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. 2001 Aug;81(2):1029–1036. doi: 10.1016/S0006-3495(01)75760-8

Assembly of a polytopic membrane protein structure from the solution structures of overlapping peptide fragments of bacteriorhodopsin.

M Katragadda 1, J L Alderfer 1, P L Yeagle 1
PMCID: PMC1301572  PMID: 11463644

Abstract

Three-dimensional structures of only a handful of membrane proteins have been solved, in contrast to the thousands of structures of water-soluble proteins. Difficulties in crystallization have inhibited the determination of the three-dimensional structure of membrane proteins by x-ray crystallography and have spotlighted the critical need for alternative approaches to membrane protein structure. A new approach to the three-dimensional structure of membrane proteins has been developed and tested on the integral membrane protein, bacteriorhodopsin, the crystal structure of which had previously been determined. An overlapping series of 13 peptides, spanning the entire sequence of bacteriorhodopsin, was synthesized, and the structures of these peptides were determined by NMR in dimethylsulfoxide solution. These structures were assembled into a three-dimensional construct by superimposing the overlapping sequences at the ends of each peptide. Onto this construct were written all the distance and angle constraints obtained from the individual solution structures along with a limited number of experimental inter-helical distance constraints, and the construct was subjected to simulated annealing. A three-dimensional structure, determined exclusively by the experimental constraints, emerged that was similar to the crystal structure of this protein. This result suggests an alternative approach to the acquisition of structural information for membrane proteins consisting of helical bundles.

