Skip to main content
NCBI home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1993 Apr;2(4):588–596. doi: 10.1002/pro.5560020410

Crevice-forming mutants of bovine pancreatic trypsin inhibitor: stability changes and new hydrophobic surface.

K S Kim 1, F Tao 1, J Fuchs 1, A T Danishefsky 1, D Housset 1, A Wlodawer 1, C Woodward 1
PMCID: PMC2142357  PMID: 7686069

Abstract

Four mutants of bovine pancreatic trypsin inhibitor (BPTI) with replacements in the rigid core result in the creation of deep crevices on the surface of the protein. Other than crevices at the site of the mutation, few other differences are observed in the crystal structures of wild-type BPTI and the mutants F22A, Y23A, N43G, and F45A. These mutants are highly destabilized relative to wild type (WT). The differences between WT and mutants in the free energy change associated with cooperative folding/unfolding, delta delta G0 (WT-->mut), have been measured by calorimetry, and they are in good agreement with delta delta G0(WT-->mut) values from hydrogen exchange rates. For F22A the change in free energy difference is about 1.7 kcal/mol at 25 degrees C; for the other three mutants it is in the range of 5-7 kcal/mol at 25 degrees C. The experimental delta delta G0(WT-->mut) values of F22A, Y23A, and F45A are reasonably well accounted for as the sum of two terms: the difference in transfer free energy change, and a contribution from exposure to solvent of new surface (Eriksson, A.E., et al., 1992, Science 255, 178-183), if the recently corrected transfer free energies and surface hydrophobicities (De Young, L. & Dill, K., 1990, J. Phys. Chem. 94, 801-809; Sharp, K.A., et al., 1991a, Science 252, 106-109) are used and only nonpolar surface is taken into account. In N43G, three protein-protein hydrogen bonds are replaced by protein-water hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (928.5 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bowie J. U., Lüthy R., Eisenberg D. A method to identify protein sequences that fold into a known three-dimensional structure. Science. 1991 Jul 12;253(5016):164–170. doi: 10.1126/science.1853201. [DOI] [PubMed] [Google Scholar]
  2. Chothia C. Hydrophobic bonding and accessible surface area in proteins. Nature. 1974 Mar 22;248(446):338–339. doi: 10.1038/248338a0. [DOI] [PubMed] [Google Scholar]
  3. Connelly P., Ghosaini L., Hu C. Q., Kitamura S., Tanaka A., Sturtevant J. M. A differential scanning calorimetric study of the thermal unfolding of seven mutant forms of phage T4 lysozyme. Biochemistry. 1991 Feb 19;30(7):1887–1891. doi: 10.1021/bi00221a022. [DOI] [PubMed] [Google Scholar]
  4. Connolly M. L. Solvent-accessible surfaces of proteins and nucleic acids. Science. 1983 Aug 19;221(4612):709–713. doi: 10.1126/science.6879170. [DOI] [PubMed] [Google Scholar]
  5. Danishefsky A. T., Housset D., Kim K. S., Tao F., Fuchs J., Woodward C., Wlodawer A. Crevice-forming mutants in the rigid core of bovine pancreatic trypsin inhibitor: crystal structures of F22A, Y23A, N43G, and F45A. Protein Sci. 1993 Apr;2(4):577–587. doi: 10.1002/pro.5560020409. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eigenbrot C., Randal M., Kossiakoff A. A. Structural effects induced by removal of a disulfide-bridge: the X-ray structure of the C30A/C51A mutant of basic pancreatic trypsin inhibitor at 1.6 A. Protein Eng. 1990 Jul;3(7):591–598. doi: 10.1093/protein/3.7.591. [DOI] [PubMed] [Google Scholar]
  7. Eisenberg D., McLachlan A. D. Solvation energy in protein folding and binding. Nature. 1986 Jan 16;319(6050):199–203. doi: 10.1038/319199a0. [DOI] [PubMed] [Google Scholar]
  8. Eriksson A. E., Baase W. A., Wozniak J. A., Matthews B. W. A cavity-containing mutant of T4 lysozyme is stabilized by buried benzene. Nature. 1992 Jan 23;355(6358):371–373. doi: 10.1038/355371a0. [DOI] [PubMed] [Google Scholar]
  9. Eriksson A. E., Baase W. A., Zhang X. J., Heinz D. W., Blaber M., Baldwin E. P., Matthews B. W. Response of a protein structure to cavity-creating mutations and its relation to the hydrophobic effect. Science. 1992 Jan 10;255(5041):178–183. doi: 10.1126/science.1553543. [DOI] [PubMed] [Google Scholar]
  10. Hilton B. D., Trudeau K., Woodward C. K. Hydrogen exchange rates in pancreatic trypsin inhibitor are not correlated to thermal stability in urea. Biochemistry. 1981 Aug 4;20(16):4697–4703. doi: 10.1021/bi00519a027. [DOI] [PubMed] [Google Scholar]
