e200 01NaturePublishingGrouphttp://structbio.nature.com news and views Department of Biologi sciences Columbia University, New York, New York & Chepowish , Net al. Structure 9. 571-86( fneT, KF.etal.ce101,789800(2000) A.L. 15. Jones, P. 10027, USA. Correspondence should be addressed to PJ.T email: philip thomas@ Liu, P.O. Liu. C E.& Ames. G F.J. BioL utsouthwestern. edu or J.EH. email: hunt@ 8. Mourez. M Hofnung M.& Dassa, E. EMBO J16 Schneider E.J. Biol. Chem. 2. Dean, M- Hamon, y& Chimini, G.J. 4=学做 Clarke. D.M. J Bio Chem 276 3. c0an-1G 8 c 3.1793-1800(2001) tz, C.R. J. Biol. 13. Gaudet, R.& wiley. D.C. EMBO J. 20, 4964- 22. Qu, Q.& Sharon, F. Biochemistry history The way to NMR structures of proteins 5 Kurt Wuthrich In 1998 Kurt Wuthrich was awarded the Bell Telephone Laboratories in Murray active centers of hemoproteins-6, and I Kyoto Prize in Advanced Technology for Hill, New Jersey, where a superconducting developed a mild infatuation with having developed a method of determining high resolution H NMR spectrometer polypeptide chains only in connection the conformations of proteins, nucleic acids operating at 220 MHz was available for with the discovery of aromatic ring flip- and other biomacromolecules in solutions research on protein structure and func- ping?. My primary research interest or biomembranes, where they exhibit their tion At that time I was aware of exactly 10 changed in 1975, when I took some time to papers on NMR observations of proteins write a monograph on the early years of Wuthrich has used nuclear magnetic res- and nucleic acids, which had all been pub- biomacromolecular NMR2. These reflec- onance(NMR)techniques to study proteins lished during the period of 1957-19652. tions on the state of the field turned out e and nucleic acids since 1967. In a series of Prominent figures in the small community have been the starting point for our subse for NMR structure determination of pro- NMR observation of biological macromol- determination by nmr teins in 1982, and in 1984 the first de novo ecules were William D. Phillips, Oleg Four principal elements are combined in structure of a globular protein in solution Jardetzky and Robert G. Shulman. Based the NMR method for protein structure was determined. The Wuthrich group went on the observation of empirical correla- determination& 9: () the nuclear Over- on to solve more than 60 protein structures tions between protein unfolding and nMr hauser effect (NOE)as an experimentally in solution, including the Antennapedia spectra?-, there was much enthusiasm accessible NMr parameter in proteins that homeodomain, the cyclophilin A-cyclo- about the future of NMR for de novo pro- can yield the information needed for de orin A complex, and the human and tein structure determination. Nonetheless, novo global fold determination of a poly bovine prion proteins What follows is a personal recollection by on the metal ion coordination in the active ment of the many hundred to sever Kurt Wuthrich of how he and his associates sites of hemoproteins and on the electronic thousand NMR peaks from a protein; (ii) arrived at the first view of a protein struc- structure of the heme groups computational tools for the structural ture through the NMR eye At the time, Swiss scientists who landed a interpretation of the NMr data and the job at the famous Bell Telephone evaluation of the resulting molecular struc- oToscopy evolved into a useful tool in ered prime candidates for academic posi- techniques for efficient data collection. iod of tions back home. In 1969, I moved to the during the period 1976-1980, my research 1962-1967, my graduate and postdoctoral Eidgenossiche Technische Hochschule group at the ETH Zurich had grown to research, with Professor Silvio Fallab at the (ETH) in Zurich, where my startup pack more than 20 scientists, all of whom made University of Basel and Professor Robert E. age included an EPR and three NMR spec- great contributions toward the structure Connick at the University of California, trometers- all the instrumentation that determination method. In particular, I Berkeley, focused on the use of electron had been available to me at Bell Telephone worked with Regula M. Keller, Sidney L. paramagnetic resonance (EPR)and Laboratories. I assembled a small research Gordon and Gerhard Wagner on develop nuclear magnetic resonance (NMR) spin group, and, with time, I was promoted to ing techniques to measure NOEs for the relaxation measurements to study metal Professor of Biophysics, which is also my collection of conformational constraints in complexes in solution. With this back- present position at ETH. During the first proteins, and with Martin Billeter, Werner ground he Biophysics years at Zurich, my research continued to Braun and gerhard Wagner on the sequen Department of Dr. Robert G. Shulman at focus primarily on the metal ions in the tial resonance assignment strategy and nature structural biology volume 8 number 11. november 2001
