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

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得主要资金， 因此可以在其他CRISP条目中表示。列出的机构是 中心，不一定是研究者的机构。 一种开发良好的光响应系统，光活性黄蛋白（PYP），含有对香豆酸作为发色团，其通过硫酯键与Cys 69残基共价连接（1），并在446 nm处具有最大吸光度（2）。发色团通过氢键和疏水相互作用与附近的残基相互作用，并且这种氢键网络作为胶水，使得holo-PYP（具有发色团的PYP）比apo-PYP（不具有发色团的PYP）更紧凑和稳定。因此，从holo-PYP中去除发色团可诱导去折叠过程形成apo-PYP，并为研究蛋白质去折叠过程提供了独特的机会。由于与PYP的共价连接，发色团不会通过变性或煮沸而被去除，但可以通过与羟胺反应而不可逆地分离，羟胺特异性地与发色团共价键反应。羟胺与holo-PYP反应产生apo-PYP的反应以几小时的缓慢速率常数发生。X-射线溶液散射是研究蛋白质在溶液相中去折叠过程的有效手段，因为它可以提供蛋白质的近似大小、形状和构象变化的信息。小角X射线散射（SAXS）和广角X射线溶液散射（WAXS）的组合将被用来研究在去折叠过程中的形状和大小的整体变化，通过SAXS和蛋白质的构象变化通过WAXS。我们还将研究浓度对holo-PYP和apo-PYP的SAXS和WAXS模式的影响，以检查拥挤程度是否取决于发色团的存在（4，5）。

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).