OFF-PATHWAY MISFOLDED FOLDING INTERMEDIATES OF CHEY AND VARIANTS

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来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 这一建议是确定氨基酸与蛋白质相互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 二级和三级结构。 黄氧还蛋白折叠的机制 蛋白质已经被几个小组充分研究。 他们的一个有趣的特点是， 折叠能量景观是一个偏离途径的折叠中间体已被观察到， 所有黄素氧还蛋白折叠蛋白动力学重折叠实验，包括CheY。 在 最近的一份出版物，我们已经证明了实验和计算 该非途径物质是一种专性中间体，其形成于 停流仪表 该中间体具有相当大的二级结构， 稳定性，并保持填充约100 s。来自我们合作者的Go建模结果 和氢交换质谱数据（未发表的结果）表明，这种能量挫折的来源是蛋白质的N-和C-末端半部分之间（a4和a3之间以及b4和b3之间）的接触的早期形成，这导致非生产性能量陷阱，为了形成生产性过渡态中间体，必须解除该陷阱。 我们的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，并探测其对残基的二级结构和包装的影响。 的 平衡和时间分辨的小角X射线散射将补充这些研究，监测产生的 专性途径外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remains populated for ~100 s. Go-modeling results from our collaborators and hydrogen-exchange mass spectrometry data (unpublished results) suggest that the source of this energetic frustration is the early formation of contacts between the N- and C- terminal halves of the protein (between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.