news and views Department of Biological Sciences, Columbia University, New York, New York 10027, USA. Correspondence should be addressed to P.J.T. email: philip.thomas@ utsouthwestern.edu or J.F.H. email: hunt@ sid.bio.columbia.edu 1. Young J. & Holland I.B. Biochim. Biophys. Acta 1461, 177–200 (1999). 2. Dean, M., Hamon, Y. & Chimini, G. J. Lipid Res. 42, 1007–1017 (2001). 3. Chang G. & Roth C.B. Science 293, 1793–1800 (2001). 4. Doerrler, W.T., Reedy, M.C. & Raetz, C.R. J. Biol. Chem. 276, 11461–11464 (2001). 5. Karpowich, N. et al. Structure 9, 571–86 (2001). 6. Chen, J., Sharma, S., Quiocho, F.A. & Davidson, A.L. Proc. Natl. Acad. Sci. USA 98, 1525–1530 (2001). 7. Liu, P.Q., Liu, C.E. & Ames, G.F. J. Biol. Chem. 274, 18310–18318 (1999). 8. Mourez, M., Hofnung, M. & Dassa, E. EMBO J. 16, 3066–3077 (1997). 9. Hunke, S., Mourez, M., Jehanno, M., Dassa E. & Schneider, E. J. Biol. Chem. 275, 15526–15534 (2000). 10. Hung, L.W. et al. Nature. 396, 703–707 (1998). 11. Diederichs, K. et al. EMBO J. 19, 5951–5961 (2000). 12. Yuan, Y.R. et al. J. Biol. Chem. 276, 32313–32321 (2001). 13. Gaudet, R. & Wiley, D.C. EMBO J. 20, 4964–4972. (2001). 14. Hopfner, K.P. et al. Cell 101, 789–800 (2000). 15. Jones, P.M. & George, A.M. FEMS Microbiol. Lett. 179, 187–202. (1999). 16. Sprang, S.R. Annu. Rev. Biochem. 66, 639–678 (1997). 17. Subramaniam, S. & Henderson, R. Nature 406, 653–657 (2000). 18. Iwata, S., Ostermeier, C., Ludwig, B. & Michel, H. Nature 376, 660–669 (1995). 19. Janin, J. Nature Struct. Biol. 4, 973–974 (1997). 20. Rosenberg, M.F. et al. J. Biol. Chem. 276, 16076–16082 (2001). 21. Loo, T.W. & Clarke, D.M. J. Biol. Chem. 276, 36877–36880 (2001). 22. Qu, Q. & Sharom, F.J. Biochemistry 40, 1413–1422 (2001). nature structural biology • volume 8 number 11 • november 2001 923 history The way to NMR structures of proteins Kurt Wüthrich In 1998 Kurt Wüthrich was awarded the Kyoto Prize in Advanced Technology for having “developed a method of determining the conformations of proteins, nucleic acids and other biomacromolecules in solutions or biomembranes, where they exhibit their function”1. Wüthrich has used nuclear magnetic resonance (NMR) techniques to study proteins and nucleic acids since 1967. In a series of four papers his group outlined a framework for NMR structure determination of proteins in 1982, and in 1984 the first de novo structure of a globular protein in solution was determined. The Wüthrich group went on to solve more than 60 protein structures in solution, including the Antennapedia homeodomain, the cyclophillin A–cyclosporin A complex, and the human and bovine prion proteins. What follows is a personal recollection by Kurt Wüthrich of how he and his associates arrived at the first view of a protein structure through the NMR eye. In the 1950s, magnetic resonance spectroscopy evolved into a useful tool in chemistry. During the period of 1962–1967, my graduate and postdoctoral research, with Professor Silvio Fallab at the University of Basel and Professor Robert E. Connick at the University of California, Berkeley, focused on the use of electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) spin relaxation measurements to study metal complexes in solution. With this background, I joined the Biophysics Department of Dr. Robert G. Shulman at Bell Telephone Laboratories in Murray Hill, New Jersey, where a superconducting high resolution 1H NMR spectrometer operating at 220 MHz was available for ‘research on protein structure and function’. At that time I was aware of exactly 10 papers on NMR observations of proteins and nucleic acids, which had all been published during the period of 1957–19652. Prominent figures in the small community