Effects of interfacial binding kinetics on two-dimensional streptavidin crystallization

The two-dimensional crystallization of streptavidin at biotinylated interfa ces provides a model system for elucidating the role of interfacial binding dynamics in determining crystal morphologies and phases. In this study, a library of eight well-characterized site-directed mutants with increased bi otin dissociation rates has been compared to core streptavidin (dissociatio n half-life of 59 h). The W79F, W120F, Y43F, and S27A mutants, with half-li ves of 9.8 h, 38 min, 20 min, and 10 min, respectively, all displayed the " X"-shaped crystal morphology that is characteristic of core streptavidin. A sharp change in morphology is observed with the N23E (7.2 min) and N23A (4 .8 min) mutants. The N23E mutant crystallizes in rectangular shapes, and th e N23A displays square crystal morphology. The D128A mutant (1.7 min) cryst allizes in elongated needles, but this is the one mutant which displays sig nificant three-dimensional structural alterations. The W120A (<1 min) mutan t did not display significant interfacial binding and did not crystallize. Quantitative Brewster angle microscopy was used to characterize the crystal lization process. The noncrystalline background remained in equilibrium wit h the crystalline regions, and thus all the mutants crystallized as a first -order phase transition. The critical surface concentration for crystalliza tion remained constant until the half-life reached 10.4 min for S27A. The s quare-shaped N23A crystal displayed the lowest critical surface concentrati on, which was equivalent to the values observed previously for square-shape d crystals obtained via metal binding. Fourier analysis of transmission ele ctron microscopy images demonstrated that all of the mutants crystallized w ith the same C-222 space group characteristic of core streptavidin. The cha nge in crystal shape over a sharp range of dissociation rates is consistent with a change in a rate-limiting microscopic kinetic step that underlies m acroscopic morphology. Alternatively, the altered shapes observed with the Asn 23 mutants could be the result of changing structural and energetic cou pling between biotin binding and the directly adjacent Thr 20 and Tyr 22 re sidues which hydrogen bond to each other across the protein-protein crystal contacts.