Skip to main content
PMC home page
Protein Science : A Publication of the Protein Society logoLink to Protein Science : A Publication of the Protein Society
. 1994 Feb;3(2):211–226. doi: 10.1002/pro.5560030206

Do salt bridges stabilize proteins? A continuum electrostatic analysis.

Z S Hendsch 1, B Tidor 1
PMCID: PMC2142793  PMID: 8003958

Abstract

The electrostatic contribution to the free energy of folding was calculated for 21 salt bridges in 9 protein X-ray crystal structures using a continuum electrostatic approach with the DELPHI computer-program package. The majority (17) were found to be electrostatically destabilizing; the average free energy change, which is analogous to mutation of salt bridging side chains to hydrophobic isosteres, was calculated to be 3.5 kcal/mol. This is fundamentally different from stability measurements using pKa shifts, which effectively measure the strength of a salt bridge relative to 1 or more charged hydrogen bonds. The calculated effect was due to a large, unfavorable desolvation contribution that was not fully compensated by favorable interactions within the salt bridge and between salt-bridge partners and other polar and charged groups in the folded protein. Some of the salt bridges were studied in further detail to determine the effect of the choice of values for atomic radii, internal protein dielectric constant, and ionic strength used in the calculations. Increased ionic strength resulted in little or no change in calculated stability for 3 of 4 salt bridges over a range of 0.1-0.9 M. The results suggest that mutation of salt bridges, particularly those that are buried, to "hydrophobic bridges" (that pack at least as well as wild type) can result in proteins with increased stability. Due to the large penalty for burying uncompensated ionizable groups, salt bridges could help to limit the number of low free energy conformations of a molecule or complex and thus play a role in determining specificity (i.e., the uniqueness of a protein fold or protein-ligand binding geometry).

Full Text

The Full Text of this article is available as a PDF (4.8 MB).