of spectroscopists that ventured into direct NMR observation of biological macromolecules were William D. Phillips3, Oleg Jardetzky4 and Robert G. Shulman5. Based on the observation of empirical correlations between protein unfolding and NMR spectra2–4, there was much enthusiasm about the future of NMR for de novo protein structure determination. Nonetheless, true to my background, I initially focused on the metal ion coordination in the active sites of hemoproteins and on the electronic structure of the heme groups6. At the time, Swiss scientists who landed a job at the famous Bell Telephone Laboratories were automatically considered prime candidates for academic positions back home. In 1969, I moved to the Eidgenössiche Technische Hochschule (ETH) in Zürich, where my startup package included an EPR and three NMR spectrometers — all the instrumentation that had been available to me at Bell Telephone Laboratories. I assembled a small research group, and, with time, I was promoted to Professor of Biophysics, which is also my present position at ETH. During the first years at Zürich, my research continued to focus primarily on the metal ions in the active centers of hemoproteins2–6, and I developed a mild infatuation with polypeptide chains only in connection with the discovery of aromatic ring flipping2. My primary research interest changed in 1975, when I took some time to write a monograph on the early years of biomacromolecular NMR2. These reflections on the state of the field turned out to have been the starting point for our subsequent work on de novo protein structure determination by NMR7. Four principal elements are combined in the NMR method for protein structure determination8,9: (i) the nuclear Overhauser effect (NOE) as an experimentally accessible NMR parameter in proteins that can yield the information needed for de novo global fold determination of a polymer chain; (ii) sequence-specific assignment of the many hundred to several thousand NMR peaks from a protein; (iii) computational tools for the structural interpretation of the NMR data and the evaluation of the resulting molecular structures; and (iv) multidimensional NMR techniques for efficient data collection. During the period 1976–1980, my research group at the ETH Zurich had grown to more than 20 scientists, all of whom made great contributions toward the structure determination method. In particular, I worked with Regula M. Keller, Sidney L. Gordon and Gerhard Wagner on developing techniques to measure NOEs for the collection of conformational constraints in proteins, and with Martin Billeter, Werner Braun and Gerhard Wagner on the sequential resonance assignment strategy and © 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com
e200 01NaturePublishingGrouphttp://structbio.nature.com history the University of Zurich. In June 1985 I pre- sented the structure of rabbit metallo thionein at Yale University, where I learned about a manuscript accepted for publica tion in proc. Nat. acad. Sci. USA. which described a completely different metalloth ionein NMR structure and at the University of Pittsburgh, where I was con- fronted with a rat metallothionein cryst structure that was again very different fro our nmr structure. In both instances the structural differences were very clearcut, since they involved different polypeptic Fig. 1 The first protein structure determined by NMR. a, All heavy-atom presentation of the NMr folds as well as different coordinating lig structu n) with the ands to the metals metallothionein had polypeptide segment in the X-ray crystal structure of the homologous porcine reto been a tough challenge for all of us nates involved, and my initial reaction was to obtained in refs 12, 13 spend two nights on the phone in my US motel room rechecking step by step the sequential resonance assignments with algorithms for structure calculation from protein PSTI (porcine pancreatic secretory Gerhard Wagner in Zurich. All the assign- NMr data. This technology passed its ini- trypsin inhibitor)3 it was suggested that ments were, of course, correct, and I am tial tests when we obtained partial structure our structure must have been modeled afraid that Gerhard still bears a grudge determinations of the bovine pancreatic after this crystal structure. In a discussion against me for ever having