Folding and Stability of TIM Barrel Proteins

项目概要 从分子水平理解蛋白质氨基酸序列的机制 将其快速有效的折叠引导至其天然的功能性构象仍然是杰出的结构之一 分子生物物理学的挑战。尽管计算机模拟成功折叠了小蛋白质 结构域，氨基酸序列在定义物种结构和稳定性中的精确作用 填充折叠自由能表面的物质仍然难以捉摸。我们假设分支簇 脂肪族侧链、异亮氨酸、亮氨酸和缬氨酸 (ILV) 是折叠中间体稳定性的核心 以及 TIM 桶蛋白的天然状态，这是生物学中最常见的基序之一。我们提出了一个多 对三种吲哚-3-甘油磷酸合酶 (IGPS) 直向同源物进行了这一假设的多面检验，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 将探测填充折叠自由能表面的部分折叠态的结构并测试它们 与这些饱和烃簇的关系。 CD、FRET 和 SAXS 技术将评估二级 结构并提供从微秒时间范围开始的成对和全局尺寸信息， 氢交换质谱和核磁共振方法将绘制稳定的氢键网络 出现在毫秒到秒时间范围内的部分折叠状态，以及这些状态中的侧链埋藏 将使用新型氧化标记方法对同一物种进行评估。结果将用于验证 使用以原生为中心的 GM 模型模拟来预测部分折叠状态的结构，该模型能够 定义这些直系同源物的整个折叠反应坐标。在一项令人兴奋的新事业中，我们将评估 一组详尽的氨基酸替换对所有 8 个 ¿ 链和前面的 ¿/¿ 环稳定性的影响 在缺乏其直系同源物的酵母菌株的生长竞争测定中，元素对每个直向同源物的相对适合度的影响 内在的 IGPS 基因。健身提供体内稳定性估计的假设将通过以下方法进行验证 对天然状态和中间状态稳定性扰动的体外定量评估 SsIGPS 直向同源物中相同稳定性元件中约 100 个定点突变的子集。并行 CD 和 酶活性测定将测量突变对结构和功能的影响 酶作为健康下降的另一种解释。这些体内和体外测量的输出 稳定性扰动将作为生物信息学分析的输入，旨在提供统计- 对突变的背景依赖性进行重要评估。突变影响的比较 ILV 集群内部和外部将提供公正且稳健的方法来确定其 在定义球状蛋白稳定性核心方面具有重要意义。 验证了我们的假设，即支化脂肪族侧链簇在 稳定部分折叠状态并引导 TIM 桶蛋白折叠，有可能具有非常大的潜力 对生物学、生物技术和医学产生广泛影响。

Project Summary 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 folding of TIM barrel proteins, has the potential to have a very broad impact on biology, biotechnology and medicine.