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. 1996 Oct;5(10):2009–2019. doi: 10.1002/pro.5560051007

Surface point mutations that significantly alter the structure and stability of a protein's denatured state.

C K Smith 1, Z Bu 1, K S Anderson 1, J M Sturtevant 1, D M Engelman 1, L Regan 1
PMCID: PMC2143264  PMID: 8897601

Abstract

Significantly different m values (1.9-2.7 kcal mol-1 M-1) were observed for point mutations at a single, solvent-exposed site (T53) in a variant of the B1 domain of streptococcal Protein G using guanidine hydrochloride (GuHCl) as a denaturant. This report focuses on elucidating the energetic and structural implications of these m-value differences in two Protein G mutants, containing Ala and Thr at position 53. These two proteins are representative of the high (m+) and low (m-) m-value mutants studied. Differential scanning calorimetry revealed no evidence of equilibrium intermediates. A comparison of GuHCl denaturation monitored by fluorescence and circular dichroism showed that secondary and tertiary structure denatured concomitantly. The rates of folding (286 S-1 for the m+ mutant and 952 S-1 for the m- mutant) and the rates of unfolding (11 S-1 for m+ mutant and 3 S-1 for the m- mutant) were significantly different, as determined by stopped-flow fluorescence. The relative solvation free energies of the transition states were identical for the two proteins (alpha ++ = 0.3). Small-angle X-ray scattering showed that the radius of gyration of the denatured state (Rgd) of the m+ mutant did not change with increasing denaturant concentrations (Rgd approximately 23 A); whereas, the Rgd of the m- mutant increased from approximately 17 A to 23 A with increasing denaturant concentration. The results indicate that the mutations exert significant effects in both the native and GuHCl-induced denatured state of these two proteins.