doubted his psin inhibitor(BPTD, cytochrome b following a seminar in Munich on May 14, spectral analysis. The crystal structure, and the polypeptide hormone glucagon 1984, Robert Huber (Nobel Prize in which included erroneous chain tracing based on data collection with one-dimen- Chemistry, 1988)proposed that we settle and identification of 1l out of a total of 20 sional(ID)NMR experiments. the matter by independently solving a new metal-coordinating amino acid residues Richard R. Ernst (Nobel Prize in and by NMR. For this purpose, eacs phy eventually appeared as a feature article in e Science, whereas Nature rejected our NMR 8 Chemistry, 1991), who also worked at the of us received an ample supply of the a- structure paper. In 1992, the crystal struc ETH Zurich, and I joined forces to develop amylase inhibitor tendamistat from scien- ture of rat metallothionein was redeter- two-dimensional(2D) NMR techniques tists at the Hoechst company. Virtually mined, a correction of the first structure for applications with biological macromol- identical three-dimensional structures of was published, and the correct cryst ecules Kuniaki Nagayama used 2D correla- tendamistat were obtained in our labora- structure was found to be identical with the tion spectroscopy for amino acid spin tory by Nmr in solution and in Robert NMR structures of the rabbit, rat and stem identification in a protein, and Anil Huber's laboratory by X-ray diffraction in human metallothioneins that we had mar recorded the first 2D NOE spectra single crystals. olved from 1985 to 19906 during the Christmas break 1979, when he The refined tendamistat structure was Over the years a variety of applications was allotted two weeks of the precious published in Journal of Molecular Biologyas of the MR structure determination measuring time on our highest-field spec- a 50-page report A, and the addendum to method have been pursued in my labora- trometer operating at a proton NMR fre- that paper clearly illustrated the impact of tory. The following three examples may quency of 360 MHzio. By 1981 we routinely structure determination by NMR. I quote: convey some of the excitement that was applied a group of four homonuclear 2D" Editor's Note: We have taken the step of thus generated in our professional life and H NMR experiments, known under the publishing this paper with full supporting further indicates the wide range of NMR acronyms COsY, SECSY, FOCSY and data since it is the first high resolution applications in structural biology. Studies NOESY, in the protein structure determi- structure worked out in detail by 2D NMR. on the structural foundations of transcrip- nation project. This resulted in complete We therefore think that in this one instance tional regulation in higher organisms pur- teins in 1982 and 1983 the first it does not set a precedent, since it is hoped Gehring at the Biocenter of the University de novo atomic resolution NMR structure that in the future, such supporting data can of Basel, Switzerland, yielded the nI determination of a globular protein, the be deposited in a data bank, as is the prac- structure of the Antennapedia home- bull seminal protease inhibitor(BUSD2, tice in X-ray protein crystallography". domain 7, and provided entirely novel by Timothy F Havel and Michael P. Considering that over 2,000 NMR struc- insights into the role of hydration water in Williamson in 1984 tures have since been deposited in the protein-DNa recs s mg of the human The completion of the first protein Protein Data Bank, the NMR structure brought new, unexpected commended for his vision. cyclophilin A-cyclosporin A complex was challenges. When I presented the structure At that time his kind comments were obtained in collaboration with two of my of BUSI(Fig. 1a) 2in some lectures in the comforting in the context of our structure former graduate students, Hans Senn and spring of 1984, the reaction was one of dis- determinations of mammalian metallo- Hans Widmer, who had subsequently belief, and because of the close coincidence thionein, which are a class of small, metal- joined the Sandoz company in Basel, Fig. 1b)with results from an independent rich proteins that we studied in Switzerland. This structure detern stallographic study of the homologous collaboration with Jeremias H.R. Kagi at not only introduced me to the field nature structural biology. volume 8 number 11november 2001
