WAXS AS A PROBE FOR THE STUDY OF PROTEIN STRUCTURE, DYNAMICS AND FUNCTION

Illinois Institute Of Technology, Chicago IL

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Wide-angle x-ray solution scattering (WAXS) has significant potential for characterizing the structure, dynamics, and function of proteins without requiring crystallization (Fischetti et al., 2003). To assess the relationships among the structures of hemoglobins in different liganded forms in solution, WAXS patterns were collected from solutions of human normal adult carbonmonoxy-Hb A (HbCO A), the carbonmonoxy-form in the presence of 3 mM inositol hexaphosphate (IHP), di-[unreadable]-rHbCO (a variant in which the two [unreadable]-chains are covalently linked by a single glycine residue), a recombinant rHbCO ([unreadable]V96W-[unreadable]N108K), and bovine Hb in the met- and deoxy- forms. Comparisons were made between the observed patterns and those predicted on the basis of atomic coordinate sets. We conclude that, in solution, the structures of Hb in different liganded forms exhibit clear differences from known crystal structures: (i) The structural difference between deoxy-Hb and HbCO A in solution is larger than predicted from the crystal structures. (ii) The structure of met-Hb in solution corresponds more closely to that of deoxy-Hb than HbCO. (iii) The structure of di-[unreadable]-rHbCO is very similar to that of HbCO A. (iv) IHP binds to two sites located ~43 [unreadable] apart on the surface of HbCO A. Based on the responsiveness of their WAXS patterns to changes in protein concentration: (i) Deoxy-Hb exhibits much larger structural fluctuations than HbCO A or met-Hb. (ii) IHP induces a modest increase in the magnitude of structural fluctuations in HbCO A. (iii) di-[unreadable]-rHbCO exhibits no observable structural fluctuations in solution. (iv) rHbCO ([unreadable]V96W/[unreadable]N108K) exhibits fluctuations of an amplitude indistinguishable from those of HbCO A.