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Selected References

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  1. Adler M., Seto M. H., Nitecki D. E., Lin J. H., Light D. R., Morser J. The structure of a 19-residue fragment from the C-loop of the fourth epidermal growth factor-like domain of thrombomodulin. J Biol Chem. 1995 Oct 6;270(40):23366–23372. doi: 10.1074/jbc.270.40.23366. [DOI] [PubMed] [Google Scholar]
  2. Altenbach C., Cai K., Khorana H. G., Hubbell W. L. Structural features and light-dependent changes in the sequence 306-322 extending from helix VII to the palmitoylation sites in rhodopsin: a site-directed spin-labeling study. Biochemistry. 1999 Jun 22;38(25):7931–7937. doi: 10.1021/bi9900121. [DOI] [PubMed] [Google Scholar]
  3. Barsukov I. L., Nolde D. E., Lomize A. L., Arseniev A. S. Three-dimensional structure of proteolytic fragment 163-231 of bacterioopsin determined from nuclear magnetic resonance data in solution. Eur J Biochem. 1992 Jun 15;206(3):665–672. doi: 10.1111/j.1432-1033.1992.tb16972.x. [DOI] [PubMed] [Google Scholar]
  4. Behrends H. W., Folkers G., Beck-Sickinger A. G. A new approach to secondary structure evaluation: secondary structure prediction of porcine adenylate kinase and yeast guanylate kinase by CD spectroscopy of overlapping synthetic peptide segments. Biopolymers. 1997 Feb;41(2):213–231. doi: 10.1002/(SICI)1097-0282(199702)41:2<213::AID-BIP8>3.0.CO;2-W. [DOI] [PubMed] [Google Scholar]
  5. Blanco F. J., Serrano L. Folding of protein G B1 domain studied by the conformational characterization of fragments comprising its secondary structure elements. Eur J Biochem. 1995 Jun 1;230(2):634–649. doi: 10.1111/j.1432-1033.1995.tb20605.x. [DOI] [PubMed] [Google Scholar]
  6. Blumenstein M., Matsueda G. R., Timmons S., Hawiger J. A beta-turn is present in the 392-411 segment of the human fibrinogen gamma-chain. Effects of structural changes in this segment on affinity to antibody 4A5. Biochemistry. 1992 Nov 10;31(44):10692–10698. doi: 10.1021/bi00159a008. [DOI] [PubMed] [Google Scholar]
  7. Callihan D. E., Logan T. M. Conformations of peptide fragments from the FK506 binding protein: comparison with the native and urea-unfolded states. J Mol Biol. 1999 Feb 5;285(5):2161–2175. doi: 10.1006/jmbi.1998.2440. [DOI] [PubMed] [Google Scholar]
  8. Campbell A. P., McInnes C., Hodges R. S., Sykes B. D. Comparison of NMR solution structures of the receptor binding domains of Pseudomonas aeruginosa pili strains PAO, KB7, and PAK: implications for receptor binding and synthetic vaccine design. Biochemistry. 1995 Dec 19;34(50):16255–16268. doi: 10.1021/bi00050a005. [DOI] [PubMed] [Google Scholar]
  9. Chandrasekhar K., Profy A. T., Dyson H. J. Solution conformational preferences of immunogenic peptides derived from the principal neutralizing determinant of the HIV-1 envelope glycoprotein gp120. Biochemistry. 1991 Sep 24;30(38):9187–9194. doi: 10.1021/bi00102a009. [DOI] [PubMed] [Google Scholar]
  10. Chopra A., Yeagle P. L., Alderfer J. A., Albert A. D. Solution structure of the sixth transmembrane helix of the G-protein-coupled receptor, rhodopsin. Biochim Biophys Acta. 2000 Jan 15;1463(1):1–5. doi: 10.1016/s0005-2736(99)00212-6. [DOI] [PubMed] [Google Scholar]
  11. Cox J. P., Evans P. A., Packman L. C., Williams D. H., Woolfson D. N. Dissecting the structure of a partially folded protein. Circular dichroism and nuclear magnetic resonance studies of peptides from ubiquitin. J Mol Biol. 1993 Nov 20;234(2):483–492. doi: 10.1006/jmbi.1993.1600. [DOI] [PubMed] [Google Scholar]
  12. Dmitriev O., Jones P. C., Jiang W., Fillingame R. H. Structure of the membrane domain of subunit b of the Escherichia coli F0F1 ATP synthase. J Biol Chem. 1999 May 28;274(22):15598–15604. doi: 10.1074/jbc.274.22.15598. [DOI] [PubMed] [Google Scholar]
