Skip to main content
NCBI home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 Jun;82(6):3289–3304. doi: 10.1016/s0006-3495(02)75670-1

Experimental pK(a) values of buried residues: analysis with continuum methods and role of water penetration.

Carolyn A Fitch 1, Daniel A Karp 1, Kelly K Lee 1, Wesley E Stites 1, Eaton E Lattman 1, Bertrand García-Moreno E 1
PMCID: PMC1302117  PMID: 12023252

Abstract

Lys-66 and Glu-66, buried in the hydrophobic interior of staphylococcal nuclease by mutagenesis, titrate with pK(a) values of 5.7 and 8.8, respectively (Dwyer et al., Biophys. J. 79:1610-1620; García-Moreno E. et al., Biophys. Chem. 64:211-224). Continuum calculations with static structures reproduced the pK(a) values when the protein interior was treated with a dielectric constant (epsilon(in)) of 10. This high apparent polarizability can be rationalized in the case of Glu-66 in terms of internal water molecules, visible in crystallographic structures, hydrogen bonded to Glu-66. The water molecules are absent in structures with Lys-66; the high polarizability cannot be reconciled with the hydrophobic environment surrounding Lys-66. Equilibrium thermodynamic experiments showed that the Lys-66 mutant remained folded and native-like after ionization of the buried lysine. The high polarizability must therefore reflect water penetration, minor local structural rearrangement, or both. When in pK(a) calculations with continuum methods, the internal water molecules were treated explicitly, and allowed to relax in the field of the buried charged group, the pK(a) values of buried residues were reproduced with epsilon(in) in the range 4-5. The calculations show that internal waters can modulate pK(a) values of buried residues effectively, and they support the hypothesis that the buried Lys-66 is in contact with internal waters even though these are not seen crystallographically. When only the one or two innermost water molecules were treated explicitly, epsilon(in) of 5-7 reproduced the pK(a) values. These values of epsilon(in) > 4 imply that some conformational reorganization occurs concomitant with the ionization of the buried groups.

Full Text

The Full Text of this article is available as a PDF (279.6 KB).

