Folding and Stability of TIM Barrel Proteins

项目摘要 从分子水平理解蛋白质的氨基酸序列 将其快速有效地折叠到其天然的功能构象仍然是最突出的 分子生物物理学的挑战。尽管计算机模拟成功地折叠了小蛋白质， 结构域，氨基酸序列在定义物种的结构和稳定性中的精确作用 折叠自由能表面上的分子仍然难以捉摸。我们假设， 脂肪族侧链，异亮氨酸，亮氨酸和缬氨酸（ILV），作为折叠中间体的稳定核心 以及TIM桶蛋白的天然状态，TIM桶蛋白是生物学中最常见的基序之一。我们提出了一个多- 在三个吲哚-3-甘油磷酸合酶（IGPS）直系同源物上进行了这一假设的多面检验， 序列同一性导致其驻留ILV簇的大小和位置不同。一系列的技术 将探测填充折叠自由能表面的部分折叠状态的结构，并测试它们的 与这些饱和烃簇的关系。CD、FRET和SAXS技术将评估继发性 结构化并提供在微秒时间范围内开始的成对和全局维度信息， 氢交换质谱和核磁共振方法将绘制稳定的氢键网络， 部分折叠状态出现在毫秒到秒的时间范围内，侧链埋在这些 将用新的氧化标记方法评估相同的物种。结果将用于验证 使用以本地为中心的GM模型模拟预测部分折叠状态下的结构， 定义这些直向同源物的整个折叠反应坐标。在一个令人兴奋的新企业，我们将评估 在所有8 <$链和前<$/<$环稳定性中的一组详尽的氨基酸置换的影响 元素对缺乏其同源物的酵母菌株中的生长竞争测定中每个直系同源物的相对适合度的影响。 内源性IGPS基因。拟合度提供了体内稳定性估计值的假设将通过以下方法进行验证： 在体外定量评估扰动的天然和中间状态的稳定性， SsIGPS直系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蛋白质结构和功能的影响。 酶作为适应性下降的另一种解释。这些体内和体外测量的输出 将作为生物信息学分析的输入，旨在提供统计学- 对突变的上下文依赖性的显著评估。突变效应的比较 内部和外部的ILV集群将提供一个公正和强大的方法来确定他们的 在定义球状蛋白质的稳定核心方面具有重要意义。 验证了我们的假设，即支链脂肪族侧链簇在 稳定部分折叠状态并引导TIM桶蛋白的折叠，具有非常大的潜力。 对生物学、生物技术和医学的广泛影响。

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.