COMPARISON OF STREPTAVIDIN WxF AND WxA MUTANTS WITH THE NATIVE PROTEIN. Stefanie Freitag^a, Isolde Le Trong^a, Ronald E. Stenkamp^a, Ashutosh Chilkoti^b, Patrick S. Stayton^b, Dept. of Biological Structure^a and Center of Bioengineering^b, University of Washington, Seattle, WA 98195

The high affinity binding of biotin to streptavidin (K_a ~ 10^13-15 M^-1) is hypothesized to be highly dependent on three different binding motifs [1]. These three classes of interactions include four tryptophan side-chains that mediate aromatic contacts to biotin, an extensive hydrogen-binding network, and a flexible binding loop that becomes ordered upon biotin association. Single site-directed mutants replacing Trp residues with Phe and Ala at positions 79, 108 and 120 have been prepared, and their binding properties studied by ELISA experiments, isothermal titrating calorimetry, and kinetic analysis [2].

Four of the mutants and the native protein crystallize in the monoclinic space group P2₁. Xray crystallographic analyses of the unliganded mutants as well as their complexes formed with biotin and HABA have been carried out. Diffraction data were collected to high resolution limits between 1.7 and 2.0 Å. Refinement of the structural models were performed with the programs XPLOR and SHELXL-93. Our current structural models showing the interactions giving rise to biotin binding will be presented. (This work is supported by NIH grant DK49655.)

[1] a) Hendrickson et al., Proc. Natl. Acad. Sci. USA 86, 1989, 2190 - 2194;

b) Weber et al., Science 243, 1989, 85 - 88.

[2] A. Chilkoti and P. S. Stayton, JACS 117, 1995, 10622 - 10628.