Selected References

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

  1. Anderson D. E., Becktel W. J., Dahlquist F. W. pH-induced denaturation of proteins: a single salt bridge contributes 3-5 kcal/mol to the free energy of folding of T4 lysozyme. Biochemistry. 1990 Mar 6;29(9):2403–2408. doi: 10.1021/bi00461a025. [DOI] [PubMed] [Google Scholar]
  2. Barlow D. J., Thornton J. M. Ion-pairs in proteins. J Mol Biol. 1983 Aug 25;168(4):867–885. doi: 10.1016/s0022-2836(83)80079-5. [DOI] [PubMed] [Google Scholar]
  3. Bashford D., Karplus M. pKa's of ionizable groups in proteins: atomic detail from a continuum electrostatic model. Biochemistry. 1990 Nov 6;29(44):10219–10225. doi: 10.1021/bi00496a010. [DOI] [PubMed] [Google Scholar]
  4. Beamer L. J., Pabo C. O. Refined 1.8 A crystal structure of the lambda repressor-operator complex. J Mol Biol. 1992 Sep 5;227(1):177–196. doi: 10.1016/0022-2836(92)90690-l. [DOI] [PubMed] [Google Scholar]
  5. Bell J. A., Wilson K. P., Zhang X. J., Faber H. R., Nicholson H., Matthews B. W. Comparison of the crystal structure of bacteriophage T4 lysozyme at low, medium, and high ionic strengths. Proteins. 1991;10(1):10–21. doi: 10.1002/prot.340100103. [DOI] [PubMed] [Google Scholar]
  6. Bernstein F. C., Koetzle T. F., Williams G. J., Meyer E. F., Jr, Brice M. D., Rodgers J. R., Kennard O., Shimanouchi T., Tasumi M. The Protein Data Bank: a computer-based archival file for macromolecular structures. J Mol Biol. 1977 May 25;112(3):535–542. doi: 10.1016/s0022-2836(77)80200-3. [DOI] [PubMed] [Google Scholar]
  7. Beroza P., Fredkin D. R., Okamura M. Y., Feher G. Protonation of interacting residues in a protein by a Monte Carlo method: application to lysozyme and the photosynthetic reaction center of Rhodobacter sphaeroides. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5804–5808. doi: 10.1073/pnas.88.13.5804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blake C. C., Mair G. A., North A. C., Phillips D. C., Sarma V. R. On the conformation of the hen egg-white lysozyme molecule. Proc R Soc Lond B Biol Sci. 1967 Apr 18;167(1009):365–377. doi: 10.1098/rspb.1967.0034. [DOI] [PubMed] [Google Scholar]
  9. Brünger A. T., Karplus M. Polar hydrogen positions in proteins: empirical energy placement and neutron diffraction comparison. Proteins. 1988;4(2):148–156. doi: 10.1002/prot.340040208. [DOI] [PubMed] [Google Scholar]
  10. Cohen G. H., Silverton E. W., Davies D. R. Refined crystal structure of gamma-chymotrypsin at 1.9 A resolution. Comparison with other pancreatic serine proteases. J Mol Biol. 1981 Jun 5;148(4):449–479. doi: 10.1016/0022-2836(81)90186-8. [DOI] [PubMed] [Google Scholar]
  11. Corfield P. W., Lee T. J., Low B. W. The crystal structure of erabutoxin a at 2.0-A resolution. J Biol Chem. 1989 Jun 5;264(16):9239–9242. doi: 10.2210/pdb5ebx/pdb. [DOI] [PubMed] [Google Scholar]
  12. Diamond R. Real-space refinement of the structure of hen egg-white lysozyme. J Mol Biol. 1974 Jan 25;82(3):371–391. doi: 10.1016/0022-2836(74)90598-1. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. Erwin C. R., Barnett B. L., Oliver J. D., Sullivan J. F. Effects of engineered salt bridges on the stability of subtilisin BPN'. Protein Eng. 1990 Oct;4(1):87–97. doi: 10.1093/protein/4.1.87. [DOI] [PubMed] [Google Scholar]
  15. Fersht A. R. Conformational equilibria in -and -chymotrypsin. The energetics and importance of the salt bridge. J Mol Biol. 1972 Mar 14;64(2):497–509. doi: 10.1016/0022-2836(72)90513-x. [DOI] [PubMed] [Google Scholar]
  16. Gao J., Kuczera K., Tidor B., Karplus M. Hidden thermodynamics of mutant proteins: a molecular dynamics analysis. Science. 1989 Jun 2;244(4908):1069–1072. doi: 10.1126/science.2727695. [DOI] [PubMed] [Google Scholar]
  17. Gilson M. K., Honig B. H. Calculation of electrostatic potentials in an enzyme active site. Nature. 1987 Nov 5;330(6143):84–86. doi: 10.1038/330084a0. [DOI] [PubMed] [Google Scholar]
  18. Gilson M. K., Honig B. Calculation of the total electrostatic energy of a macromolecular system: solvation energies, binding energies, and conformational analysis. Proteins. 1988;4(1):7–18. doi: 10.1002/prot.340040104. [DOI] [PubMed] [Google Scholar]
  19. Gilson M. K. Multiple-site titration and molecular modeling: two rapid methods for computing energies and forces for ionizable groups in proteins. Proteins. 1993 Mar;15(3):266–282. doi: 10.1002/prot.340150305. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Hill C. P., Anderson D. H., Wesson L., DeGrado W. F., Eisenberg D. Crystal structure of alpha 1: implications for protein design. Science. 1990 Aug 3;249(4968):543–546. doi: 10.1126/science.2382133. [DOI] [PubMed] [Google Scholar]
  22. KAUZMANN W. Some factors in the interpretation of protein denaturation. Adv Protein Chem. 1959;14:1–63. doi: 10.1016/s0065-3233(08)60608-7. [DOI] [PubMed] [Google Scholar]
  23. Kabsch W., Sander C. Dictionary of protein secondary structure: pattern recognition of hydrogen-bonded and geometrical features. Biopolymers. 1983 Dec;22(12):2577–2637. doi: 10.1002/bip.360221211. [DOI] [PubMed] [Google Scholar]
  24. Klapper I., Hagstrom R., Fine R., Sharp K., Honig B. Focusing of electric fields in the active site of Cu-Zn superoxide dismutase: effects of ionic strength and amino-acid modification. Proteins. 1986 Sep;1(1):47–59. doi: 10.1002/prot.340010109. [DOI] [PubMed] [Google Scholar]