history algorithms for structure calculation from NMR data. This technology passed its initial tests when we obtained partial structure determinations of the bovine pancreatic trypsin inhibitor (BPTI), cytochrome b5 and the polypeptide hormone glucagon based on data collection with one-dimensional (1D) NMR experiments. In a parallel project from 1976 to 1980, Richard R. Ernst (Nobel Prize in Chemistry, 1991), who also worked at the ETH Zürich, and I joined forces to develop two-dimensional (2D) NMR techniques for applications with biological macromolecules. Kuniaki Nagayama used 2D correlation spectroscopy for amino acid spin system identification in a protein, and Anil Kumar recorded the first 2D NOE spectra during the Christmas break 1979, when he was allotted two weeks of the precious measuring time on our highest-field spectrometer operating at a proton NMR frequency of 360 MHz10. By 1981 we routinely applied a group of four homonuclear 2D 1H NMR experiments, known under the acronyms COSY, SECSY, FOCSY and NOESY9, in the protein structure determination project. This resulted in complete resonance assignments of several small proteins in 1982 and 198311, and in the first de novo atomic resolution NMR structure determination of a globular protein, the bull seminal protease inhibitor (BUSI)12, by Timothy F. Havel and Michael P. Williamson in 1984. The completion of the first protein NMR structure brought new, unexpected challenges. When I presented the structure of BUSI (Fig. 1a)12 in some lectures in the spring of 1984, the reaction was one of disbelief, and because of the close coincidence (Fig. 1b) with results from an independent crystallographic study of the homologous protein PSTI (porcine pancreatic secretory trypsin inhibitor)13 it was suggested that our structure must have been modeled after this crystal structure. In a discussion following a seminar in Munich on May 14, 1984, Robert Huber (Nobel Prize in Chemistry, 1988) proposed that we settle the matter by independently solving a new protein structure by X-ray crystallography and by NMR. For this purpose, each one of us received an ample supply of the α- amylase inhibitor tendamistat from scientists at the Hoechst company. Virtually identical three-dimensional structures of tendamistat were obtained in our laboratory by NMR in solution and in Robert Huber’s laboratory by X-ray diffraction in single crystals. The refined tendamistat structure was published in Journal of Molecular Biology as a 50-page report14, and the addendum to that paper clearly illustrated the impact of structure determination by NMR. I quote: “Editor’s Note: We have taken the step of publishing this paper with full supporting data since it is the first high resolution structure worked out in detail by 2D NMR. We therefore think that in this one instance everything should be published in full, but it does not set a precedent, since it is hoped that in the future, such supporting data can be deposited in a data bank, as is the practice in X-ray protein crystallography”. Considering that over 2,000 NMR structures have since been deposited in the Protein Data Bank, the Editor should be commended for his vision. At that time his kind comments were comforting in the context of our structure determinations of mammalian metallothioneins, which are a class of small, metalrich proteins that we studied in collaboration with Jeremias H.R. Kägi at the University of Zürich. In June 1985 I presented the structure of rabbit metallothionein at Yale University, where I learned about a manuscript accepted for publication in Proc. Nat. Acad. Sci. USA, which described a completely different metallothionein ‘NMR structure’, and at the University of Pittsburgh, where I was confronted with a rat metallothionein crystal structure that was again very different from our NMR structure. In both instances the structural differences were very clearcut, since they involved different polypeptide folds as well as different coordinating ligands to the metals. Metallothionein had been a tough challenge for all of us involved15, and my initial reaction was to spend two nights on the phone in my US motel room rechecking step by step the sequential resonance assignments with Gerhard Wagner in Zürich. All the