  11. Hurle M. R., Eads C. D., Pearlman D. A., Seibel G. L., Thomason J., Kosen P. A., Kollman P., Anderson S., Kuntz I. D. Comparison of solution structures of mutant bovine pancreatic trypsin inhibitor proteins using two-dimensional nuclear magnetic resonance. Protein Sci. 1992 Jan;1(1):91–106. doi: 10.1002/pro.5560010110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hynes T. R., Randal M., Kennedy L. A., Eigenbrot C., Kossiakoff A. A. X-ray crystal structure of the protease inhibitor domain of Alzheimer's amyloid beta-protein precursor. Biochemistry. 1990 Oct 30;29(43):10018–10022. doi: 10.1021/bi00495a002. [DOI] [PubMed] [Google Scholar]
  13. Jackson W. M., Brandts J. F. Thermodynamics of protein denaturation. A calorimetric study of the reversible denaturation of chymotrypsinogen and conclusions regarding the accuracy of the two-state approximation. Biochemistry. 1970 May 26;9(11):2294–2301. doi: 10.1021/bi00813a011. [DOI] [PubMed] [Google Scholar]
  14. Lee B., Richards F. M. The interpretation of protein structures: estimation of static accessibility. J Mol Biol. 1971 Feb 14;55(3):379–400. doi: 10.1016/0022-2836(71)90324-x. [DOI] [PubMed] [Google Scholar]
  15. Moses E., Hinz H. J. Basic pancreatic trypsin inhibitor has unusual thermodynamic stability parameters. J Mol Biol. 1983 Nov 5;170(3):765–776. doi: 10.1016/s0022-2836(83)80130-2. [DOI] [PubMed] [Google Scholar]
  16. Murphy K. P., Bhakuni V., Xie D., Freire E. Molecular basis of co-operativity in protein folding. III. Structural identification of cooperative folding units and folding intermediates. J Mol Biol. 1992 Sep 5;227(1):293–306. doi: 10.1016/0022-2836(92)90699-k. [DOI] [PubMed] [Google Scholar]
  17. Nicholls A., Sharp K. A., Honig B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins. 1991;11(4):281–296. doi: 10.1002/prot.340110407. [DOI] [PubMed] [Google Scholar]
  18. Ooi T., Oobatake M. Effects of hydrated water on protein unfolding. J Biochem. 1988 Jan;103(1):114–120. doi: 10.1093/oxfordjournals.jbchem.a122215. [DOI] [PubMed] [Google Scholar]
  19. Ooi T., Oobatake M., Némethy G., Scheraga H. A. Accessible surface areas as a measure of the thermodynamic parameters of hydration of peptides. Proc Natl Acad Sci U S A. 1987 May;84(10):3086–3090. doi: 10.1073/pnas.84.10.3086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Pace C. N. Contribution of the hydrophobic effect to globular protein stability. J Mol Biol. 1992 Jul 5;226(1):29–35. doi: 10.1016/0022-2836(92)90121-y. [DOI] [PubMed] [Google Scholar]
  21. Privalov P. L., Griko YuV, Venyaminov SYu, Kutyshenko V. P. Cold denaturation of myoglobin. J Mol Biol. 1986 Aug 5;190(3):487–498. doi: 10.1016/0022-2836(86)90017-3. [DOI] [PubMed] [Google Scholar]
  22. Privalov P. L. Stability of proteins: small globular proteins. Adv Protein Chem. 1979;33:167–241. doi: 10.1016/s0065-3233(08)60460-x. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Richarz R., Sehr P., Wagner G., Wüthrich K. Kinetics of the exchange of individual amide protons in the basic pancreatic trypsin inhibitor. J Mol Biol. 1979 May 5;130(1):19–30. doi: 10.1016/0022-2836(79)90549-7. [DOI] [PubMed] [Google Scholar]
  25. Sharp K. A., Nicholls A., Fine R. F., Honig B. Reconciling the magnitude of the microscopic and macroscopic hydrophobic effects. Science. 1991 Apr 5;252(5002):106–109. doi: 10.1126/science.2011744. [DOI] [PubMed] [Google Scholar]
  26. Shirley B. A., Stanssens P., Hahn U., Pace C. N. Contribution of hydrogen bonding to the conformational stability of ribonuclease T1. Biochemistry. 1992 Jan 28;31(3):725–732. doi: 10.1021/bi00118a013. [DOI] [PubMed] [Google Scholar]
  27. Spolar R. S., Livingstone J. R., Record M. T., Jr Use of liquid hydrocarbon and amide transfer data to estimate contributions to thermodynamic functions of protein folding from the removal of nonpolar and polar surface from water. Biochemistry. 1992 Apr 28;31(16):3947–3955. doi: 10.1021/bi00131a009. [DOI] [PubMed] [Google Scholar]
  28. Vriend G., Berendsen H. J., van der Zee J. R., van den Burg B., Venema G., Eijsink V. G. Stabilization of the neutral protease of Bacillus stearothermophilus by removal of a buried water molecule. Protein Eng. 1991 Dec;4(8):941–945. doi: 10.1093/protein/4.8.941. [DOI] [PubMed] [Google Scholar]
  29. Wagner G. Characterization of the distribution of internal motions in the basic pancreatic trypsin inhibitor using a large number of internal NMR probes. Q Rev Biophys. 1983 Feb;16(1):1–57. doi: 10.1017/s0033583500004911. [DOI] [PubMed] [Google Scholar]

Articles from Protein Science : A Publication of the Protein Society are provided here courtesy of The Protein Society

RESOURCES