TIME-RESOLVED SAXS/WAXS OF PHOTOACTIVE YELLOW PROTEIN (PYP)

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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A well-developed light-responsive system, photoactive yellow protein (PYP), contains p- coumaric acid as the chromophore, which is covalently linked to the Cys69 residue by a thioester bond (1) and has a maximum absorbance at 446 nm (2). The chromophore interacts with nearby residues through hydrogen bonds and hydrophobic interactions, and this hydrogen bond networking acts as the glue which makes holo-PYP (PYP with the chromophore) more compact and stable than apo-PYP (PYP without the chromophore). Therefore removal of the chromophore from holo-PYP induces unfolding process into apo-PYP and provides a unique opportunity to study protein unfolding process. Due to the covalent linking to PYP, the chromophore is not removed by denaturing or boiling, but can be irreversibly detached by reacting with hydroxylamine, which specifically reacts with the chromophore covalent bond The reaction of hydroxylamine with holo-PYP to produce apo-PYP occurs with a slow rate constant of a few hours. X-ray solution scattering is useful for studying protein unfolding process in solution phase because it provides information concerning the approximate size, shape, and conformational changes of protein. A combination of both small-angle (SAXS) and wide-angle X-ray solution scattering (WAXS) will be used to study overall changes of shape and size by SAXS and conformational changes of protein by WAXS during the unfolding process. We will also investigate the effects of concentration on SAXS and WAXS patterns for both holo-PYP and apo-PYP to check whether the degree of crowding depends on the presence of the chromophore (4, 5).