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. 1998 Feb;7(2):358–368. doi: 10.1002/pro.5560070216

A folding pathway for betapep-4 peptide 33mer: from unfolded monomers and beta-sheet sandwich dimers to well-structured tetramers.

K H Mayo 1, E Ilyina 1
PMCID: PMC2143938  PMID: 9521112

Abstract

It was recently reported that a de novo designed peptide 33mer, betapep-4, can form well-structured beta-sheet sandwich tetramers (Ilyina E, Roongta V, Mayo KH, 1997b, Biochemistry 36:5245-5250). For insight into the pathway of betapep-4 folding, the present study investigates the concentration dependence of betapep-4 self-association by using 1H-NMR pulsed-field gradient (PFG)-NMR diffusion measurements, and circular dichroism. Downfield chemically shifted alphaH resonances, found to arise only from the well-structured betapep-4 tetramer state, yield the fraction of tetramer within the oligomer equilibrium distribution. PFG-NMR-derived diffusion coefficients, D, provide a means for deriving the contribution of monomer and other oligomer states to this distribution. These data indicate that tetramer is the highest oligomer state formed, and that inclusion of monomer and dimer states in the oligomer distribution is sufficient to explain the concentration dependence of D values for betapep-4. Equilibrium constants calculated from these distributions [2.5 x 10(5) M(-1) for M-D and 1.2 x 10(4) M(-1) for D-T at 313 K] decrease only slightly, if at all, with decreasing temperature indicating a hydrophobically mediated, entropy-driven association/folding process. Conformational analyses using NMR and CD provide a picture where "random coil" monomers associate to form molten globule-like beta-sheet sandwich dimers that further associate and fold as well-structured tetramers. Betapep-4 folding is thermodynamically linked to self-association. As with folding of single-chain polypeptides, the final folding step to well-structured tetramer betapep-4 is rate limiting.