  13. Dyson H. J., Merutka G., Waltho J. P., Lerner R. A., Wright P. E. Folding of peptide fragments comprising the complete sequence of proteins. Models for initiation of protein folding. I. Myohemerythrin. J Mol Biol. 1992 Aug 5;226(3):795–817. doi: 10.1016/0022-2836(92)90633-u. [DOI] [PubMed] [Google Scholar]
  14. Fan J. S., Cheng H. C., Zhang M. A peptide corresponding to residues Asp177 to Asn208 of human cyclin A forms an alpha-helix. Biochem Biophys Res Commun. 1998 Dec 30;253(3):621–627. doi: 10.1006/bbrc.1998.9828. [DOI] [PubMed] [Google Scholar]
  15. Gao J., Li Y., Yan H. NMR solution structure of domain 1 of human annexin I shows an autonomous folding unit. J Biol Chem. 1999 Jan 29;274(5):2971–2977. doi: 10.1074/jbc.274.5.2971. [DOI] [PubMed] [Google Scholar]
  16. Gegg C. V., Bowers K. E., Matthews C. R. Probing minimal independent folding units in dihydrofolate reductase by molecular dissection. Protein Sci. 1997 Sep;6(9):1885–1892. doi: 10.1002/pro.5560060909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ghiara J. B., Stura E. A., Stanfield R. L., Profy A. T., Wilson I. A. Crystal structure of the principal neutralization site of HIV-1. Science. 1994 Apr 1;264(5155):82–85. doi: 10.1126/science.7511253. [DOI] [PubMed] [Google Scholar]
  18. Gouaux E. It's not just a phase: crystallization and X-ray structure determination of bacteriorhodopsin in lipidic cubic phases. Structure. 1998 Jan 15;6(1):5–10. doi: 10.1016/s0969-2126(98)00002-1. [DOI] [PubMed] [Google Scholar]
  19. Goudreau N., Cornille F., Duchesne M., Parker F., Tocqué B., Garbay C., Roques B. P. NMR structure of the N-terminal SH3 domain of GRB2 and its complex with a proline-rich peptide from Sos. Nat Struct Biol. 1994 Dec;1(12):898–907. doi: 10.1038/nsb1294-898. [DOI] [PubMed] [Google Scholar]
  20. Grigorieff N., Ceska T. A., Downing K. H., Baldwin J. M., Henderson R. Electron-crystallographic refinement of the structure of bacteriorhodopsin. J Mol Biol. 1996 Jun 14;259(3):393–421. doi: 10.1006/jmbi.1996.0328. [DOI] [PubMed] [Google Scholar]
  21. Hamada D., Kuroda Y., Tanaka T., Goto Y. High helical propensity of the peptide fragments derived from beta-lactoglobulin, a predominantly beta-sheet protein. J Mol Biol. 1995 Dec 8;254(4):737–746. doi: 10.1006/jmbi.1995.0651. [DOI] [PubMed] [Google Scholar]
  22. Henderson R., Baldwin J. M., Ceska T. A., Zemlin F., Beckmann E., Downing K. H. Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol. 1990 Jun 20;213(4):899–929. doi: 10.1016/S0022-2836(05)80271-2. [DOI] [PubMed] [Google Scholar]
  23. Hunt J. F., Earnest T. N., Bousché O., Kalghatgi K., Reilly K., Horváth C., Rothschild K. J., Engelman D. M. A biophysical study of integral membrane protein folding. Biochemistry. 1997 Dec 9;36(49):15156–15176. doi: 10.1021/bi970146j. [DOI] [PubMed] [Google Scholar]
  24. Jimenez M. A., Evangelio J. A., Aranda C., Lopez-Brauet A., Andreu D., Rico M., Lagos R., Andreu J. M., Monasterio O. Helicity of alpha(404-451) and beta(394-445) tubulin C-terminal recombinant peptides. Protein Sci. 1999 Apr;8(4):788–799. doi: 10.1110/ps.8.4.788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Katragadda M., Alderfer J. L., Yeagle P. L. Solution structure of the loops of bacteriorhodopsin closely resembles the crystal structure. Biochim Biophys Acta. 2000 Jun 1;1466(1-2):1–6. doi: 10.1016/s0005-2736(00)00167-x. [DOI] [PubMed] [Google Scholar]
  26. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  27. Lomize A. L., Pervushin K. V., Arseniev A. S. Spatial structure of (34-65)bacterioopsin polypeptide in SDS micelles determined from nuclear magnetic resonance data. J Biomol NMR. 1992 Jul;2(4):361–372. doi: 10.1007/BF01874814. [DOI] [PubMed] [Google Scholar]
  28. Ludvigsen S., Andersen K. V., Poulsen F. M. Accurate measurements of coupling constants from two-dimensional nuclear magnetic resonance spectra of proteins and determination of phi-angles. J Mol Biol. 1991 Feb 20;217(4):731–736. doi: 10.1016/0022-2836(91)90529-f. [DOI] [PubMed] [Google Scholar]
