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Localized thermodynamic coupling between hydrogen bonding and microenvironment polarity substantially stabilizes proteins

Nature Structural & Molecular Biology volume 16, pages 684–690 (2009)Cite this article

Abstract

The energetic contributions of hydrogen bonding to protein folding are still unclear, despite more than 70 years of study. This is due partly to the difficulty of extracting thermodynamic information about specific interactions from protein mutagenesis data and partly to the context dependence of hydrogen bond strengths. Herein, we test the hypothesis that hydrogen bond strengths depend on the polarity of their microenvironment, with stronger hydrogen bonds forming in nonpolar surroundings. Double-mutant cycle analysis using a combination of amide-to-ester backbone mutagenesis and traditional side chain mutagenesis revealed that hydrogen bonds can be stronger by up to 1.2 kcal mol−1 when they are sequestered in hydrophobic surroundings than when they are solvent exposed. Such large coupling energies between hydrogen bond strengths and local polarity suggest that the context dependence of hydrogen bond strengths must be accounted for in any comprehensive account of the forces responsible for protein folding.

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Figure 1: Double-mutant cycle design.
Figure 2: Environmental dependence of hydrogen bond strength in the loop 1–modified Pin WW domain at Tyr23.
Figure 3: Environmental dependence of hydrogen bond strength in protein A* at Phe31.
Figure 4: Environmental dependence of hydrogen bond strength in the Pin WW domain at Trp11.

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References

  1. Dill, K.A. Dominant forces in protein folding. Biochemistry 29, 7133–7155 (1990).

    Article  CAS  Google Scholar 

  2. Pauling, L., Corey, R.B. & Branson, H.R. The structure of proteins: two hydrogen-bonded helical configurations of the polypeptide chain. Proc. Natl. Acad. Sci. USA 37, 205–211 (1951).

    Article  CAS  Google Scholar 

  3. Kauzmann, W. Some factors in the interpretation of protein denaturation. Adv. Protein Chem. 14, 1–63 (1959).

    Article  CAS  Google Scholar 

  4. Bolen, D.W. & Rose, G.D. Structure and energetics of the hydrogen-bonded backbone in protein folding. Annu. Rev. Biochem. 77, 339–362 (2008).

    Article  CAS  Google Scholar 

  5. Avbelj, F. & Baldwin, R.L. Limited validity of group additivity for the folding energetics of the peptide group. Proteins 63, 283–289 (2006).

    Article  CAS  Google Scholar 

  6. Avbelj, F. & Baldwin, R.L. Origin of the change in solvation enthalpy of the peptide group when neighboring peptide groups are added. Proc. Natl. Acad. Sci. USA 106, 3137–3141 (2009).

    Article  CAS  Google Scholar 

  7. Klotz, I.M. & Franzen, J.S. Hydrogen bonds between model peptide groups in solution. J. Am. Chem. Soc. 84, 3461–3466 (1962).

    Article  CAS  Google Scholar 

  8. Krescheck, G.C. & Klotz, I.M. Thermodynamics of transfer of amides from an apolar to an aqueous solution. Biochemistry 8, 8–12 (1969).

    Article  CAS  Google Scholar 

  9. Umeyama, H. & Morokuma, K. The origin of hydrogen bonding: an energy decomposition study. J. Am. Chem. Soc. 99, 1316–1332 (1977).

    Article  CAS  Google Scholar 

  10. Némethy, G., Steinberg, I.Z. & Scheraga, H.A. Influence of water structure and of hydrophobic interactions on the strength of side-chain hydrogen bonds in proteins. Biopolymers 1, 43–69 (1963).

    Article  Google Scholar 

  11. Fernández, A. & Berry, R.S. Extent of hydrogen-bond protection in folded proteins: a constraint on packing architectures. Biophys. J. 83, 2475–2481 (2002).

    Article  Google Scholar 

  12. Fernandez, A., Zhang, X. & Chen, J.P. Folding and wrapping soluble proteins: Exploring the molecular basis of cooperativity and aggregation. Prog. Nucleic Acid Res. Mol. Biol. 83, 53–87 (2008).

    CAS  Google Scholar 

  13. Joh, N.H. et al. Modest stabilization by most hydrogen-bonded side-chain interactions in membrane proteins. Nature 453, 1266–1270 (2008).