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Selected References

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  1. Beasty A. M., Hurle M. R., Manz J. T., Stackhouse T., Onuffer J. J., Matthews C. R. Effects of the phenylalanine-22----leucine, glutamic acid-49----methionine, glycine-234----aspartic acid, and glycine-234----lysine mutations on the folding and stability of the alpha subunit of tryptophan synthase from Escherichia coli. Biochemistry. 1986 May 20;25(10):2965–2974. doi: 10.1021/bi00358a035. [DOI] [PubMed] [Google Scholar]
  2. Behe M. J., Lattman E. E., Rose G. D. The protein-folding problem: the native fold determines packing, but does packing determine the native fold? Proc Natl Acad Sci U S A. 1991 May 15;88(10):4195–4199. doi: 10.1073/pnas.88.10.4195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bierzynski A., Kim P. S., Baldwin R. L. A salt bridge stabilizes the helix formed by isolated C-peptide of RNase A. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2470–2474. doi: 10.1073/pnas.79.8.2470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Briggs M. S., Roder H. Early hydrogen-bonding events in the folding reaction of ubiquitin. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2017–2021. doi: 10.1073/pnas.89.6.2017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bryson J. W., Betz S. F., Lu H. S., Suich D. J., Zhou H. X., O'Neil K. T., DeGrado W. F. Protein design: a hierarchic approach. Science. 1995 Nov 10;270(5238):935–941. doi: 10.1126/science.270.5238.935. [DOI] [PubMed] [Google Scholar]
  6. Greenfield N., Fasman G. D. Computed circular dichroism spectra for the evaluation of protein conformation. Biochemistry. 1969 Oct;8(10):4108–4116. doi: 10.1021/bi00838a031. [DOI] [PubMed] [Google Scholar]
  7. Harbury P. B., Zhang T., Kim P. S., Alber T. A switch between two-, three-, and four-stranded coiled coils in GCN4 leucine zipper mutants. Science. 1993 Nov 26;262(5138):1401–1407. doi: 10.1126/science.8248779. [DOI] [PubMed] [Google Scholar]
  8. Jackson S. E., Fersht A. R. Folding of chymotrypsin inhibitor 2. 2. Influence of proline isomerization on the folding kinetics and thermodynamic characterization of the transition state of folding. Biochemistry. 1991 Oct 29;30(43):10436–10443. doi: 10.1021/bi00107a011. [DOI] [PubMed] [Google Scholar]
  9. Johnson W. C., Jr Protein secondary structure and circular dichroism: a practical guide. Proteins. 1990;7(3):205–214. doi: 10.1002/prot.340070302. [DOI] [PubMed] [Google Scholar]
  10. Kaumaya P. T., Berndt K. D., Heidorn D. B., Trewhella J., Kezdy F. J., Goldberg E. Synthesis and biophysical characterization of engineered topographic immunogenic determinants with alpha alpha topology. Biochemistry. 1990 Jan 9;29(1):13–23. doi: 10.1021/bi00453a002. [DOI] [PubMed] [Google Scholar]
  11. Lumb K. J., Carr C. M., Kim P. S. Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator GCN4. Biochemistry. 1994 Jun 14;33(23):7361–7367. doi: 10.1021/bi00189a042. [DOI] [PubMed] [Google Scholar]
  12. Marion D., Wüthrich K. Application of phase sensitive two-dimensional correlated spectroscopy (COSY) for measurements of 1H-1H spin-spin coupling constants in proteins. Biochem Biophys Res Commun. 1983 Jun 29;113(3):967–974. doi: 10.1016/0006-291x(83)91093-8. [DOI] [PubMed] [Google Scholar]
  13. Matouschek A., Kellis J. T., Jr, Serrano L., Fersht A. R. Mapping the transition state and pathway of protein folding by protein engineering. Nature. 1989 Jul 13;340(6229):122–126. doi: 10.1038/340122a0. [DOI] [PubMed] [Google Scholar]
  14. Mayo K. H., Ilyina E., Park H. A recipe for designing water-soluble, beta-sheet-forming peptides. Protein Sci. 1996 Jul;5(7):1301–1315. doi: 10.1002/pro.5560050709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Munson M., O'Brien R., Sturtevant J. M., Regan L. Redesigning the hydrophobic core of a four-helix-bundle protein. Protein Sci. 1994 Nov;3(11):2015–2022. doi: 10.1002/pro.5560031114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Nölting B., Golbik R., Neira J. L., Soler-Gonzalez A. S., Schreiber G., Fersht A. R. The folding pathway of a protein at high resolution from microseconds to seconds. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):826–830. doi: 10.1073/pnas.94.3.826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Quinn T. P., Tweedy N. B., Williams R. W., Richardson J. S., Richardson D. C. Betadoublet: de novo design, synthesis, and characterization of a beta-sandwich protein. Proc Natl Acad Sci U S A. 1994 Sep 13;91(19):8747–8751. doi: 10.1073/pnas.91.19.8747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Radford S. E., Dobson C. M., Evans P. A. The folding of hen lysozyme involves partially structured intermediates and multiple pathways. Nature. 1992 Jul 23;358(6384):302–307. doi: 10.1038/358302a0. [DOI] [PubMed] [Google Scholar]
  19. Rajarathnam K., Clark-Lewis I., Sykes B. D. 1H NMR solution structure of an active monomeric interleukin-8. Biochemistry. 1995 Oct 10;34(40):12983–12990. doi: 10.1021/bi00040a008. [DOI] [PubMed] [Google Scholar]
  20. Rajarathnam K., Sykes B. D., Kay C. M., Dewald B., Geiser T., Baggiolini M., Clark-Lewis I. Neutrophil activation by monomeric interleukin-8. Science. 1994 Apr 1;264(5155):90–92. doi: 10.1126/science.8140420. [DOI] [PubMed] [Google Scholar]
  21. Richardson J. S., Richardson D. C. The de novo design of protein structures. Trends Biochem Sci. 1989 Jul;14(7):304–309. doi: 10.1016/0968-0004(89)90070-4. [DOI] [PubMed] [Google Scholar]
  22. Richardson J. S., Richardson D. C., Tweedy N. B., Gernert K. M., Quinn T. P., Hecht M. H., Erickson B. W., Yan Y., McClain R. D., Donlan M. E. Looking at proteins: representations, folding, packing, and design. Biophysical Society National Lecture, 1992. Biophys J. 1992 Nov;63(5):1185–1209. [PMC free article] [PubMed] [Google Scholar]
  23. Shoemaker K. R., Kim P. S., Brems D. N., Marqusee S., York E. J., Chaiken I. M., Stewart J. M., Baldwin R. L. Nature of the charged-group effect on the stability of the C-peptide helix. Proc Natl Acad Sci U S A. 1985 Apr;82(8):2349–2353. doi: 10.1073/pnas.82.8.2349. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Squire P. G., Himmel M. E. Hydrodynamics and protein hydration. Arch Biochem Biophys. 1979 Aug;196(1):165–177. doi: 10.1016/0003-9861(79)90563-0. [DOI] [PubMed] [Google Scholar]
  25. Tanimura R., Kidera A., Nakamura H. Determinants of protein side-chain packing. Protein Sci. 1994 Dec;3(12):2358–2365. doi: 10.1002/pro.5560031220. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Taniyama Y., Ogasahara K., Yutani K., Kikuchi M. Folding mechanism of mutant human lysozyme C77/95A with increased secretion efficiency in yeast. J Biol Chem. 1992 Mar 5;267(7):4619–4624. [PubMed] [Google Scholar]
  27. Teller D. C., Swanson E., de Haën C. The translational friction coefficient of proteins. Methods Enzymol. 1979;61:103–124. doi: 10.1016/0076-6879(79)61010-8. [DOI] [PubMed] [Google Scholar]
  28. Waterhous D. V., Johnson W. C., Jr Importance of environment in determining secondary structure in proteins. Biochemistry. 1994 Mar 1;33(8):2121–2128. doi: 10.1021/bi00174a019. [DOI] [PubMed] [Google Scholar]
  29. Wishart D. S., Sykes B. D., Richards F. M. The chemical shift index: a fast and simple method for the assignment of protein secondary structure through NMR spectroscopy. Biochemistry. 1992 Feb 18;31(6):1647–1651. doi: 10.1021/bi00121a010. [DOI] [PubMed] [Google Scholar]
  30. Yan Y., Erickson B. W. Engineering of betabellin 14D: disulfide-induced folding of a beta-sheet protein. Protein Sci. 1994 Jul;3(7):1069–1073. doi: 10.1002/pro.5560030709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Zitzewitz J. A., Bilsel O., Luo J., Jones B. E., Matthews C. R. Probing the folding mechanism of a leucine zipper peptide by stopped-flow circular dichroism spectroscopy. Biochemistry. 1995 Oct 3;34(39):12812–12819. doi: 10.1021/bi00039a042. [DOI] [PubMed] [Google Scholar]
  32. de Alba E., Jiménez M. A., Rico M., Nieto J. L. Conformational investigation of designed short linear peptides able to fold into beta-hairpin structures in aqueous solution. Fold Des. 1996;1(2):133–144. doi: 10.1016/s1359-0278(96)00022-3. [DOI] [PubMed] [Google Scholar]

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