  29. Luecke H., Schobert B., Richter H. T., Cartailler J. P., Lanyi J. K. Structure of bacteriorhodopsin at 1.55 A resolution. J Mol Biol. 1999 Aug 27;291(4):899–911. doi: 10.1006/jmbi.1999.3027. [DOI] [PubMed] [Google Scholar]
  30. Padmanabhan S., Jiménez M. A., Rico M. Folding propensities of synthetic peptide fragments covering the entire sequence of phage 434 Cro protein. Protein Sci. 1999 Aug;8(8):1675–1688. doi: 10.1110/ps.8.8.1675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Palczewski K., Kumasaka T., Hori T., Behnke C. A., Motoshima H., Fox B. A., Le Trong I., Teller D. C., Okada T., Stenkamp R. E. Crystal structure of rhodopsin: A G protein-coupled receptor. Science. 2000 Aug 4;289(5480):739–745. doi: 10.1126/science.289.5480.739. [DOI] [PubMed] [Google Scholar]
  32. Pebay-Peyroula E., Rummel G., Rosenbusch J. P., Landau E. M. X-ray structure of bacteriorhodopsin at 2.5 angstroms from microcrystals grown in lipidic cubic phases. Science. 1997 Sep 12;277(5332):1676–1681. doi: 10.1126/science.277.5332.1676. [DOI] [PubMed] [Google Scholar]
  33. Pervushin K. V., Orekhov VYu, Popov A. I., Musina LYu, Arseniev A. S. Three-dimensional structure of (1-71)bacterioopsin solubilized in methanol/chloroform and SDS micelles determined by 15N-1H heteronuclear NMR spectroscopy. Eur J Biochem. 1994 Jan 15;219(1-2):571–583. doi: 10.1111/j.1432-1033.1994.tb19973.x. [DOI] [PubMed] [Google Scholar]
  34. Popot J. L., Engelman D. M. Helical membrane protein folding, stability, and evolution. Annu Rev Biochem. 2000;69:881–922. doi: 10.1146/annurev.biochem.69.1.881. [DOI] [PubMed] [Google Scholar]
  35. Ramírez-Alvarado M., Serrano L., Blanco F. J. Conformational analysis of peptides corresponding to all the secondary structure elements of protein L B1 domain: secondary structure propensities are not conserved in proteins with the same fold. Protein Sci. 1997 Jan;6(1):162–174. doi: 10.1002/pro.5560060119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Reymond M. T., Merutka G., Dyson H. J., Wright P. E. Folding propensities of peptide fragments of myoglobin. Protein Sci. 1997 Mar;6(3):706–716. doi: 10.1002/pro.5560060320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Wilce J. A., Salvatore D., Wade J. D., Craik D. J. 1H-NMR structural studies of a cystine-linked peptide containing residues 71-93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy. Eur J Biochem. 1999 Jun;262(2):586–594. doi: 10.1046/j.1432-1327.1999.00423.x. [DOI] [PubMed] [Google Scholar]
  38. Wüthrich K., Billeter M., Braun W. Pseudo-structures for the 20 common amino acids for use in studies of protein conformations by measurements of intramolecular proton-proton distance constraints with nuclear magnetic resonance. J Mol Biol. 1983 Oct 5;169(4):949–961. doi: 10.1016/s0022-2836(83)80144-2. [DOI] [PubMed] [Google Scholar]
  39. Yang A. S., Hitz B., Honig B. Free energy determinants of secondary structure formation: III. beta-turns and their role in protein folding. J Mol Biol. 1996 Jun 21;259(4):873–882. doi: 10.1006/jmbi.1996.0364. [DOI] [PubMed] [Google Scholar]
  40. Yang K., Farrens D. L., Hubbell W. L., Khorana H. G. Structure and function in rhodopsin. Single cysteine substitution mutants in the cytoplasmic interhelical E-F loop region show position-specific effects in transducin activation. Biochemistry. 1996 Sep 24;35(38):12464–12469. doi: 10.1021/bi960848t. [DOI] [PubMed] [Google Scholar]
  41. Yeagle P. L., Alderfer J. L., Albert A. D. Structure of the carboxy-terminal domain of bovine rhodopsin. Nat Struct Biol. 1995 Oct;2(10):832–834. doi: 10.1038/nsb1095-832. [DOI] [PubMed] [Google Scholar]
  42. Yeagle P. L., Alderfer J. L., Salloum A. C., Ali L., Albert A. D. The first and second cytoplasmic loops of the G-protein receptor, rhodopsin, independently form beta-turns. Biochemistry. 1997 Apr 1;36(13):3864–3869. doi: 10.1021/bi962403a. [DOI] [PubMed] [Google Scholar]

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