Sequence and Environmental Determinants of the Protein Energy Landscape

 描述(申请人提供)：破译蛋白质初级序列中的信息是现代生物学中最基本的挑战之一。蛋白质的序列不仅编码天然结构，它还编码整个能量景观--一组能量和动力学都经过微调的构象。这一建议的目标是对序列和能量景观之间的关系以及环境如何调节这一景观进行分子、定量和预测性的理解。从序列到功能的一个主要障碍是我们缺乏对景观中非自然或高能区域的理解，以及它们是如何被环境调制的。高能构象对蛋白质的稳定性和折叠起着重要的指导作用，对高能构象的调控在错误折叠、蛋白质信号转导、催化活性和变构等方面发挥着重要作用。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因景观的变化而显著不同。序列中的微小变化可能会产生从无法检测到的到病理性的影响。很快，我们将获得数千个人类基因组，如果没有我们解释变异的能力，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，我们必须对顺序和能源格局之间的关系有一个理解和控制。由于高能物种的种群数量较少，且具有暂时性，因此不容易检测到能量格局的变化。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格局。目的1：复杂环境中蛋白质折叠和稳定性的定量测量a.建立核糖体和其他复杂混合物上蛋白质稳定性的定量台式测量。B.测量核糖体结合多肽链的构象动力学c.以HaloTag为模型系统监测翻译偶联折叠目标2：通过进化和序列调制探索能量图景a.使用祖先序列重建(ASR)来探索RNaseH图景随时间的变化。我们将评估这些复活蛋白质的能量图景，以确定能量图景的优化，从而确定功能/适应性是如何随着进化时间的推移而发生的。B.使用祖先序列重建来评估限速步骤，使用两者动力学稳定的α-裂解酶家族 热力学稳定的蛋白水解酶的目标3：通过单分子机械展开探索能量格局。单分子力学研究探索蛋白质折叠中的能量障碍

 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 or high- energy regions of the landscape and how they are modulated by the environment. 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 - due to changes in the landscape. Small variations in a sequence can have effects that range from undetectable to pathological. 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: Quantitative measures of protein folding and stability in complex environments a. Develop a quantitative bench-top measure of protein stability on the ribosome and other complex mixtures. b. Measure conformational dynamics of ribosome-bound polypeptide chains c. Monitor translational coupled folding using HaloTag as a model system Aim 2: Probe the energy landscape through evolution and sequence modulation a. Use Ancestral Sequence Reconstruction (ASR) to explore changes in the landscape of RNase H over time. We will evaluate the energy landscapes of these resurrected proteins to determine how optimizations of the energy landscape, and thus function/fitness, occur over evolutionary time. b. Use Ancestral Sequence Reconstruction to evaluate the rate-limiting step by using the alpha-lytic protease family of both kinetically stable and thermodynamically stable proteases Aim 3: Probe the energy landscape through single molecule mechanical unfolding a. Single molecule mechanical studies to probe the energy barriers in protein folding