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Folding and Stability of TIM Barrel Proteins

TIM 桶蛋白的折叠和稳定性

基本信息

批准号：
9194405
负责人：
Osman Bilsel
金额：
$ 37.26万
依托单位：
UNIV OF MASSACHUSETTS MED SCH WORCESTER
依托单位国家：
美国
项目类别：
财政年份：
1976
资助国家：
美国
起止时间：
1976-05-01 至 2018-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9194405
关键词：
Adverse effects Amino Acid Sequence Amino Acids Bioinformatics Biological Assay Biological Process Biology Biophysics Biotechnology Burial Cereals Chemicals Collaborations Collection Complement Complex Computer Simulation DNA sequencing Data Analyses Dependence Deuterium Dimensions Elements Energy Transfer Enzymes Event Fluorescence Resonance Energy Transfer Free Energy Genes Glycerophosphates Gold Grant Growth Hydration status Hydrocarbons Hydrogen Hydrogen Bonding In Vitro Indoles Isoleucine Kinetics Knock-out Label Leucine Literature Location Maps Mass Spectrum Analysis Measures Medicine Methods Michigan Molecular Molecular Conformation Mutation Mutation Analysis Ontario Orthologous Gene Output Pathway interactions Pattern Peptides Play Population Process Property Protein Conformation Protein Engineering Proteins Pulse Radiolysis Reaction Roentgen Rays Role Side Site Solvents Source Structure Surface Survivors System Techniques Tertiary Protein Structure Testing Time Tryptophan Validation Valine Variant Work Yeasts alpha-glycerophosphoric acid computer studies design exhaustion experimental study fitness globular protein human disease in vivo insight interest melting millisecond models and simulation novel protein aggregation public health relevance simulation three dimensional structure tool

项目摘要

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.

描述（由申请人提供）：对蛋白质的氨基酸序列将其快速有效折叠成其天然功能构象的机制的分子水平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的蛋白质和结构域，但氨基酸序列在定义填充折叠自由能表面的物质的结构和稳定性方面的精确作用仍然难以捉摸。我们假设，集群的分支脂肪族侧链，异亮氨酸，亮氨酸和缬氨酸（ILV），作为稳定的折叠中间体和TIM桶蛋白，生物学中最常见的图案之一的天然状态的核心。我们提出了一个多方面的测试，这一假设的三个吲哚-3-甘油磷酸合酶（IGPS）的直系同源，其低序列的同一性导致不同的大小和位置的居民ILV集群。一组技术将探测填充折叠自由能表面的部分折叠状态的结构，并测试它们的性质。与这些饱和烃簇的关系。CD，FRET和SAXS技术将评估二级结构，并提供在微秒时间范围内开始的成对和全局尺寸信息，氢交换质谱和NMR方法将映射在毫秒到秒的时间范围内出现的部分折叠状态下的稳定氢键网络，并将用一种新的氧化标记方法评估这些相同物种的侧链掩埋。这些结果将用于验证使用能够定义这些直系同源物的整个折叠反应坐标的以天然为中心的GM模型模拟的部分折叠状态下的结构预测。在一个令人兴奋的新的冒险，我们将评估一个详尽的一套氨基酸替换在所有8 ′链和前面的环稳定性元素的相对健身的每个直系同源物在生长竞争试验中缺乏其内在的IGPS基因的酵母菌株的影响。将通过对SsIGPS直系同源物中相同稳定性元件中约100个定点突变的子集中天然和中间状态稳定性扰动的体外定量评估来验证适合性提供体内稳定性估计的假设。平行CD和酶活性测定将测量突变对酶的结构和功能的影响，作为适应性降低的替代解释。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，该分析旨在提供突变背景依赖性的显著评估。比较ILV簇内部和外部突变的影响将提供一种公正且稳健的方法来确定它们在定义球状蛋白稳定性核心方面的重要性。验证我们的假设，即支链脂肪族侧链簇在稳定部分折叠状态和指导TIM桶蛋白的折叠中起着至关重要的作用，有可能对生物学，生物技术和医学产生非常广泛的影响。