assignments were, of course, correct, and I am afraid that Gerhard still bears a grudge against me for ever having doubted his spectral analysis. The crystal structure, which included erroneous chain tracing and identification of 11 out of a total of 20 metal-coordinating amino acid residues, eventually appeared as a feature article in Science, whereas Nature rejected our NMR structure paper. In 1992, the crystal structure of rat metallothionein was redetermined, a correction of the first structure was published, and the correct crystal structure was found to be identical with the NMR structures of the rabbit, rat and human metallothioneins that we had solved from 1985 to 199016. Over the years a variety of applications of the NMR structure determination method have been pursued in my laboratory. The following three examples may convey some of the excitement that was thus generated in our professional life and further indicates the wide range of NMR applications in structural biology. Studies on the structural foundations of transcriptional regulation in higher organisms pursued in collaboration with Walter J. Gehring at the Biocenter of the University of Basel, Switzerland, yielded the NMR structure of the Antennapedia homeodomain17, and provided entirely novel insights into the role of hydration water in protein–DNA recognition18. An NMR structure determination of the human cyclophilin A–cyclosporin A complex was obtained in collaboration with two of my former graduate students, Hans Senn and Hans Widmer, who had subsequently joined the Sandoz company in Basel, Switzerland. This structure determination not only introduced me to the field of 924 nature structural biology • volume 8 number 11 • november 2001 Fig. 1 The first protein structure determined by NMR. a, All heavy-atom presentation of the NMR structure of the proteinase inhibitor IIA from bull seminal plasma (BUSI IIA)12. b, Superposition of the core region of residues 23–42 in the NMR structure of BUSI IIA (green) with the corresponding polypeptide segment in the X-ray crystal structure of the homologous porcine pancreatic secretory trypsin inhibitor (PSTI) (blue)13. The drawings were prepared from the atomic coordinates obtained in refs 12,13. a b © 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com
e200 01NaturePublishingGrouphttp://structbio.nature.com histo immune suppression but also had an i scrapie form. With the introduc- 4. Jardetzky, o &Roberts, GCKNMR in Molecular immediate practical impact on cycle TROSY(transverse relaxation-opti- s shulman. . e a. senice 165, 251-257(1969 research, since the structure of the spectroscopy)22, the molecular 6. Wuthrich, K. Structure and Bonding 8,53-121 ug molecule was found to be weight limit for solution NMR spec- 7. wuthrich, K. NMR in Structural Biology-A inside-out when compared with the struc- troscopy has extended to-500 k Da, and we ollection of Papers by Kurt Wuthrich (World ture of free cyclosporin A9. Barely 10 days may soon be able to obtain information on 8wuthrich, K,Wider,G, Wagner,G&Br after the bovine spongiform encephalopa- the structure of the disease-related, aggre- wuthrich, kNmr of Proteins and Nucleic Acids ly(BSe) crisis in Great Britain had broken gated form of the prion protein nrich. K. Biochem. the Nmr structure determination of the Kurt Wuthrich is Professor of Biophysics at 11. Wagner. G.& wuthrich, murine prion protein20 in a collaboration the Institute of Molecular Biology and ith Rudi Glockshuber, who had joined Biophysics, ETH Zurich, CH-8093 Zurich,Biol. 182, 295-315 avel, .E&wuthrich,K1Mol our institute at the ETH Zurich as an Switzerland, Fax: 41 1-633-1151, and Cecil 14. Kling. M et at 1 mol Biol 162. 839-86 Assistant Professor in 1994. The observa- H. and lda M. green visiting Professor of s 204 675 4 19%l Biol 187.125-12901986 llustration of the unique power of NMR to Jolla, CA 92037, USA, Far: 1858-784-8014. 17. ian r 1z28(p Prac. Nati tinsel presents on the one hand a striking Institute, 10550 North Torrey Pines Road, La 10124- characterize partially structured polypep- ae5 tide chains in physiological milieus, and on and Inamori Grants 1998, 13(The 19. wuthrich, K the other hand indicates novel possible 20.Riek,R.eta. Nature382.180-182(1996 avenues for the transition of the benign cel lular form of prion proteins to the disease- C.