Folding and Stability of TIM Barrel Proteins

描述(申请人提供)：在分子水平上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的机制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的蛋白质和结构域，但氨基酸序列在定义折叠自由能表面物种的结构和稳定性方面的确切作用仍然难以捉摸。我们假设，支链脂肪族侧链的簇，异亮氨酸，亮氨酸和缬氨酸(ILV)，作为折叠中间体和TIM桶蛋白的天然状态的稳定核心，这是生物学中最常见的基序之一。我们对这一假设提出了一个多方面的测试对三个吲哚-3-甘油磷酸合成酶(IGPS)同源基因，其低序列同源性导致其驻留ILV簇的大小和位置不同。一系列技术将探测填充在折叠自由能表面上的部分折叠状态的结构，并测试它们的 与这些饱和碳氢化合物簇的关系。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维度信息，氢交换质谱仪和核磁共振方法将在毫秒到秒的时间范围内绘制部分折叠状态下稳定的氢键网络，并将使用一种新的氧化标记方法评估这些相同物种中的侧链埋藏。这些结果将被用来验证部分折叠状态下的结构预测，使用以自然为中心的GM模型模拟，该模拟能够定义这些直系物的整个折叠反应坐标。在一项令人兴奋的新冒险中，我们将评估所有8条链和之前的/环稳定元件中的一组详尽的氨基酸替换对缺乏固有IGPS基因的酵母菌株的生长竞争分析中每个直系同源物的相对适合度的影响。适合性提供了体内稳定性估计的假设将通过对SsIGPS同源基因中相同稳定性元件中的~100个定点突变的子集中天然和中间态稳定性的扰动进行体外定量评估来验证。平行的CD和酶活性分析将衡量突变对酶结构和功能的影响，作为适应性下降的另一种解释。这些体内和体外稳定性扰动测量的输出将作为生物信息学分析的输入，该分析旨在提供对突变背景相关性的统计上有意义的评估。ILV簇内部和外部突变的影响的比较将提供一种公正和稳健的方法来确定它们在确定球状蛋白稳定核心方面的重要性。我们的假设得到验证，即分支脂肪族侧链簇在稳定部分折叠状态和指导Tim桶蛋白的折叠方面发挥着关键作用，这可能会对生物学、生物技术和医学产生非常广泛的影响。

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.