Protein Folding Modules and Cooperativity

描述（由申请人提供）：解码蛋白质一级序列中的信息是现代生物学中最基本的挑战之一。蛋白质的序列编码的不仅仅是天然结构；它编码了整个能量景观——一个能量学和动力学被精细调整的构象集合。该提案的目标是对序列和能源景观之间的关系进行分子、定量和预测性的理解，以及对环境如何调节这种景观的理解。从序列到功能的一个主要障碍是我们对景观的非原生区域缺乏了解。高能构象对于指导蛋白质的稳定性和折叠是重要的，并且这种组合的调节在错误折叠，蛋白质信号传导，催化活性和变构中起作用。虽然许多序列可以编码相同的结构，但它们的功能和动态可能会有很大差异。序列的微小变化可能会产生从无法检测到病理的影响。这些差异往往是景观中非原生区域细微变化的结果。很快我们就能接触到成千上万的人类基因组，如果我们没有解释变异的能力，这些数据影响医学和人类健康的潜力将永远不会得到充分认识。因此，我们必须理解和控制序列和能量景观之间的关系。由于高能量物种的小种群和瞬态特性，能量景观的调节不容易被探测到。这里概述的实验旨在了解序列和环境的变化如何影响能源景观。目标1：通过单分子力学研究确定应变如何影响能量格局。a.确定拉动的几何形状如何影响过渡态，或蛋白质折叠的屏障。b.确定展开的限速屏障如何随力变化，以及这些屏障如何与体内机械过程（如蛋白质展开酶）相关。目标2：通过演化和序列调制探索能量景观。a.利用祖先序列重建技术探索能源格局的演化变化。b.通过选择具有改变的结合特异性的dna结合蛋白的核心变体来评估疏水核心在蛋白质功能中的作用。目标3：绘制能源景观中未开发区域的地图。a.发展硫醇交换方法来绘制折叠反应坐标上的构象，并利用这种动力学分配方法来表征过渡态原生侧的波动。b.利用快速混合技术和早期折叠中间体的平衡模型，绘制出折叠中最早事件的序列依赖性。

DESCRIPTION (provided by applicant): Decoding the information in the primary sequence of a protein is one of the most fundamental challenges in modern biology. A protein's sequence encodes more than just the native structure; it encodes the entire energy landscape - an ensemble of conformations whose energetics and dynamics are finely tuned. The goal of this proposal is a molecular, quantitative, and predictive understanding of the relationship between sequence and the energy landscape together with an understanding of how the environment modulates this landscape. A major hurdle in going from sequence to function is our lack of understanding of the non-native regions of the landscape. High-energy conformations are important for directing the stability and folding of a protein, and modulations of this ensemble play a role in misfolding, protein signaling, catalytic activity, and allostery. While many sequences can encode the same structure, their function and dynamics can vary dramatically. Small variations in a sequence can have effects that range from undetectable to pathological. These differences are often a consequence of subtle changes in the non-native regions of the landscape. Soon we will have access to thousands of human genomes, and without our ability to interpret variation, the potential of these data to impact medicine and human health will never be fully appreciated. It is imperative, therefore, that we have an understanding and control over the relationship between sequence and the energy landscape. Modulations of the energy landscape are not easily detected due to the small populations and transient nature of the high-energy species. The experiments outlined here are aimed at understanding how changes in the sequence and the environment affect the energy landscape. Aim 1: Determine how strain influences the energy landscape via single molecule mechanical studies. a. Determine how the geometry of pulling affects the transition state, or barrier for protein folding. b. Determine how the rate-limiting barrier to unfolding changes with force, and how these barriers relate to in vivo mechanical processes such as protein unfoldases. Aim 2: Probe the energy landscape through evolution and sequence modulation. a. Use Ancestral Sequence Reconstruction to probe evolutionary changes in the energy landscape. b. Evaluate the role of the hydrophobic core in protein function by selecting for core variants of DNA-binding proteins with altered binding specificities. Aim 3: Map unexplored regions of the energy landscape. a. Develop thiol exchange methods to map conformations on the folding reaction coordinate and utilize this kinetic partitioning method to characterize fluctuations on the native side of the transition state. b. Map out the sequence dependence of the earliest events in folding using both rapid mixing techniques and equilibrium models of early folding intermediates.