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Cooperative hydrogen bond interactions in the streptavidin–biotin system

¹ Department of Bioengineering and ² Department of Biological Structure and the Biomolecular Structure Center, University of Washington, Seattle, Washington 98195, USA
³ Department of Physical Biochemistry and ⁴ Department of Mathematical Biology, National Institutes for Medical Research, Mill Hill, London NW7 1AA, United Kingdom

(RECEIVED November 10, 2005; FINAL REVISION November 22, 2005; ACCEPTED December 1, 2005)

The thermodynamic and structural cooperativity between the Ser45– and D128–biotin hydrogen bonds was measured by calorimetric and X-ray crystallographic studies of the S45A/D128A double mutant of streptavidin. The double mutant exhibits a binding affinity ~2 x 10⁷ times lower than that of wild-type streptavidin at 25°C. The corresponding reduction in binding free energy (G) of 10.1 kcal/mol was nearly completely due to binding enthalpy losses at this temperature. The loss of binding affinity is 11-fold greater than that predicted by a linear combination of the single-mutant energetic perturbations (8.7 kcal/mol), indicating that these two mutations interact cooperatively. Crystallographic characterization of the double mutant and comparison with the two single mutant structures suggest that structural rearrangements at the S45 position, when the D128 carboxylate is removed, mask the true energetic contribution of the D128–biotin interaction. Taken together, the thermodynamic and structural analyses support the conclusion that the wild-type hydrogen bond between D128–OD and biotin–N2 is thermodynamically stronger than that between S45–OG and biotin–N1.

Keywords: molecular recognition; cooperativity; hydrogen bond; streptavidin; X-ray crystallography; structure; thermodynamics; hydrodynamics; calorimetry

Abbreviations: ITC, isothermal calorimetry • MD, molecular dynamics • SPR, surface plasmon resonance • StAv, streptavidin • 45/128 S45A/D128A, double mutant of streptavidin

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051970306.

Reprint requests to: Patrick S. Stayton, Box 351721, Department of Bioengineering, University of Washington, Seattle, WA 98195-1721, USA; e-mail: stayton{at}u.washington.edu; fax: (206) 685-8256; or Ronald E. Stenkamp, Box 357420, Department of Biological Structure, University of Washington, Seattle, WA 98195-7420, USA; e-mail: stenkamp{at}u.washington.edu; fax: (206) 543-1524.

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