& Phillips, W. D.J. Am. Chem. Soc. 22. Pervushin, K, Riel roc. Nat. Acad. Sci. USA 94. 12366-12371(1997) picture story A force to be reckoned with 8 Bacteriophage DNA is packaged into addition of ATP, and the beads move allowing them to quantitatively estimate protein capsids to near crystalline densi- closer together. The experiment can be the internal force produced by the dNA It was originally thought that the dna done in a constant force feedback as it is condensed and packaged (right) was condensed first and the protein shell mode keeping a predetermined tension Interestingly, the internal force is quite was built around it, until about 30 years in the dna by moving the bead posi- small until% of the genome is pack go when empty phage capsids, or pro- tion, or the ' no feedback mode, where aged, indicating that the dNA is initially heads, were found to form first. This dis- the force is allowed to change but the packed fairly loosely, not in its con- ery presented the difficult question: beads are held in place. densed final state. The force then how does a virus force its DNA into the The authors show that packaging is increases, reaching-50 pN as the entire tiny capsid? For the well-studied Bacillus highly processive and efficient, with few genome is packaged and making the subtilis bacteriophage 29, -19 kilobases pauses and slips. Despite this efficiency, packaging machinery one of the of double stranded DNA (6.6 um long) the rate of packaging decreases as more strongest molecular motors reported As must fit into a prohead of 42 x 54 nm. DNA is packed into the head (middle), the authors point out, building up so The portal complex, the ATP-dependent suggesting that pressure builds up inside much internal force may be useful for protein and RNA motor responsible for the capsid. Using the ' no feedback the phage during infection; the pressure his feat, must overcome substantial mode the authors measured the may be used to partially inject the dna energetic barriers to package the dna so decreasing rate of packaging as the ten- into the host cell htly, but exactly how this is accom- sion between the tethered ends built up, Julie hollien hed is not clear As reported in a recent issue of Nature 413, 748-752: 2001), Bustamante and optical trap colleagues use optical tweezers to mea- sure the rates and forces involved in strap avin packaging 29 DNA into individual phage heads. The unpackaged end of the DNA is attached to a polystyrene bead, comple which is held in an optical trap(left).At antbody the other end of the DNA, the partly packaged phage head is attached to ano ld in place with a pipette. Packaging resumes upon the tAgYMiwNo of genome packag nature structural biology volume 8 number 11. november 2001
history picture story immune suppression but also had an immediate practical impact on cyclosporin research, since the structure of the bound drug molecule was found to be turned inside-out when compared with the structure of free cyclosporin A19. Barely 10 days after the bovine spongiform encephalopathy (BSE) crisis in Great Britain had broken into the open in March 1996, we completed the NMR structure determination of the murine prion protein20 in a collaboration with Rudi Glockshuber, who had joined our institute at the ETH Zürich as an Assistant Professor in 1994. The observation of a long flexible tail in prion proteins21 presents on the one hand a striking illustration of the unique power of NMR to characterize partially structured polypeptide chains in physiological milieus, and on the other hand indicates novel possible avenues for the transition of the benign cellular form of prion proteins to the diseaserelated scrapie form. With the introduction of TROSY (transverse relaxation-optimized spectroscopy)22, the molecular weight limit for solution NMR spectroscopy has extended to ∼500 kDa, and we may soon be able to obtain information on the structure of the disease-related, aggregated form of the prion protein. Kurt Wüthrich is Professor of Biophysics at the Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland, Fax: 41 1-633-1151, and Cecil H. and Ida M. Green Visiting Professor of Structural Biology at The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA, Fax: 1 858-784-8014. 1. Kyoto Prizes and Inamori Grants 1998, 13 (The Inamori Foundation, Kyoto; 1999). 