Protein Folding Modules and Cooperativity

描述（由申请人提供）：本提案中概述的实验旨在鉴定和控制包含蛋白质天然状态的构象集合。为了理解蛋白质序列如何编码折叠的功能分子，我们需要了解的不仅仅是它的天然结构。编码在主序列中的是整个能量景观。景观的非天然区域对于指导蛋白质的稳定性和折叠非常重要，并且在该集合中的调节在错误折叠、信号传导、变构和周转中起着至关重要的作用。我们从序列到功能的能力的一个主要障碍是我们缺乏对这些非原生状态的理解。许多不同的序列可以编码相同的三维结构，但它们的功能和动力学变化很大;微小的变化可能产生从无法检测到病理性的影响。这些差异往往是非原生地区景观发生微妙变化的结果。在这里，我们提出的实验，旨在确定和控制不同的功能的景观，使用新的和建立的实验技术和系统的组合。具体而言，本提案的目标是：具体目标1：蛋白质构象变化的单分子研究a。探索非本征态B的不寻常的力学性质。从这些固有的非平衡研究中获得能量信息。确定重复蛋白质中的机械耦合具体目标2：检查自由能景观的扰动。a.探索嗜热菌B的能量景观。使用诱变和MPAX探测RNase H的能量景观：错误掺入质子烷基交换特定目标3：通过构象集合的调节进行功能的从头设计。a.可控变构开关B的设计。通过配体结合和机械张力演示控制

DESCRIPTION (provided by applicant): The experiments outlined in this proposal are aimed at identifying and controlling the ensemble of conformations that comprise the native state of a protein. In order to appreciate how a protein's sequence encodes a folded, functional molecule, we need to understand much more than just its native structure. Encoded in the primary sequence is the entire energy landscape. Non-native regions of the landscape are important for directing the stability and folding of a protein, and modulations in this ensemble play a crucial role in misfolding, signaling, allostery, and turnover. A major hurdle in our ability to go from sequence to function is our lack of understanding of these non-native states. Many different sequences can encode the same three dimensional structure, yet their function and dynamics vary dramatically; small variations can have effects that range from undetectable to pathological. These differences are often a consequence of subtle changes in non-native regions of the landscape. Here, we propose experiments directed at determining and controlling different features of the landscape using a mix of new and established experimental techniques and systems. Specifically, the aims of this proposal are: Specific aim 1: Single molecule studies of protein conformational changes a. Explore the unusual mechanical properties of non-native states b. Obtain energetic information from these inherently non-equilibrium studies c. Determine the mechanical coupling in repeat proteins Specific aim 2: Examine perturbations of the free energy landscape. a. Explore the energy landscape of thermophiles b. Probe the energy landscape of RNase H using mutagenesis and MPAX:mis-incorporation proton alkyl exchange Specific Aim 3: The de novo design of function through modulation of conformational ensembles. a. Design of a controllable allosteric switch b. Demonstrate control by ligand binding and mechanical tension