OFF-PATHWAY MISFOLDED FOLDING INTERMEDIATES OF CHEY AND VARIANTS

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。 这项提议是确定氨基酸的作用机制的总体目标的一部分。 α/β/α类蛋白质的序列和基序指导它们获得 折叠过程中的二级和三级结构。黄还蛋白折叠的折叠机制 蛋白质已经被几个小组很好地研究过。他们的一个有趣的特点是 折叠能量图景是已观察到的偏离路径的折叠中间体 包括Chey在内的所有黄毒素折叠蛋白的动力学复性实验。在……里面 我们通过实验和计算证明了最近的一份出版物 旁路物种是一种在死亡时间内形成的专有中间体 停流仪表。中间体具有相当大的二级结构和 稳定，并保持人口约100 S。GO建模结果来自我们的合作者 氢交换质谱仪数据(未发表的结果)表明，这种能量受挫的来源是蛋白质的N端和C端之间(a4和a3之间以及b4和b3之间)的接触的早期形成，这导致了一个非生产性的能量陷阱，必须解除该陷阱才能形成生产性的过渡态中间体。 我们实验室正在进行突变研究，以取代a4和b4中的疏水残基。 区域，并探讨它们对残留物二级结构和堆积的影响。这个 平衡和时间分辨SAXS将通过监测由此产生的 专有的旁路中间体的大小和形状的变化。

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. This proposal is part of an overall goal of determining the mechanism by which the amino acid sequences and motifs of the alpha/beta/alpha class of proteins direct their acquisition of secondary and tertiary structure during folding. The folding mechanism of flavodoxin fold proteins has been well studied by several groups. An interesting feature of their folding energy landscape is that an off-pathway folding intermediate has been observed in kinetic refolding experiments for all flavodoxin fold proteins, including CheY. In a recent publication we have demonstrated experimentally and computationally that the off-pathway species is an obligate intermediate that forms within the dead time of stopped-flow instrumentation. The intermediate has considerable secondary structure and stability and remains populated for ~100 s. Go-modeling results from our collaborators and hydrogen-exchange mass spectrometry data (unpublished results) suggest that the source of this energetic frustration is the early formation of contacts between the N- and C- terminal halves of the protein (between a4 and a3 and between b4 and b3) which result in a non-productive energetic trap that must be undone in order for the productive transition state intermediate to form. Mutagenesis studies are underway in our lab to replace hydrophobic residues in the a4 and b4 regions and probe their effect on the secondary structure and packing of residues. The equilibrium and time-resolved SAXS will complement these studies by monitoring the resulting changes in the size and shape of the obligate off-pathway intermediate.