    Article  CAS  Google Scholar 

  14. Myers, J.K. & Pace, C.N. Hydrogen bonding stabilizes globular proteins. Biophys. J. 71, 2033–2039 (1996).

    Article  CAS  Google Scholar 

  15. Albeck, S., Unger, R. & Schreiber, G. Evaluation of direct and cooperative contributions towards the strength of buried hydrogen bonds and salt bridges. J. Mol. Biol. 298, 503–520 (2000).

    Article  CAS  Google Scholar 

  16. Blankenship, J.W., Balambika, R. & Dawson, P.E. Probing backbone hydrogen bonds in the hydrophobic core of GCN4. Biochemistry 41, 15676–15684 (2002).

    Article  CAS  Google Scholar 

  17. Takano, K., Scholtz, J.M., Sacchettini, J.C. & Pace, C.N. The contribution of polar group burial to protein stability is strongly context-dependent. J. Biol. Chem. 278, 31790–31795 (2003).

    Article  CAS  Google Scholar 

  18. Scheike, J.A. et al. Amide-to-ester substitution in coiled coils: the effect of removing hydrogen bonds on protein structure. Angew. Chem. Int. Edn Engl. 46, 7766–7769 (2007).

    Article  CAS  Google Scholar 

  19. Deechongkit, S., Dawson, P.E. & Kelly, J.W. Toward assessing the position-dependent contributions of backbone hydrogen bonding to β-sheet folding thermodynamics employing amide-to-ester perturbations. J. Am. Chem. Soc. 126, 16762–16771 (2004).

    Article  CAS  Google Scholar 

  20. Deechongkit, S. et al. Context-dependent contributions of backbone hydrogen bonding to β-sheet folding energetics. Nature 430, 101–105 (2004).

    Article  CAS  Google Scholar 

  21. Powers, E.T., Deechongkit, S. & Kelly, J.W. Backbone-backbone H-bonds make context-dependent contributions to protein folding kinetics and thermodynamics: Lessons from amide-to-ester mutations. Adv. Protein Chem. 72, 39–78 (2006).

    Article  CAS  Google Scholar 

  22. Ferguson, N., Johnson, C.M., Macias, M., Oschkinat, H. & Fersht, A. Ultrafast folding of WW domains without structured aromatic clusters in the denatured state. Proc. Natl. Acad. Sci. USA 98, 13002–13007 (2001).

    Article  CAS  Google Scholar 

  23. Ferguson, N. et al. Using flexible loop mimetics to extend φ-value analysis to secondary structure interactions. Proc. Natl. Acad. Sci. USA 98, 13008–13013 (2001).

    Article  CAS  Google Scholar 

  24. Socolich, M. et al. Evolutionary information for specifying a protein fold. Nature 437, 512–518 (2005).

    Article  CAS  Google Scholar 

  25. Jäger, M., Nguyen, H., Crane, J.C., Kelly, J.W. & Gruebele, M. The folding mechanism of a β-sheet: the WW domain. J. Mol. Biol. 311, 373–393 (2001).

    Article  Google Scholar 

  26. Jäger, M. et al. Structure-function-folding relationship in a WW domain. Proc. Natl. Acad. Sci. USA 103, 10648–10653 (2006).

    Article  Google Scholar 

  27. Deechongkit, S. et al. β-sheet folding mechanisms from perturbation energetics. Curr. Opin. Struct. Biol. 16, 94–101 (2006).

    Article  CAS  Google Scholar 

  28. Nguyen, H., Jager, M., Kelly, J.W. & Gruebele, M. Engineering a β-sheet protein toward the folding speed limit. J. Phys. Chem. B 109, 15182–15186 (2005).

    Article  CAS  Google Scholar 

  29. Ranganathan, R., Lu, K.P., Hunter, T. & Noel, J.P. Structural and functional analysis of the mitotic rotamase Pin1 suggests substrate recognition is phosphorylation dependent. Cell 89, 875–886 (1997).

    Article  CAS  Google Scholar 

  30. Bai, Y., Karimi, A., Dyson, H.J. & Wright, P.E. Absence of a stable intermediate on the folding pathway of protein A. Protein Sci. 6, 1449–1457 (1997).

