heart of the reaction center crystals of Rh. viridis. He published his consists of four molecules of chlorophyll, results in the Journal of Molecular Biology two of pheophytin, and two quinones. The in 1982.6 work of the laureates helped clarib the role

Table 2: SCP research frorrtafrom 1974to 198Sin which MUMSbv J. Deiseahofer. H. Mkhel. and R. Huber ..” - nrcur as core dnrumerrts. A = number of core papers. B = number of citing papers for that year

Number Name A B

74-0408 Proteinase inhibitors 14 I20 76-1369 Trypsin-trypsin inhibitor complexes 3 49 77-0362 Protein structure 51 492 770455 Human platelet glycoproteins 12 162 78-0151 Structure and conformation of inurwnoglobulins 10 126 78-0537 X-ray StfUCNre of proteins 5 89 7s-0594 Structural basis of action of trypsin 2 41 78-1119 NMR spectra of macromolecules 5 67 78-1633 Structure of globular pruteins and nucleic acids 3 46 79-0124 Light energy conversion in Halobacterium halobium 32 528 79-1123 NMR of pancreatic trypsin inhibition 9 126 79-1725 Activity and inhibition of bovine trypsin 3 44 79-1726 Three-dimensional structure of inrmunoglobtdins 4 42 81-0365 PrOtcin-prOtein interactions 4 58 82-1042 Crystallographic and NMR studies of the serin+ proteases 4 68 82-1799 Structural amlysis of immunoglobulin binding to protein A receptors preduccd by 2 27 stapbylwmcci and streptumrxi 83-1221 Moleeular dynamics of prntein 11 245 83-5197 Smdies of protein A binding to irnmunoglobulins and other amibndies 5 117 84-289I Photosynthetic reactinn centers in bacteria 6 73 85-2777 Peptide synthetic chemistry 3 35 85-4691 Study of confortnatiorral states of proteins 5 79 S5-4692 Photosynthetic reaction centers 3 76 8643853 Photosynthetic membrane 40 532 86-2054 Photosynthetic light reactions 10 200 86-6704 Enzyme catatysis in trypsin 3 47 87-11S2 Photosynthetic electron transport chain 47 665 87-3576 NMR studies on conforrnational properties of the Fe-fragments of bunran 2 44 inununoglobrdins 87-6S01 Folding and association of proteins 2 32 g8-0623 Photosynthetic bacteria and photosynthetic reaction centers 35 572 88-34S2 Structure and function of Fc-reeeptors 4 69

150 F@sre 1: Hktoriugraph of research on the photosynthetic reaction center. Numbers of core/citing papers are indicated at the bottom of each box. Asterisks (*) indicate research fronts in which J. Dcisenhofer is a core or citing aurhor; dollar signs ($) indicate rescarcb fronts in which H. Michel is a core or citing author.

82-1072 Structure and function of the radicalpair primarystate of photo synthetic bacteria reaction centers iEiiJ/ 84-0422 end electron 10/73 Isolation of / trensfer i n photosystem II photosystem II 82-0509 83-0397 reaction canter 7/1 06 Structure of proteins in Organization complex photosystem I and of photosyn. 14/206 r photosystem II thetic protein 26/239 complexes $84-2891 271343 ] Photosynthetic 82-0924 reaction centers 81-1542 Functional subunit structure in bacteria Bacterial of polypeptidas of photo- 6/73 photosyrrthesis rl svstem I EI L__2!E11’ 2/35

