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

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 光活性黄蛋白(PYP)是一种成熟的光响应系统，它以对香豆酸为发色团，通过硫代酯键(1)共价连接到Cys69残基上，在446 nm(2)处有最大吸收。生色团通过氢键和疏水相互作用与附近的残基相互作用，这种氢键网络起到了胶粘剂的作用，使得holo-PYP(有生色团的PYP)比apo-PYP(没有生色团的PYP)更紧密和稳定。因此，从全息PYP中去除发色团可以将蛋白质的去折叠过程诱导为apo-PYP，从而为研究蛋白质去折叠过程提供了一个独特的机会。由于与PYP的共价连接，生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，羟胺与全息PYP发生反应生成apo-PYP的反应以几个小时的慢速常数进行。X射线溶液散射可用于研究蛋白质在溶液相中的展开过程，因为它提供了有关蛋白质的大致大小、形状和构象变化的信息。采用小角X射线溶液散射(SAXS)和广角X射线溶液散射(WAXS)相结合的方法，研究了SAXS在蛋白质去折叠过程中形状和大小的整体变化以及WAXS对蛋白质构象的影响。我们还将研究浓度对全息PYP和APO-PYP的SAXS和WAXS图案的影响，以检查拥挤程度是否取决于生色团(4，5)的存在。

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).