    Article  CAS  Google Scholar 

  31. Sato, S., Religa, T.L., Daggett, V. & Fersht, A.R. Testing protein-folding simulations by experiment: B domain of protein A. Proc. Natl. Acad. Sci. USA 101, 6952–6956 (2004).

    Article  CAS  Google Scholar 

  32. Fersht, A.R., Matouschek, A. & Serrano, L. The folding of an enzyme. I. Theory of protein engineering analysis of stability and pathway of protein folding. J. Mol. Biol. 224, 771–782 (1992).

    Article  CAS  Google Scholar 

  33. Carter, P.J., Winter, G., Wilkinson, A.J. & Fersht, A.R. The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus). Cell 38, 835–840 (1984).

    Article  CAS  Google Scholar 

  34. Horovitz, A. & Fersht, A.R. Co-operative interactions during protein folding. J. Mol. Biol. 224, 733–740 (1992).

    Article  CAS  Google Scholar 

  35. Seebach, D., Beck, A.K. & Bierbaum, D.J. The world of β- and γ-peptides comprised of homologated proteinogenic amino acids and other components. Chem. Biodivers. 1, 1111–1239 (2004).

    Article  CAS  Google Scholar 

  36. Horne, W.S., Price, J.L. & Gellman, S.H. Interplay among side chain sequence, backbone composition, and residue rigidification in polypeptide folding and assembly. Proc. Natl. Acad. Sci. USA 105, 9151–9156 (2008).

    Article  CAS  Google Scholar 

  37. Price, J.L., Horne, W.S. & Gellman, S.H. Discrete heterogeneous quaternary structure formed by α/β-peptide foldamers and α-peptides. J. Am. Chem. Soc. 129, 6376–6377 (2007).

    Article  CAS  Google Scholar 

  38. Chatterjee, S., Roy, R.S. & Balaram, P. Expanding the polypeptide backbone: hydrogen-bonded conformations in hybrid polypeptides containing the higher homologues of α-amino acids. J. R. Soc. Interface 4, 587–606 (2007).

    Article  CAS  Google Scholar 

  39. Hann, M.M., Sammes, P.G., Kennewell, P.D. & Taylor, J.B. On double bond isosteres of the peptide bond; an enkephalin analogue. J. Chem. Soc. Chem. Commun. 5, 234–235 (1980).

    Article  Google Scholar 

  40. Xiao, J., Weisblum, B. & Wipf, P. Trisubstituted (E)-alkene dipeptide isosteres as β-turn promoters in the gramicidin S cyclodecapeptide scaffold. Org. Lett. 8, 4731–4734 (2006).

    Article  CAS  Google Scholar 

  41. Jenkins, C.L., Vasbinder, M.M., Miller, S.J. & Raines, R.T. Peptide bond isosteres: ester or E-alkene in the backbone of the collagen triple helix. Org. Lett. 7, 2619–2622 (2005).

    Article  CAS  Google Scholar 

  42. Fu, Y., Gao, J.M., Bieschke, J., Dendle, M.A. & Kelly, J.W. Amide-to-E-olefin versus amide-to-ester backbone H-bond perturbations: Evaluating the O-O repulsion for extracting H-bond energies. J. Am. Chem. Soc. 128, 15948–15949 (2006).

    Article  CAS  Google Scholar 

  43. Gao, J. & Kelly, J.W. Toward quantification of protein backbone-backbone hydrogen bonding energies: An energetic analysis of an amide-to-ester mutation in an α-helix within a protein. Protein Sci. 17, 1096–1101 (2008).

    Article  CAS  Google Scholar 

  44. Stigers, K.D., Soth, M.J. & Nowick, J.S. Designed molecules that fold to mimic protein secondary structures. Curr. Opin. Chem. Biol. 3, 714–723 (1999).

    Article  CAS  Google Scholar 

  45. Yang, X., Wang, M. & Fitzgerald, M.C. Analysis of protein folding and function using backbone modified proteins. Bioorg. Chem. 32, 438–449 (2004).

    Article  CAS  Google Scholar 

  46. Myers, J.K. & Oas, T.G. Preorganized secondary structure as an important determinant of fast protein folding. Nat. Struct. Biol. 8, 552–558 (2001).

    Article  CAS  Google Scholar 

  47. Dawson, P.E. & Kent, S.B. Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 69, 923–960 (2000).

    Article  CAS  Google Scholar 

  48. Bai, Y., Milne, J.S., Mayne, L. & Englander, S.W. Primary structure effects on peptide group hydrogen exchange. Proteins 17, 75–86 (1993).

    Article  CAS  Google Scholar 

  49. Dyson, H.J., Rance, M., Houghten, R.A., Lerner, R.A. & Wright, P.E. Folding of immunogenic peptide fragments of proteins in water solution. I. Sequence requirements for the formation of a reverse turn. J. Mol. Biol. 201, 161–200 (1988).

    Article  CAS  Google Scholar 

  50. Wright, P.E., Dyson, H.J. & Lerner, R.A. Conformation of peptide fragments of proteins in aqueous solution: implications for initiation of protein folding. Biochemistry 27, 7167–7175 (1988).

    Article  CAS  Google Scholar 

  51. Yi, Q., Scalley-Kim, M.L., Alm, E.J. & Baker, D. NMR characterization of residual structure in the denatured state of protein L. J. Mol. Biol. 299, 1341–1351 (2000).

    Article  CAS  Google Scholar 

  52. Zhang, O. & Forman-Kay, J.D. NMR studies of unfolded states of an SH3 domain in aqueous solution and denaturing conditions. Biochemistry 36, 3959–3970 (1997).

    Article  CAS  Google Scholar 

  53. Anil, B., Li, Y., Cho, J.H. & Raleigh, D.P. The unfolded state of NTL9 is compact in the absence of denaturant. Biochemistry 45, 10110–10116 (2006).

    Article  CAS  Google Scholar 

  54. Blaber, M. et al. Energetic cost and structural consequences of burying a hydroxyl group within the core of a protein determined from Ala → Ser and Val → Thr substitutions in T4 lysozyme. Biochemistry 32, 11363–11373 (1993).

    Article  CAS  Google Scholar 

  55. Alber, T. et al. Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme. Nature 330, 41–46 (1987).

    Article  CAS  Google Scholar 

  56. McDonald, I.K. & Thornton, J.M. Satisfying hydrogen bonding potential in proteins. J. Mol. Biol. 238, 777–793 (1994).

    Article  CAS  Google Scholar 

  57. Fleming, P.J. & Rose, G.D. Do all backbone polar groups in proteins form hydrogen bonds? Protein Sci. 14, 1911–1917 (2005).

    Article  CAS  Google Scholar 

  58. Bai, Y. & Englander, S.W. Hydrogen bond strength and β-sheet propensities—the role of a side chain blocking effect. Proteins 18, 262–266 (1994).

    Article  CAS  Google Scholar 

  59. Franzen, J.S. & Stephens, R.E. Effect of a dipolar solvent system on interamide hydrogen bonds. Biochemistry 2, 1321–1327 (1963).

    Article  CAS  Google Scholar 

  60. Pace, C.N. The stability of globular proteins. CRC Crit. Rev. Biochem. 3, 1–43 (1975).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by a grant from the US National Institutes of Health (GM051105), the Lita Annenberg Hazen Foundation and The Skaggs Institute for Chemical Biology. D.A.B. thanks the National Institutes of Health for a postdoctoral fellowship (NS047024).

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Authors and Affiliations

  1. Departments of Chemistry and Molecular and Experimental Medicine and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA

    Jianmin Gao, Daryl A Bosco, Evan T Powers & Jeffery W Kelly

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  1. Jianmin Gao
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  2. Daryl A Bosco
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  3. Evan T Powers
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Contributions

J.W.K., E.T.P. and J.G. designed the project, analyzed the data and wrote the manuscript; J.G. performed the protein folding studies; D.A.B. and J.G. obtained and analyzed the NMR data.

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Correspondence to Evan T Powers or Jeffery W Kelly.

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Gao, J., Bosco, D., Powers, E. et al. Localized thermodynamic coupling between hydrogen bonding and microenvironment polarity substantially stabilizes proteins. Nat Struct Mol Biol 16, 684–690 (2009). https://doi.org/10.1038/nsmb.1610

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