of two chlorophyll molecules-known as the and Herbert A. Hauptman special pair—that sit at the head of the com- shared the chemistry prize for their mathe- plex with one molecule each of chlorophyll, matical’ ‘direct methods” designed to solve pheophytin, and quinone arranged in sym- the three-dimensional crystal structures of metrical chains on either side around a single important molecules. We dkcussed the work iron atom. However, for reasons that remain of Hauptman and Karle in a 1986 essay.9 unclear, the electron transfer that is set in Nobel committees have recognized a num- motion by the absorption of light proceeds ber of discoveries based on X-ray diffrac- along just one of these chains.s tion methods. The elucidation of the chem- As the Swedish Academy points out in ical structure of DNA, for which American their announcement, the determination of molecular biologist James D. Watson and this structure will aid in our understanding his English colleagues Francis H .C. Crick of photosynthetic processes in higher plants. and Maurice H. F. Wilkins shared the phys- The work also has many ramifications be- iology or medicine prize in 1962, is one ex- yond the immediate study of photosynthe- ample. Another is the structural studies of sis. Other biological functions involving globular proteins by English biochemists Sir membrane proteins include the transport of John C. Kendrew and Max F. Pertatz, chemical substances between cells, hormone honored with the 1962 chemistry prize. action, and nerve impulses. I The 1961 chemistry prize also recognized The prizewinning work of Michel, Huber, research into photosynthetic processes. Or- and Deisenhofer followed the related con- ganic was honored tributions of several previous NoM lau- for his work at the University of Califor- reates. The German physicist Max von nia, Berkeley, on carbon dioxide assimila- Laue, for exampie, won the physics prize tion in plants. The cycle by which carbon in 1914 for his investigations into tbe diffrac- dioxide is converted to carbohydrates bears tion of X rays by crystals. In 1915 the En- Calvin’s name. Also applicable is the work glish physicists Sir William Henry Bragg of English biochemist Sir Peter D. Mitchell, (father) and Sir William Lawrence Bragg who received the chemistry prize in 1978 (son) shared the physics prize for their meth- for his chemiosmotic theory, which sought ods of studying crystrd structures by means to explain energy coupling in the mecha- of X rays. Seventy years later, Americans nisms of oxidative and photosynthetic phos-

151 transport chain J, ‘h”’”s’n’hesis,,,,,,I —$86-2054 471665 ‘$ 88-0623 Photosynthetic Photosynthetic I light reactions bacteria and ●$ 854692 1 Photosynthetic 0/200 photosynthetic reaction centers reaction centen 3/76 35/572 L I__-__l

phorylation-the means by which aerobic pearing in Table 1 with A. Ruehlmam as organisms and plants obtain their energy. first author, with approximately 260 cites), is one of 14 core documents in the 1974 front ‘‘Proteinase inhibitors, ” #74-0408. Most-Cited Papers Table 2 is a complete listing of the research Table 1 lists the most-cited papers by the fronts in which their work appears as core three laureates. At the top of the list is the documents. As can be seen, the fronts cover 1984 paper from the Journal of Molecuiar a variety of research on protein structure and Biology announcing the structure of the activity. membrane protein in Rh. viridis. 7 This pa- Also among the most-cited papers in Ta- per has attracted over 360 citations. It is one ble 1, with over 280 citations, is the 1985 of three core papers in the 1985 research paper from Nature giving a complete de- front “Photosynthetic reaction centers, ” scription of the reaction center. s ‘flsis paper #85-4692. As we’ve noted previously, re- also appears in Figure 1, one of 35 core search fronts are formed when papers in a papers in a 1988 front, “Photosynthetic bac- given year collectively cite a common, core teria and photosynthetic reaction centers, ” group of older papers. Research front #88-0623. Figure 2 is a graph of year-by- #85-4692 and others are shown in Figure 1, year citations to this paper and to the 1984 a histonograph of research on the photosyn- Journal of Molecular Biology paper in which thetic reaction center. the researchers initiafly announced the The second most-cited paper in Table 1, structure. 7 with approximately 340 citations, is a 1975 paper from Acts Crystallographic Section Figure 2: Year-by-year citations to J. Deiaenhofer et al.. Nafure 318:618-24, 1985 (black bar) and J. B—Structural Science by Dekenhofer and Deisenhofer et al., J. Mol. Eiol. 180:385-98. 1984 his colleague, W. Steigemann. 10 This (whke bar] paper, discussing the crystallographic struc- 200, ture of bovine pancreatic trypsin inhibitor, 175 is one of five core documents in the 1985 research front “Study of conformationaf 15!2 states of proteins, ” #85-4691. 125I The work of Huber, D&senhofer, and Michel can be found in several ISI” re- search fronts, as far back as 1974. For ex- ample, a 1973 Journal of Molecular Biology 50 paper coauthored by Huber, “Structure of 25 the complex formed by bovine trypsin and 0I.._cLl bovine pancreatic trypsin inhibitor” 11(ap- 1985 1$ 6 1587

152 ......