Selected References

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

  1. Alexandrescu A. T., Mills D. A., Ulrich E. L., Chinami M., Markley J. L. NMR assignments of the four histidines of staphylococcal nuclease in native and denatured states. Biochemistry. 1988 Mar 22;27(6):2158–2165. doi: 10.1021/bi00406a051. [DOI] [PubMed] [Google Scholar]
  2. Alexandrescu A. T., Ulrich E. L., Markley J. L. Hydrogen-1 NMR evidence for three interconverting forms of staphylococcal nuclease: effects of mutations and solution conditions on their distribution. Biochemistry. 1989 Jan 10;28(1):204–211. doi: 10.1021/bi00427a028. [DOI] [PubMed] [Google Scholar]
  3. Alexov E. G., Gunner M. R. Calculated protein and proton motions coupled to electron transfer: electron transfer from QA- to QB in bacterial photosynthetic reaction centers. Biochemistry. 1999 Jun 29;38(26):8253–8270. doi: 10.1021/bi982700a. [DOI] [PubMed] [Google Scholar]
  4. Alexov E. G., Gunner M. R. Incorporating protein conformational flexibility into the calculation of pH-dependent protein properties. Biophys J. 1997 May;72(5):2075–2093. doi: 10.1016/S0006-3495(97)78851-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Antosiewicz J., McCammon J. A., Gilson M. K. Prediction of pH-dependent properties of proteins. J Mol Biol. 1994 May 6;238(3):415–436. doi: 10.1006/jmbi.1994.1301. [DOI] [PubMed] [Google Scholar]
  6. Antosiewicz J., McCammon J. A., Gilson M. K. The determinants of pKas in proteins. Biochemistry. 1996 Jun 18;35(24):7819–7833. doi: 10.1021/bi9601565. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. Bhattacharya S., Lecomte J. T. Temperature dependence of histidine ionization constants in myoglobin. Biophys J. 1997 Dec;73(6):3241–3256. doi: 10.1016/S0006-3495(97)78349-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cocco M. J., Kao Y. H., Phillips A. T., Lecomte J. T. Structural comparison of apomyoglobin and metaquomyoglobin: pH titration of histidines by NMR spectroscopy. Biochemistry. 1992 Jul 21;31(28):6481–6491. doi: 10.1021/bi00143a018. [DOI] [PubMed] [Google Scholar]
  10. Collins K. D. Charge density-dependent strength of hydration and biological structure. Biophys J. 1997 Jan;72(1):65–76. doi: 10.1016/S0006-3495(97)78647-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Del Buono G. S., Figueirido F. E., Levy R. M. Intrinsic pKas of ionizable residues in proteins: an explicit solvent calculation for lysozyme. Proteins. 1994 Sep;20(1):85–97. doi: 10.1002/prot.340200109. [DOI] [PubMed] [Google Scholar]
  12. Denisov V. P., Halle B. Protein hydration dynamics in aqueous solution. Faraday Discuss. 1996;(103):227–244. doi: 10.1039/fd9960300227. [DOI] [PubMed] [Google Scholar]
  13. Dwyer J. J., Gittis A. G., Karp D. A., Lattman E. E., Spencer D. S., Stites W. E., García-Moreno E B. High apparent dielectric constants in the interior of a protein reflect water penetration. Biophys J. 2000 Sep;79(3):1610–1620. doi: 10.1016/S0006-3495(00)76411-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Evans P. A., Kautz R. A., Fox R. O., Dobson C. M. A magnetization-transfer nuclear magnetic resonance study of the folding of staphylococcal nuclease. Biochemistry. 1989 Jan 10;28(1):362–370. doi: 10.1021/bi00427a050. [DOI] [PubMed] [Google Scholar]
  15. Fox R. O., Evans P. A., Dobson C. M. Multiple conformations of a protein demonstrated by magnetization transfer NMR spectroscopy. Nature. 1986 Mar 13;320(6058):192–194. doi: 10.1038/320192a0. [DOI] [PubMed] [Google Scholar]
  16. García-Moreno B., Dwyer J. J., Gittis A. G., Lattman E. E., Spencer D. S., Stites W. E. Experimental measurement of the effective dielectric in the hydrophobic core of a protein. Biophys Chem. 1997 Feb 28;64(1-3):211–224. doi: 10.1016/s0301-4622(96)02238-7. [DOI] [PubMed] [Google Scholar]
  17. García A. E., Hummer G. Water penetration and escape in proteins. Proteins. 2000 Feb 15;38(3):261–272. [PubMed] [Google Scholar]
  18. Gibas C. J., Subramaniam S. Explicit solvent models in protein pKa calculations. Biophys J. 1996 Jul;71(1):138–147. doi: 10.1016/S0006-3495(96)79209-3. [DOI] [PMC free article] [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. Gunner M. R., Alexov E. A pragmatic approach to structure based calculation of coupled proton and electron transfer in proteins. Biochim Biophys Acta. 2000 May 12;1458(1):63–87. doi: 10.1016/s0005-2728(00)00060-8. [DOI] [PubMed] [Google Scholar]
  21. Harvey S. C., Hoekstra P. Dielectric relaxation spectra of water adsorbed on lysozyme. J Phys Chem. 1972 Oct 12;76(21):2987–2994. doi: 10.1021/j100665a011. [DOI] [PubMed] [Google Scholar]
  22. Havranek J. J., Harbury P. B. Tanford-Kirkwood electrostatics for protein modeling. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):11145–11150. doi: 10.1073/pnas.96.20.11145. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Hynes T. R., Fox R. O. The crystal structure of staphylococcal nuclease refined at 1.7 A resolution. Proteins. 1991;10(2):92–105. doi: 10.1002/prot.340100203. [DOI] [PubMed] [Google Scholar]
  24. Kao Y. H., Fitch C. A., Bhattacharya S., Sarkisian C. J., Lecomte J. T., García-Moreno E B. Salt effects on ionization equilibria of histidines in myoglobin. Biophys J. 2000 Sep;79(3):1637–1654. doi: 10.1016/S0006-3495(00)76414-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. 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]
  26. Krishtalik L. I., Kuznetsov A. M., Mertz E. L. Electrostatics of proteins: description in terms of two dielectric constants simultaneously. Proteins. 1997 Jun;28(2):174–182. [PubMed] [Google Scholar]
  27. Langen R., Jensen G. M., Jacob U., Stephens P. J., Warshel A. Protein control of iron-sulfur cluster redox potentials. J Biol Chem. 1992 Dec 25;267(36):25625–25627. [PubMed] [Google Scholar]
