COFACTOR ROLE IN BETA-SHEET PROTEIN FOLDING

描述：（改编自申请人摘要）辅因子可能很重要 决定簇，作为限制构象搜索的成核点， 辅因子结合蛋白的折叠速率。迄今为止，折叠动力学 具有b折叠结构的蛋白质的结构还没有像 螺旋蛋白质。本研究项目旨在探讨 两个无机和一个有机辅因子在三种蛋白质的折叠中， 主要是B片结构。靶蛋白是天青蛋白，一种b桶蛋白 与铜离子辅因子，黄素氧还蛋白，一种具有a/B双缠绕的蛋白质 拓扑结构协调有机黄素单胞苷肽（FMN），和细胞色素f， 一种与血红素共价连接的b片层蛋白。平衡生物物理学 表征（圆二色性、荧光、吸收、EXAFS、NMR和 各种生物化学方法）将有助于揭示每个辅因子的作用 其相应的蛋白质稳定性和未折叠的多肽结构。一 一种通过光化学引发折叠的新技术 电子转移将被用来探测形成过程中的快速事件。 蛋白质的自然状态。允许宽变性剂和时间范围 研究（并研究载脂蛋白），时间分辨实验将 也可以使用停流混合进行。具体目标是：1. 描述辅因子的协调和剩余结构， 在未折叠状态下的辅因子，2.研究铜FMN和血红素 （与未折叠的多肽结合）影响多肽折叠动力学 最后，3。探测早期事件（从美国时间尺度开始）， 天然天青蛋白、黄素氧还蛋白和细胞色素的形成。

DESCRIPTION: (Adapted from applicant's abstract) Cofactors may be important determinants, acting as nucleation points limiting the conformational search, for the folding rates of cofactor-binding proteins. To date, folding kinetics of proteins with b-sheet structure has not been as thoroughly investigated as for helical proteins. This research program aims towards probing the role of two inorganic and one organic cofactor in the folding of three proteins with mostly b-sheet structure. The targeted proteins are azurin, a b-barrel protein with a copper-ion cofactor, flavodoxin, a protein with an a/b doubly-wound topology coordinating an organic flavin mononucleotide (FMN), and cytochrome f, a b-sheet protein covalently linked to a heme. Equilibrium biophysical characterization (circular dichroism, fluorescence, absorption, EXAFS, NMR, and various biochemical methods) will aid in revealing the effect of each cofactor on its corresponding protein stability and unfolded polypeptide structure. A recent technique in which folding is initiated by photochemical electron-transfer will be used to probe rapid events during formation of the native-states of the proteins. To allow wide denaturant- and time- ranges to be investigated (and to study the apo proteins), time-resolved experiments will also be performed using stopped-flow mixing. The specific aims are to: 1. Characterize cofactor- coordination and residual structures created by the cofactors in the unfolded states, 2. Investigate how copper, FMN and heme (bound to the unfolded polypeptides) affect the polypeptide folding kinetics and, finally, 3. Probe early events (starting on the us time scale) during the formation -of native azurin, flavodoxin and cytochrome.