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
. 2000 May;9(5):878–885. doi: 10.1110/ps.9.5.878

Ser45 plays an important role in managing both the equilibrium and transition state energetics of the streptavidin-biotin system.

D E Hyre 1, I Le Trong 1, S Freitag 1, R E Stenkamp 1, P S Stayton 1
PMCID: PMC2144626  PMID: 10850797

Abstract

The contribution of the Ser45 hydrogen bond to biotin binding activation and equilibrium thermodynamics was investigated by biophysical and X-ray crystallographic studies. The S45A mutant exhibits a 1,700-fold greater dissociation rate and 907-fold lower equilibrium affinity for biotin relative to wild-type streptavidin at 37 degrees C, indicating a crucial role in binding energetics. The crystal structure of the biotin-bound mutant reveals only small changes from the wild-type bound structure, and the remaining hydrogen bonds to biotin retain approximately the same lengths. No additional water molecules are observed to replace the missing hydroxyl, in contrast to the previously studied D128A mutant. The equilibrium deltaG degrees, deltaH degrees, deltaS degrees, deltaC degrees(p), and activation deltaG++ of S45A at 37 degrees C are 13.7+/-0.1 kcal/mol, -21.1+/-0.5 kcal/mol, -23.7+/-1.8 cal/mol K, -223+/-12 cal/mol K, and 20.0+/-2.5 kcal/mol, respectively. Eyring analysis of the large temperature dependence of the S45A off-rate resolves the deltaH++ and deltaS++ of dissociation, 25.8+/-1.2 kcal/mol and 18.7+/-4.3 cal/mol K. The large increases of deltaH++ and deltaS++ in the mutant, relative to wild-type, indicate that Ser45 could form a hydrogen bond with biotin in the wild-type dissociation transition state, enthalpically stabilizing it, and constraining the transition state entropically. The postulated existence of a Ser45-mediated hydrogen bond in the wild-type streptavidin transition state is consistent with potential of mean force simulations of the dissociation pathway and with molecular dynamics simulations of biotin pullout, where Ser45 is seen to form a hydrogen bond with the ureido oxygen as biotin slips past this residue after breaking the native hydrogen bonds.

Full Text

The Full Text of this article is available as a PDF (379.0 KB).

Selected References

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

  1. Chilkoti A., Tan P. H., Stayton P. S. Site-directed mutagenesis studies of the high-affinity streptavidin-biotin complex: contributions of tryptophan residues 79, 108, and 120. Proc Natl Acad Sci U S A. 1995 Feb 28;92(5):1754–1758. doi: 10.1073/pnas.92.5.1754. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chu V., Freitag S., Le Trong I., Stenkamp R. E., Stayton P. S. Thermodynamic and structural consequences of flexible loop deletion by circular permutation in the streptavidin-biotin system. Protein Sci. 1998 Apr;7(4):848–859. doi: 10.1002/pro.5560070403. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Dixon R. W., Kollman P. The free energies for mutating S27 and W79 to alanine in streptavidin and its biotin complex: the relative size of polar and nonpolar free energies on biotin binding. Proteins. 1999 Sep 1;36(4):471–473. [PubMed] [Google Scholar]
  4. Fersht A. R., Shi J. P., Knill-Jones J., Lowe D. M., Wilkinson A. J., Blow D. M., Brick P., Carter P., Waye M. M., Winter G. Hydrogen bonding and biological specificity analysed by protein engineering. Nature. 1985 Mar 21;314(6008):235–238. doi: 10.1038/314235a0. [DOI] [PubMed] [Google Scholar]
  5. Freitag S., Chu V., Penzotti J. E., Klumb L. A., To R., Hyre D., Le Trong I., Lybrand T. P., Stenkamp R. E., Stayton P. S. A structural snapshot of an intermediate on the streptavidin-biotin dissociation pathway. Proc Natl Acad Sci U S A. 1999 Jul 20;96(15):8384–8389. doi: 10.1073/pnas.96.15.8384. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Freitag S., Le Trong I., Chilkoti A., Klumb L. A., Stayton P. S., Stenkamp R. E. Structural studies of binding site tryptophan mutants in the high-affinity streptavidin-biotin complex. J Mol Biol. 1998 May 29;279(1):211–221. doi: 10.1006/jmbi.1998.1735. [DOI] [PubMed] [Google Scholar]
  7. Freitag S., Le Trong I., Klumb L., Stayton P. S., Stenkamp R. E. Structural studies of the streptavidin binding loop. Protein Sci. 1997 Jun;6(6):1157–1166. doi: 10.1002/pro.5560060604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Green N. M. Avidin and streptavidin. Methods Enzymol. 1990;184:51–67. doi: 10.1016/0076-6879(90)84259-j. [DOI] [PubMed] [Google Scholar]
  9. Hemming S. A., Bochkarev A., Darst S. A., Kornberg R. D., Ala P., Yang D. S., Edwards A. M. The mechanism of protein crystal growth from lipid layers. J Mol Biol. 1995 Feb 17;246(2):308–316. doi: 10.1006/jmbi.1994.0086. [DOI] [PubMed] [Google Scholar]
  10. Hendrickson W. A., Pähler A., Smith J. L., Satow Y., Merritt E. A., Phizackerley R. P. Crystal structure of core streptavidin determined from multiwavelength anomalous diffraction of synchrotron radiation. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2190–2194. doi: 10.1073/pnas.86.7.2190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Klumb L. A., Chu V., Stayton P. S. Energetic roles of hydrogen bonds at the ureido oxygen binding pocket in the streptavidin-biotin complex. Biochemistry. 1998 May 26;37(21):7657–7663. doi: 10.1021/bi9803123. [DOI] [PubMed] [Google Scholar]
  12. Moews P. C., Kretsinger R. H. Refinement of the structure of carp muscle calcium-binding parvalbumin by model building and difference Fourier analysis. J Mol Biol. 1975 Jan 15;91(2):201–225. doi: 10.1016/0022-2836(75)90160-6. [DOI] [PubMed] [Google Scholar]
  13. Pähler A., Hendrickson W. A., Kolks M. A., Argaraña C. E., Cantor C. R. Characterization and crystallization of core streptavidin. J Biol Chem. 1987 Oct 15;262(29):13933–13937. [PubMed] [Google Scholar]
  14. Reznik G. O., Vajda S., Sano T., Cantor C. R. A streptavidin mutant with altered ligand-binding specificity. Proc Natl Acad Sci U S A. 1998 Nov 10;95(23):13525–13530. doi: 10.1073/pnas.95.23.13525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Richardson D. C., Richardson J. S. The kinemage: a tool for scientific communication. Protein Sci. 1992 Jan;1(1):3–9. doi: 10.1002/pro.5560010102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Sano T., Cantor C. R. Intersubunit contacts made by tryptophan 120 with biotin are essential for both strong biotin binding and biotin-induced tighter subunit association of streptavidin. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3180–3184. doi: 10.1073/pnas.92.8.3180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Weber P. C., Ohlendorf D. H., Wendoloski J. J., Salemme F. R. Structural origins of high-affinity biotin binding to streptavidin. Science. 1989 Jan 6;243(4887):85–88. doi: 10.1126/science.2911722. [DOI] [PubMed] [Google Scholar]

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

RESOURCES