  28. 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]
  29. Lecomte J. T., Cocco M. J. Structural features of the protoporphyrin-apomyoglobin complex: a proton NMR spectroscopy study. Biochemistry. 1990 Dec 18;29(50):11057–11067. doi: 10.1021/bi00502a007. [DOI] [PubMed] [Google Scholar]
  30. Loll P. J., Lattman E. E. The crystal structure of the ternary complex of staphylococcal nuclease, Ca2+, and the inhibitor pdTp, refined at 1.65 A. Proteins. 1989;5(3):183–201. doi: 10.1002/prot.340050302. [DOI] [PubMed] [Google Scholar]
  31. Matthew J. B., Gurd F. R., Garcia-Moreno B., Flanagan M. A., March K. L., Shire S. J. pH-dependent processes in proteins. CRC Crit Rev Biochem. 1985;18(2):91–197. doi: 10.3109/10409238509085133. [DOI] [PubMed] [Google Scholar]
  32. Mehler E. L., Guarnieri F. A self-consistent, microenvironment modulated screened coulomb potential approximation to calculate pH-dependent electrostatic effects in proteins. Biophys J. 1999 Jul;77(1):3–22. doi: 10.1016/S0006-3495(99)76868-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rabenstein B., Knapp E. W. Calculated pH-dependent population and protonation of carbon-monoxy-myoglobin conformers. Biophys J. 2001 Mar;80(3):1141–1150. doi: 10.1016/S0006-3495(01)76091-2. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rupley J. A., Careri G. Protein hydration and function. Adv Protein Chem. 1991;41:37–172. doi: 10.1016/s0065-3233(08)60197-7. [DOI] [PubMed] [Google Scholar]
  35. Scharnagl C., Raupp-Kossmann R., Fischer S. F. Molecular basis for pH sensitivity and proton transfer in green fluorescent protein: protonation and conformational substates from electrostatic calculations. Biophys J. 1999 Oct;77(4):1839–1857. doi: 10.1016/S0006-3495(99)77028-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schutz C. N., Warshel A. What are the dielectric "constants" of proteins and how to validate electrostatic models? Proteins. 2001 Sep 1;44(4):400–417. doi: 10.1002/prot.1106. [DOI] [PubMed] [Google Scholar]
  37. Sham Y. Y., Muegge I., Warshel A. The effect of protein relaxation on charge-charge interactions and dielectric constants of proteins. Biophys J. 1998 Apr;74(4):1744–1753. doi: 10.1016/S0006-3495(98)77885-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sharp K. A. Calculation of electron transfer reorganization energies using the finite difference Poisson-Boltzmann model. Biophys J. 1998 Mar;74(3):1241–1250. doi: 10.1016/S0006-3495(98)77838-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Shortle D., Meeker A. K. Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation. Proteins. 1986 Sep;1(1):81–89. doi: 10.1002/prot.340010113. [DOI] [PubMed] [Google Scholar]
  40. Simonson T., Perahia D. Polar fluctuations in proteins: molecular-dynamic studies of cytochrome c in aqueous solution. Faraday Discuss. 1996;(103):71–90. doi: 10.1039/fd9960300071. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Ullmann G. M., Knapp E. W. Electrostatic models for computing protonation and redox equilibria in proteins. Eur Biophys J. 1999;28(7):533–551. doi: 10.1007/s002490050236. [DOI] [PubMed] [Google Scholar]
  43. Warshel A. Calculations of enzymatic reactions: calculations of pKa, proton transfer reactions, and general acid catalysis reactions in enzymes. Biochemistry. 1981 May 26;20(11):3167–3177. doi: 10.1021/bi00514a028. [DOI] [PubMed] [Google Scholar]
  44. Warshel A., Naray-Szabo G., Sussman F., Hwang J. K. How do serine proteases really work? Biochemistry. 1989 May 2;28(9):3629–3637. doi: 10.1021/bi00435a001. [DOI] [PubMed] [Google Scholar]
  45. Warshel A., Papazyan A. Electrostatic effects in macromolecules: fundamental concepts and practical modeling. Curr Opin Struct Biol. 1998 Apr;8(2):211–217. doi: 10.1016/s0959-440x(98)80041-9. [DOI] [PubMed] [Google Scholar]
  46. Warshel A., Russell S. T. Calculations of electrostatic interactions in biological systems and in solutions. Q Rev Biophys. 1984 Aug;17(3):283–422. doi: 10.1017/s0033583500005333. [DOI] [PubMed] [Google Scholar]
  47. Warshel A. What about protein polarity? Nature. 1987 Nov 5;330(6143):15–16. doi: 10.1038/330015a0. [DOI] [PubMed] [Google Scholar]
  48. Warwicker J., Watson H. C. Calculation of the electric potential in the active site cleft due to alpha-helix dipoles. J Mol Biol. 1982 Jun 5;157(4):671–679. doi: 10.1016/0022-2836(82)90505-8. [DOI] [PubMed] [Google Scholar]
  49. Whitten S. T., García-Moreno E B. pH dependence of stability of staphylococcal nuclease: evidence of substantial electrostatic interactions in the denatured state. Biochemistry. 2000 Nov 21;39(46):14292–14304. doi: 10.1021/bi001015c. [DOI] [PubMed] [Google Scholar]
  50. Wishart D. S., Bigam C. G., Yao J., Abildgaard F., Dyson H. J., Oldfield E., Markley J. L., Sykes B. D. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J Biomol NMR. 1995 Sep;6(2):135–140. doi: 10.1007/BF00211777. [DOI] [PubMed] [Google Scholar]
  51. 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]
  52. You T. J., Bashford D. Conformation and hydrogen ion titration of proteins: a continuum electrostatic model with conformational flexibility. Biophys J. 1995 Nov;69(5):1721–1733. doi: 10.1016/S0006-3495(95)80042-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Zhou H. X., Vijayakumar M. Modeling of protein conformational fluctuations in pKa predictions. J Mol Biol. 1997 Apr 11;267(4):1002–1011. doi: 10.1006/jmbi.1997.0895. [DOI] [PubMed] [Google Scholar]
  54. van Vlijmen H. W., Schaefer M., Karplus M. Improving the accuracy of protein pKa calculations: conformational averaging versus the average structure. Proteins. 1998 Nov 1;33(2):145–158. doi: 10.1002/(sici)1097-0134(19981101)33:2<145::aid-prot1>3.0.co;2-i. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES