Integrating experiment and theory for predicting protein folding pathways and structure

整合实验和理论来预测蛋白质折叠途径和结构

• 批准号: 9403133
• 负责人: KARL F FREED
• 金额: $ 39.98万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-08-01 至 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9403133
• 关键词: Aging; Amides; Amyloidosis; Antifreeze Proteins; Behavior; Binding; Biological; Biological Models; Burial; Chemicals; Communities; Comparative Study; Crystallization; Development; Disease; Equilibrium; Evolution; Fleas; Fractionation; Free Energy; Future; Glycine; Goals; Host Defense Mechanism; Hydrogen; Hydrogen Bonding; Hydrophobicity; Investigation; Iron; Machine Learning; Measures; Membrane; Membrane Proteins; Membrane Transport Proteins; Metals; Methods; Mind; Molecular Chaperones; Molecular Conformation; Monitor; Neisseria; Pathogenicity; Pathway interactions; Procedures; Property; Protein Conformation; Protein Dynamics; Proteins; Race; Sampling; Shigella; Side; Snow; Structure; System; Tertiary Protein Structure; Testing; Time; Training; Variant; Vertebral column; Yersinia; arm; base; computer studies; conformer; design; disulfide bond; experimental study; improved; learning strategy; method development; molecular dynamics; pathogen; pathogenic bacteria; polyproline; pressure; protein degradation; protein folding; protein function; quantum; simulation; theories; tool

Project Summary/Abstract Protein folding and dynamics are integral to many biological activities, including chaperone action, degradation, amyloid diseases and aging. Our goal is to combine experimental and computational studies to produce a predictive understanding of protein dynamics through the development of methods capable of simulating folding and dynamics just using physio-chemical principles. Our past studies have focused on single domain proteins. Our efforts have expanded to more complicated systems including the snow flea anti-freeze protein (sfAFP) and TonB-dependent transporters (TBDT) that are relevant to iron sequestration in pathogenic bacteria. Tying these studies together is our new molecular dynamics (MD) package, Upside, which can reversibly fold some proteins up to 97 AA to under 4 Å C-RMSD in cpu-days without the use of fragments, homology or evolution. Upside utilizes a number of unique features, including rapid side chain packing that enables simulations using only 3 backbone atoms while retaining considerable detail and avoiding side chain “rattling”, which slows all-atom methods. We will improve Upside and implement enhanced sampling methods to increase our accuracy and size range, and study protein dynamics as monitored by hydrogen exchange. sfAFP's unique structure challenges conventional wisdom regarding cooperative folding and stability. Lacking a hydrophobic core to promote folding, other factors must contribute to sfAFP's stability. We will test our quantum calculations that sfAFP's H-bonds are unusually stable by measuring amide H/D fractionation factors and NMR J-couplings. We will evaluate whether intrinsic biases in backbone dihedral angles for the PP2 basin in the unfolded state are another major stabilizing factor. This information will be used to improve the Upside simulations. Finally, we will apply our standard folding tools to characterize the folding pathway and compare it to the behavior we expect based on principles derived from proteins with hydrophobic cores. Many aspects of the transport cycle in TBDT remain unknown despite protracted study, including the conformational rearrangement of the plug domain during transport. We will provide the first structure of the plug domain outside the barrel, and so answer whether this structure matches the crystal structure in the barrel. Additionally, the study of Nakamoto's V10C-S120C variant of the BtuB plug enables the investigation of a possible folding or transport intermediate. We will characterize the plug's dynamics while it is in the barrel using HX to observe possible transport-competent states. The mechanism of plug folding and insertion into the barrel will be investigated, with comparative studies for FhuA, a TBDT whose plug domain is intrinsically disordered in solution. These studies represent an exciting combination of protein folding and function, at the interface between soluble and membrane folding, using experiments and complementary folding simulations.

项目总结/摘要 蛋白质折叠和动力学是许多生物活动的组成部分，包括分子伴侣作用， 降解、淀粉样疾病和衰老。我们的目标是将联合收割机的实验和计算研究结合起来， 通过开发能够预测蛋白质动力学的方法， 仅仅利用物理化学原理来模拟折叠和动力学。我们过去的研究集中在单一的 结构域蛋白我们的努力已经扩大到更复杂的系统，包括雪蚤防冻 蛋白（sfAFP）和TonB依赖性转运蛋白（TBDT），与致病性铁螯合相关， 细菌将这些研究联系在一起的是我们新的分子动力学（MD）软件包Upside，它可以 在cpu-天内可逆地折叠一些蛋白质高达97 AA至低于4 μ C，而不使用片段， 同源性或进化。Upside利用了许多独特的功能，包括快速侧链包装， 仅使用3个骨架原子进行模拟，同时保留相当多的细节并避免侧链 “嘎嘎作响”，减慢了全原子方法。我们将改进Upside并实施增强的采样方法 以提高我们的准确性和尺寸范围，并研究通过氢交换监测的蛋白质动力学。 sfAFP的独特结构挑战了关于协同折叠和稳定性的传统智慧。 由于缺乏促进折叠的疏水核心，其他因素必须有助于sfAFP的稳定性。我们将测试 通过测量酰胺H/D分馏，我们的量子计算表明sfAFP的H-键异常稳定 因子和NMR J耦合。我们将评估是否内在偏见的骨干二面角为 PP 2盆地处于展开状态是另一个主要的稳定因素。这些信息将用于改善 Upside模拟。最后，我们将应用我们的标准折叠工具来表征折叠途径， 将其与我们基于具有疏水核心的蛋白质的原理所预期的行为进行比较。 尽管进行了长期的研究，但TBDT的运输周期的许多方面仍然未知，包括 在运输过程中的插头结构域的构象重排。我们将提供 塞域外桶，并回答是否这种结构匹配的晶体结构在桶。 此外，对Nakamoto的BtuB插头的V10 C-S120 C变体的研究使得能够调查 可能的折叠或运输中间。我们将描述塞子在枪管中的动力学特性 使用HX观察可能的运输能力状态。插头折叠和插入的机构 桶将进行调查，与FhuA的比较研究，TBDT的插头域本质上是 在溶液中无序。这些研究代表了蛋白质折叠和功能的令人兴奋的组合， 可溶性和膜折叠之间的接口，使用实验和互补折叠模拟。