Michel’s 1982 paper from the Journal of Johann Deisenhofer was born in Zusamal- Molecular Biotbgy, reporting the successful theim, Bavaria, Germany, in 1943. He re- crystallization of Rh. vindis, also appears ceived his doctorate from the Max Planck in the most-cited list in Table 1, with ap- Institute for Biochemistry. In 1988 he came proximately 140 citations.6 This work is a to the US, accepting positions at the Howard core document in several fronts in Figure 1, Hughes Medical Institute, and the Depart- beginning with “Photosynthetic reaction ment of Biochemistry, University of Texas centers in bacteria, ” #84-2891, and contin- Southwestern Medical Center. uing through front #88-0623. Hartmut Michel was born in 1948 in Lud- As Nature noted in a wry postscript to its wigsburg, FRG, and took his doctoral de- report on the 1988 Nobel Prizes, Michel gree from the University of Wurzburg. He originally submitted his 1982 paper to Nu- worked at the Max Planck Institute for Bio- ture. However, because no structural infor- chemistry from 1979 until 1987, when he mation had yet been obtained, the joumrd became director at the Max Planck Institute declined to publish it. The 1985 publication for . of the complete description of the reaction This concludes our examination of the center in Nature, as the postscript concludes, 1988 Nobel Prize in chemistry. In forthcom- brought a happy ending to the ‘‘editor’s ing essays, as is our custom, we will ex- nightmare” of rejecting a Nobel Prize-wi- amine the 1988 Nobel laureates in physiol- nningpaper. 12 ogy or medicine, physics, economics, and literature. Biographical Notes

Robert Huber was born in 1937 in ***** Munich, Germany, and received his doctor- ate from the Te.chnicrd University in that My tharrkr to C.J. Fiscu.rand Christopher city. He became a division head at the Max King for their help in the preparation of this Planck Institute for Biochemistry in 1972 essay. and was made director in 1987. 01989 nl

REFERENCES

1. Royal Swedish Acartemy of Sciences.information. 19 Cktober 1988.5 p. (Press release.) 2. Youvan D C & Marrs B L. Molecular mechanisms of photosynthesis. .ki. Amer. 256(6):42-8, 1987. 3. Henderson R. Stnrctrrre of a bacterial photosynthetic reaction centrc. Nafure 318:598-9, 1985. 4. Mvbr R. Membrane protein holds photosynthetic secrets, Science 242:672-3, 1988. 5. Levi B G. Nobel shed light on key structure in pbotosynthesis. /’/ry$. Today 42(2):17-8, Febmsry 1989. 6. Mkhel H. Three-dimensional crystals of a membrane protein complex. The photosynthetic reaction center from Rhodopserrdomrwum vin”dis, J. IUOL Biol. 1S8:567-72, 1982. 7. Defsenftofer J, Epp O, Mikl K, Huber .R & Mkhel H. X-ray stmceureanalysis of a membraneprotein complex.Electron density map at 3 A resolution and a modelof the cbromophores of the photosynrtretic reaction center from Rhrrdopseudomonas virkfis. J. MoL BioL 180385-98, 1984. 8, .------S@+cture of the protein subunits in the photosynthetic reaction center of Rhodopseudomorras viridis at 3 A resolution, Nature 31S:61 8-24, 1985, 9. GartleldE. The 1985 Nobel chemistry prize to Jerome Karle rmd Herbert A. Hauprmsn and the physics prize IO Kfaus von Kfitzing contrast delayed versus “instant” recognition. Essays of an information scientist: rewards scientogruphy. Philadelptrix1S1Press, 1988. Vol. 9. p. 336-45. (Reprinted from: Current Contents (44): 3-12, 3 November 1986.) 10. Defsenhofer J & Stefgema+n W. CrysrrdIographic refinement of the structure of bovine pancreatic trypsin inhibitor at 1.5 A n%nhrtion. Acts Crysrallogr. J)-Sowcf. &i. 31:238-50, 1975. 11. Ruebtmann A, Kutda D, .%hwagerP, Bar@ K & Huber R. Structure of the complex formed by E@vine trypsin and bovine pancreatic trypsin inhibitor, Crystal WUCNW determination and stereochemistry of the contact re8ion, J. Mol. BioL 77:417-36, 1973. 12. 1988 Nobel prizes announced for physics and chemistry. Narure 335:752-3, 1988.

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