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Selected References

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  1. Alexander P., Fahnestock S., Lee T., Orban J., Bryan P. Thermodynamic analysis of the folding of the streptococcal protein G IgG-binding domains B1 and B2: why small proteins tend to have high denaturation temperatures. Biochemistry. 1992 Apr 14;31(14):3597–3603. doi: 10.1021/bi00129a007. [DOI] [PubMed] [Google Scholar]
  2. Arcus V. L., Vuilleumier S., Freund S. M., Bycroft M., Fersht A. R. A comparison of the pH, urea, and temperature-denatured states of barnase by heteronuclear NMR: implications for the initiation of protein folding. J Mol Biol. 1995 Nov 24;254(2):305–321. doi: 10.1006/jmbi.1995.0618. [DOI] [PubMed] [Google Scholar]
  3. Bai Y., Englander S. W. Hydrogen bond strength and beta-sheet propensities: the role of a side chain blocking effect. Proteins. 1994 Mar;18(3):262–266. doi: 10.1002/prot.340180307. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. Betz S. F., Pielak G. J. Introduction of a disulfide bond into cytochrome c stabilizes a compact denatured state. Biochemistry. 1992 Dec 15;31(49):12337–12344. doi: 10.1021/bi00164a007. [DOI] [PubMed] [Google Scholar]
  6. Bowler B. E., May K., Zaragoza T., York P., Dong A., Caughey W. S. Destabilizing effects of replacing a surface lysine of cytochrome c with aromatic amino acids: implications for the denatured state. Biochemistry. 1993 Jan 12;32(1):183–190. doi: 10.1021/bi00052a024. [DOI] [PubMed] [Google Scholar]
  7. Buck M., Radford S. E., Dobson C. M. Amide hydrogen exchange in a highly denatured state. Hen egg-white lysozyme in urea. J Mol Biol. 1994 Apr 1;237(3):247–254. doi: 10.1006/jmbi.1994.1228. [DOI] [PubMed] [Google Scholar]
  8. Carra J. H., Privalov P. L. Energetics of denaturation and m values of staphylococcal nuclease mutants. Biochemistry. 1995 Feb 14;34(6):2034–2041. doi: 10.1021/bi00006a025. [DOI] [PubMed] [Google Scholar]
  9. Chen B. L., Baase W. A., Nicholson H., Schellman J. A. Folding kinetics of T4 lysozyme and nine mutants at 12 degrees C. Biochemistry. 1992 Feb 11;31(5):1464–1476. doi: 10.1021/bi00120a025. [DOI] [PubMed] [Google Scholar]
  10. Flanagan J. M., Kataoka M., Fujisawa T., Engelman D. M. Mutations can cause large changes in the conformation of a denatured protein. Biochemistry. 1993 Oct 5;32(39):10359–10370. doi: 10.1021/bi00090a011. [DOI] [PubMed] [Google Scholar]
  11. Flanagan J. M., Kataoka M., Shortle D., Engelman D. M. Truncated staphylococcal nuclease is compact but disordered. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):748–752. doi: 10.1073/pnas.89.2.748. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Gorovits B. M., Seale J. W., Horowitz P. M. Residual structure in urea-denatured chaperonin GroEL. Biochemistry. 1995 Oct 24;34(42):13928–13933. doi: 10.1021/bi00042a026. [DOI] [PubMed] [Google Scholar]
  13. Green S. M., Meeker A. K., Shortle D. Contributions of the polar, uncharged amino acids to the stability of staphylococcal nuclease: evidence for mutational effects on the free energy of the denatured state. Biochemistry. 1992 Jun 30;31(25):5717–5728. doi: 10.1021/bi00140a005. [DOI] [PubMed] [Google Scholar]
  14. Gronenborn A. M., Filpula D. R., Essig N. Z., Achari A., Whitlow M., Wingfield P. T., Clore G. M. A novel, highly stable fold of the immunoglobulin binding domain of streptococcal protein G. Science. 1991 Aug 9;253(5020):657–661. doi: 10.1126/science.1871600. [DOI] [PubMed] [Google Scholar]
  15. Hamlin R. Multiwire area X-ray diffractometers. Methods Enzymol. 1985;114:416–452. doi: 10.1016/0076-6879(85)14029-2. [DOI] [PubMed] [Google Scholar]
  16. Kamatari Y. O., Konno T., Kataoka M., Akasaka K. The methanol-induced globular and expanded denatured states of cytochrome c: a study by CD fluorescence, NMR and small-angle X-ray scattering. J Mol Biol. 1996 Jun 14;259(3):512–523. doi: 10.1006/jmbi.1996.0336. [DOI] [PubMed] [Google Scholar]
  17. Makhatadze G. I., Privalov P. L. Protein interactions with urea and guanidinium chloride. A calorimetric study. J Mol Biol. 1992 Jul 20;226(2):491–505. doi: 10.1016/0022-2836(92)90963-k. [DOI] [PubMed] [Google Scholar]
  18. Matthews C. R., Hurle M. R. Mutant sequences as probes of protein folding mechanisms. Bioessays. 1987 Jun;6(6):254–257. doi: 10.1002/bies.950060603. [DOI] [PubMed] [Google Scholar]
  19. Monera O. D., Kay C. M., Hodges R. S. Protein denaturation with guanidine hydrochloride or urea provides a different estimate of stability depending on the contributions of electrostatic interactions. Protein Sci. 1994 Nov;3(11):1984–1991. doi: 10.1002/pro.5560031110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Myers J. K., Pace C. N., Scholtz J. M. Denaturant m values and heat capacity changes: relation to changes in accessible surface areas of protein unfolding. Protein Sci. 1995 Oct;4(10):2138–2148. doi: 10.1002/pro.5560041020. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Neri D., Billeter M., Wider G., Wüthrich K. NMR determination of residual structure in a urea-denatured protein, the 434-repressor. Science. 1992 Sep 11;257(5076):1559–1563. doi: 10.1126/science.1523410. [DOI] [PubMed] [Google Scholar]
  22. Pace C. N., Grimsley G. R., Thomson J. A., Barnett B. J. Conformational stability and activity of ribonuclease T1 with zero, one, and two intact disulfide bonds. J Biol Chem. 1988 Aug 25;263(24):11820–11825. [PubMed] [Google Scholar]
  23. Pace C. N., Vanderburg K. E. Determining globular protein stability: guanidine hydrochloride denaturation of myoglobin. Biochemistry. 1979 Jan 23;18(2):288–292. doi: 10.1021/bi00569a008. [DOI] [PubMed] [Google Scholar]
  24. Regan L., Rockwell A., Wasserman Z., DeGrado W. Disulfide crosslinks to probe the structure and flexibility of a designed four-helix bundle protein. Protein Sci. 1994 Dec;3(12):2419–2427. doi: 10.1002/pro.5560031225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Santoro M. M., Bolen D. W. Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl alpha-chymotrypsin using different denaturants. Biochemistry. 1988 Oct 18;27(21):8063–8068. doi: 10.1021/bi00421a014. [DOI] [PubMed] [Google Scholar]
  26. Shortle D., Meeker A. K. Residual structure in large fragments of staphylococcal nuclease: effects of amino acid substitutions. Biochemistry. 1989 Feb 7;28(3):936–944. doi: 10.1021/bi00429a003. [DOI] [PubMed] [Google Scholar]
  27. Shortle D. Mutational studies of protein structures and their stabilities. Q Rev Biophys. 1992 May;25(2):205–250. doi: 10.1017/s0033583500004674. [DOI] [PubMed] [Google Scholar]
  28. Shortle D., Stites W. E., Meeker A. K. Contributions of the large hydrophobic amino acids to the stability of staphylococcal nuclease. Biochemistry. 1990 Sep 4;29(35):8033–8041. doi: 10.1021/bi00487a007. [DOI] [PubMed] [Google Scholar]
  29. Smith C. K., Regan L. Guidelines for protein design: the energetics of beta sheet side chain interactions. Science. 1995 Nov 10;270(5238):980–982. doi: 10.1126/science.270.5238.980. [DOI] [PubMed] [Google Scholar]
  30. Smith C. K., Withka J. M., Regan L. A thermodynamic scale for the beta-sheet forming tendencies of the amino acids. Biochemistry. 1994 May 10;33(18):5510–5517. doi: 10.1021/bi00184a020. [DOI] [PubMed] [Google Scholar]
  31. Sosnick T. R., Trewhella J. Denatured states of ribonuclease A have compact dimensions and residual secondary structure. Biochemistry. 1992 Sep 8;31(35):8329–8335. doi: 10.1021/bi00150a029. [DOI] [PubMed] [Google Scholar]
  32. Tamura Y., Gekko K. Compactness of thermally and chemically denatured ribonuclease A as revealed by volume and compressibility. Biochemistry. 1995 Feb 14;34(6):1878–1884. doi: 10.1021/bi00006a008. [DOI] [PubMed] [Google Scholar]
  33. Tanaka A., Flanagan J., Sturtevant J. M. Thermal unfolding of staphylococcal nuclease and several mutant forms thereof studied by differential scanning calorimetry. Protein Sci. 1993 Apr;2(4):567–576. doi: 10.1002/pro.5560020408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Villafranca J. E., Howell E. E., Oatley S. J., Xuong N. H., Kraut J. An engineered disulfide bond in dihydrofolate reductase. Biochemistry. 1987 Apr 21;26(8):2182–2189. doi: 10.1021/bi00382a017. [DOI] [PubMed] [Google Scholar]

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