2. Wüthrich, K. NMR in Biological Research: Peptides and Proteins (North Holland, Amsterdam; 1976). 3. McDonald, C.C. & Phillips, W.D. J. Am. Chem. Soc. 89, 6332–6341 (1967). 4. Jardetzky, O. & Roberts, G.C.K. NMR in Molecular Biology (Academic Press, New York; 1981). 5. Shulman. R.G. et al. Science 165, 251–257 (1969). 6. Wüthrich, K. Structure and Bonding 8, 53–121 (1970). 7. Wüthrich, K. NMR in Structural Biology — A Collection of Papers by Kurt Wüthrich (World Scientific, Singapore; 1995). 8. Wüthrich, K., Wider, G., Wagner, G. & Braun, W. J. Mol. Biol. 155, 311–319 (1982). 9. Wüthrich, K. NMR of Proteins and Nucleic Acids (Wiley, New York; 1986). 10. Anil-Kumar, Ernst, R.R. & Wüthrich, K. Biochem. Biophys. Res. Comm. 95, 1–6 (1980). 11. Wagner, G. & Wüthrich, K. J. Mol. Biol. 155, 347–366 (1982). 12. Williamson, M.P., Havel, T.F. & Wüthrich, K. J. Mol. Biol. 182, 295–315 (1985). 13. Bolognesi, M. et al. J. Mol. Biol. 162, 839–868 (1992). 14. Kline, A.D., Braun, W. & Wüthrich, K. J. Mol. Biol. 204, 675–724 (1988). 15. Braun, W. et al. J. Mol. Biol. 187, 125–129 (1986). 16. Braun, W. et al. Proc. Natl. Acad. Sci. USA 89, 10124–10128 (1992). 17. Qian, Y.Q. et al. Cell 59, 573–580 (1989). 18. Billeter, M., Güntert, P., Luginbühl, P. & Wüthrich, K. Cell 85, 1057–1065 (1996). 19. Wüthrich, K. et al. Science 254, 953–954 (1991). 20. Riek, R. et al. Nature 382, 180–182 (1996). 21. Riek, R., Hornemann, S., Wider, G., Glockshuber R. & Wüthrich, K. FEBS Lett. 413, 277–281 (1997). 22. Pervushin, K., Riek, R., Wider, G. & Wüthrich, K. Proc. Natl. Acad. Sci. USA 94, 12366–12371 (1997). A force to be reckoned with Bacteriophage DNA is packaged into protein capsids to near crystalline density. It was originally thought that the DNA was condensed first and the protein shell was built around it, until about 30 years ago when empty phage capsids, or proheads, were found to form first. This discovery presented the difficult question: how does a virus force its DNA into the tiny capsid? For the well-studied Bacillus subtilis bacteriophage φ29, ∼19 kilobases of double stranded DNA (6.6 µm long) must fit into a prohead of 42 x 54 nm. The portal complex, the ATP-dependent protein and RNA motor responsible for this feat, must overcome substantial energetic barriers to package the DNA so tightly, but exactly how this is accomplished is not clear. As reported in a recent issue of Nature (413, 748–752; 2001), Bustamante and colleagues use optical tweezers to measure the rates and forces involved in packaging φ29 DNA into individual phage heads. The unpackaged end of the DNA is attached to a polystyrene bead, which is held in an optical trap (left). At the other end of the DNA, the partly packaged phage head is attached to another bead and held in place with a pipette. Packaging resumes upon the allowing them to quantitatively estimate the internal force produced by the DNA as it is condensed and packaged (right). Interestingly, the internal force is quite small until ~50% of the genome is packaged, indicating that the DNA is initially packed fairly loosely, not in its condensed final state. The force then increases, reaching ~50 pN as the entire genome is packaged and making the packaging machinery one of the strongest molecular motors reported. As the authors point out, building up so much internal force may be useful for the phage during infection; the pressure may be used to partially inject the DNA into the host cell. Julie Hollien nature structural biology • volume 8 number 11 • november 2001 925 addition of ATP, and the beads move closer together. The experiment can be done in a ‘constant force feedback’ mode, keeping a predetermined tension in the DNA by moving the bead position, or the ‘no feedback’ mode, where the force is allowed to change but the beads are held in place. The authors show that packaging is highly processive and efficient, with few pauses and slips. Despite this efficiency, the rate of packaging decreases as more DNA is packed into the head (middle), suggesting that pressure builds up inside the capsid. Using the ‘no feedback’ mode, the authors measured the decreasing rate of packaging as the tension between the tethered ends built up, © 2001 Nature Publishing Group http://structbio.nature.com © 2001 Nature Publishing Group http://structbio.nature.com