Biophysical Study of X-ray Scattering in Biological Samples

Abstract

Wide angle x-ray scattering (WAXS) from protein m solution and dry (lyophilized) proteins is characterized by the presence of two weak broad scattering peaks. The first peak corresponds to a d-spacing around I 0 A and is attributed to inter-helix packing. The second peak is at about 4.5 A and is

attributed to a-helix backbone. For a protein with P-sheets as the main secondary structure, these peaks correspond to inter-sheet packing and hydrogen bonding distance between P-strands, respectively. Since the scattering from water dominates over the scattering from protein in solution, one have to use a highly intense x-ray beam (probably from synchrotron) in order to be able to observe the scattering from protein. Otherwise, it would still be possible to unveil the protein scattering peaks through the lyophilization of protein solution into a powder form and obtaining the WAXS profile using a conventional x-ray diffractometer. The latter method is adopted in this study. Since it has been reported that wide-angle x-ray scattering would provide a means to identify induced changes in the secondary, tertiary and quaternary structure of protein, this work aims to evaluate the potential of WAXS as a probe of induced conformational changes in insulin. For such purpose, native (control) insulin is forced to unfold and breakdown either using thermal denaturation alone or in the presence of thiol (cysteine) catalysts via disulfide scrambling. Denatured products are acid-trapped and monitored using WAXS in addition to FTIR, gel