Sequence and Environmental Determinants of the Protein Energy Landscape

项目总结/摘要 解码蛋白质一级序列中的信息是生物学中最基本的挑战之一。 现代生物学蛋白质的序列编码的不仅仅是天然结构，它还编码整个能量 景观-一个整体的构象，其能量和动力学是微调。这个目标 建议是一个分子，定量，和预测的理解之间的关系序列和 能源景观，以及对环境如何调节这一景观的理解。 从序列到功能的一个主要障碍是我们缺乏对非原生或高- 景观的能量区域以及它们如何被环境调节。高能构象 对于指导蛋白质的稳定性和折叠是重要的，并且对该集合的调节在以下方面起作用： 错误折叠、蛋白质信号传导、催化活性和变构。然而，许多序列可以编码相同的 它们的结构、功能和动态可能会因景观的变化而发生巨大变化。小 序列中的变异可具有从不可检测到病理性的范围的影响。很快我们就会有 获得数千个人类基因组，如果没有我们解释变异的能力， 影响医学和人类健康的数据永远不会得到充分的重视。因此，我们必须 理解和控制序列和能量景观之间的关系。 能量景观的调制不容易被检测到，由于人口少和瞬态 高能量物种的本质。这里概述的实验旨在了解 序列和环境影响能源景观。 目标1：定量测量复杂环境中蛋白质的折叠和稳定性 a.开发一种定量的实验室测量方法，测量蛋白质在核糖体和其他复杂混合物上的稳定性。 B.测量核糖体结合多肽链的构象动力学 C.使用HaloTag作为模型系统监测平移耦合折叠 目标2：通过演化和序列调制探索能源格局 a.使用祖先序列重建（ASR）来探索RNase H随时间的变化。 我们将评估这些复活的蛋白质的能量景观，以确定如何优化的蛋白质。 能量景观，因此功能/健身，发生在进化的时间。 B.使用祖先序列重建评估使用α-裂解蛋白酶的限速步骤 动力学稳定和代谢稳定的蛋白酶家族 目标3：通过单分子机械展开探索能量格局 a.蛋白质折叠能垒的单分子力学研究

Project Summary/ Abstract 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