Folding and Stability of TIM Barrel Proteins

描述（由申请人提供）：对蛋白质的氨基酸序列如何指导其快速有效折叠到其天然功能构象的分子水平机制的理解仍然是分子生物物理学中的突出挑战之一。尽管计算机模拟成功地折叠了小蛋白质和结构域，但氨基酸序列在定义折叠自由能表面上的物质的结构和稳定性方面的确切作用仍然难以捉摸。我们假设支链脂肪侧链簇异亮氨酸和缬氨酸（ILV）是TIM桶状蛋白折叠中间体和天然状态稳定性的核心，TIM桶状蛋白是生物学中最常见的基序之一。我们在三个吲哚-3-甘油磷酸合成酶（IGPS）同源物上提出了这一假设的多方面检验，其低序列同一性导致其驻留ILV簇的大小和位置不同。一系列技术将探测部分折叠态的结构，这些结构填充了折叠自由能表面，并测试它们的结构

DESCRIPTION (provided by applicant): A molecular-level understanding of the mechanism by which the amino acid sequence of a protein directs its rapid and efficient folding to its native, functional conformation remains as one of the outstanding challenges in molecular biophysics. Although computer simulations are successfully folding small proteins and domains, the precise role of the amino acid sequence in defining the structures and stabilities of the species that populate the folding free energy surface remains elusive. We hypothesize that clusters of branched aliphatic side chains, isoleucines, leucines and valines (ILV), serve as cores of stability in folding intermediates and the native states of TIM barrel proteins, one of the most common motifs in biology. We propose a multi-faceted test of this hypothesis on a trio of indole-3-glycerolphosphate synthase (IGPS) orthologs, whose low sequence identity results in varying sizes and locations of their resident ILV clusters. A battery of techniques will probe the structures of partially-folded states that populate the folding free energy surfaces and test their relationship with these saturated hydrocarbon clusters. CD, FRET and SAXS techniques will assess secondary structure and provide pair-wise and global dimensional information beginning in the microsecond time range, hydrogen-exchange mass spectrometry and NMR methods will map the stable hydrogen bonding networks in partially-folded states that appear in the milliseconds-to-seconds time frame, and side chain burial in these same species will be assessed with a novel oxidative labeling method. The results will be used to validate the predictions of structure in partially-folded states using native-centric GM-model simulations that are capable of defining the entire folding reaction coordinate of these orthologs. In an exciting new venture, we will assess the effects of an exhaustive set of amino acid replacements in all 8 ¿-strand and preceding ¿/¿ loop stability elements on the relative fitness of each ortholog in a growth competition assay in a yeast strain lacking its intrinsic IGPS gene. The presumption that fitness provides an in vivo estimate of stability will be validated with an in vitro quantitative assessment of the perturbation of the stability of native and intermediate states in a subset of ~100 site-directed mutations in the same stability elements in the SsIGPS ortholog. Parallel CD and enzymatic activity assays will measure the effects of the mutations on the structure and the function of the enzyme as an alternative explanation for decrease in fitness. The output of these in vivo and in vitro measures of stability perturbations will serve as input for a bioinformatics analysis designed to offer a statistically-significant assessment of the context dependence of the mutations. Comparisons of the effects of mutations within and external to ILV clusters will provide an unbiased and robust approach towards determining their significance in defining cores of stability in globular proteins. Validation of our hypothesis, that clusters of branched aliphatic side chains play crucial roles in stabilizing partially-folded states and guiding the foling of TIM barrel proteins, has the potential to have a very broad impact on biology, biotechnology and medicine.