  25. Langsetmo K., Fuchs J. A., Woodward C., Sharp K. A. Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa. Biochemistry. 1991 Jul 30;30(30):7609–7614. doi: 10.1021/bi00244a033. [DOI] [PubMed] [Google Scholar]
  26. Langsetmo K., Fuchs J. A., Woodward C. The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5. Its titration produces a related shift in global stability. Biochemistry. 1991 Jul 30;30(30):7603–7609. doi: 10.1021/bi00244a032. [DOI] [PubMed] [Google Scholar]
  27. 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]
  28. Lyu P. C., Gans P. J., Kallenbach N. R. Energetic contribution of solvent-exposed ion pairs to alpha-helix structure. J Mol Biol. 1992 Jan 5;223(1):343–350. doi: 10.1016/0022-2836(92)90735-3. [DOI] [PubMed] [Google Scholar]
  29. Marqusee S., Baldwin R. L. Helix stabilization by Glu-...Lys+ salt bridges in short peptides of de novo design. Proc Natl Acad Sci U S A. 1987 Dec;84(24):8898–8902. doi: 10.1073/pnas.84.24.8898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mauguen Y., Hartley R. W., Dodson E. J., Dodson G. G., Bricogne G., Chothia C., Jack A. Molecular structure of a new family of ribonucleases. Nature. 1982 May 13;297(5862):162–164. doi: 10.1038/297162a0. [DOI] [PubMed] [Google Scholar]
  31. Mondragón A., Subbiah S., Almo S. C., Drottar M., Harrison S. C. Structure of the amino-terminal domain of phage 434 repressor at 2.0 A resolution. J Mol Biol. 1989 Jan 5;205(1):189–200. doi: 10.1016/0022-2836(89)90375-6. [DOI] [PubMed] [Google Scholar]
  32. Morize I., Surcouf E., Vaney M. C., Epelboin Y., Buehner M., Fridlansky F., Milgrom E., Mornon J. P. Refinement of the C222(1) crystal form of oxidized uteroglobin at 1.34 A resolution. J Mol Biol. 1987 Apr 20;194(4):725–739. doi: 10.1016/0022-2836(87)90250-6. [DOI] [PubMed] [Google Scholar]
  33. O'Shea E. K., Klemm J. D., Kim P. S., Alber T. X-ray structure of the GCN4 leucine zipper, a two-stranded, parallel coiled coil. Science. 1991 Oct 25;254(5031):539–544. doi: 10.1126/science.1948029. [DOI] [PubMed] [Google Scholar]
  34. O'Shea E. K., Rutkowski R., Kim P. S. Mechanism of specificity in the Fos-Jun oncoprotein heterodimer. Cell. 1992 Feb 21;68(4):699–708. doi: 10.1016/0092-8674(92)90145-3. [DOI] [PubMed] [Google Scholar]
  35. Paul C. H. Building models of globular protein molecules from their amino acid sequences. I. Theory. J Mol Biol. 1982 Feb 15;155(1):53–62. doi: 10.1016/0022-2836(82)90491-0. [DOI] [PubMed] [Google Scholar]
  36. Perutz M. F., Gronenborn A. M., Clore G. M., Fogg J. H., Shih D. T. The pKa values of two histidine residues in human haemoglobin, the Bohr effect, and the dipole moments of alpha-helices. J Mol Biol. 1985 Jun 5;183(3):491–498. doi: 10.1016/0022-2836(85)90016-6. [DOI] [PubMed] [Google Scholar]
  37. Rashin A. A., Honig B. On the environment of ionizable groups in globular proteins. J Mol Biol. 1984 Mar 15;173(4):515–521. doi: 10.1016/0022-2836(84)90394-2. [DOI] [PubMed] [Google Scholar]
  38. Regan L., DeGrado W. F. Characterization of a helical protein designed from first principles. Science. 1988 Aug 19;241(4868):976–978. doi: 10.1126/science.3043666. [DOI] [PubMed] [Google Scholar]
  39. Schmidt-Dörr T., Oertel-Buchheit P., Pernelle C., Bracco L., Schnarr M., Granger-Schnarr M. Construction, purification, and characterization of a hybrid protein comprising the DNA binding domain of the LexA repressor and the Jun leucine zipper: a circular dichroism and mutagenesis study. Biochemistry. 1991 Oct 8;30(40):9657–9664. doi: 10.1021/bi00104a013. [DOI] [PubMed] [Google Scholar]
  40. Serrano L., Horovitz A., Avron B., Bycroft M., Fersht A. R. Estimating the contribution of engineered surface electrostatic interactions to protein stability by using double-mutant cycles. Biochemistry. 1990 Oct 9;29(40):9343–9352. doi: 10.1021/bi00492a006. [DOI] [PubMed] [Google Scholar]
  41. Sharp K. A., Honig B. Electrostatic interactions in macromolecules: theory and applications. Annu Rev Biophys Biophys Chem. 1990;19:301–332. doi: 10.1146/annurev.bb.19.060190.001505. [DOI] [PubMed] [Google Scholar]
  42. Stites W. E., Gittis A. G., Lattman E. E., Shortle D. In a staphylococcal nuclease mutant the side-chain of a lysine replacing valine 66 is fully buried in the hydrophobic core. J Mol Biol. 1991 Sep 5;221(1):7–14. doi: 10.1016/0022-2836(91)80195-z. [DOI] [PubMed] [Google Scholar]
  43. Sun D. P., Sauer U., Nicholson H., Matthews B. W. Contributions of engineered surface salt bridges to the stability of T4 lysozyme determined by directed mutagenesis. Biochemistry. 1991 Jul 23;30(29):7142–7153. doi: 10.1021/bi00243a015. [DOI] [PubMed] [Google Scholar]
  44. Varadarajan R., Lambright D. G., Boxer S. G. Electrostatic interactions in wild-type and mutant recombinant human myoglobins. Biochemistry. 1989 May 2;28(9):3771–3781. doi: 10.1021/bi00435a022. [DOI] [PubMed] [Google Scholar]
  45. Vinson C. R., Hai T., Boyd S. M. Dimerization specificity of the leucine zipper-containing bZIP motif on DNA binding: prediction and rational design. Genes Dev. 1993 Jun;7(6):1047–1058. doi: 10.1101/gad.7.6.1047. [DOI] [PubMed] [Google Scholar]
  46. Yang A. S., Gunner M. R., Sampogna R., Sharp K., Honig B. On the calculation of pKas in proteins. Proteins. 1993 Mar;15(3):252–265. doi: 10.1002/prot.340150304. [DOI] [PubMed] [Google Scholar]
  47. Yang A. S., Sharp K. A., Honig B. Analysis of the heat capacity dependence of protein folding. J Mol Biol. 1992 Oct 5;227(3):889–900. doi: 10.1016/0022-2836(92)90229-d. [DOI] [PubMed] [Google Scholar]

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

RESOURCES