Sequence and Environmental Determinants of the Protein Energy Landscape

所有的蛋白质样品具有不同的构象（折叠，未折叠和激发态）， 自由能和动力学取决于环境条件。蛋白质的一级序列 编码的不仅仅是天然结构;它编码了整个能源景观-一个整体， 其能量学和动力学被精细调整和调制的构象。该提案的目标是 定量和预测的理解之间的关系序列和景观，一起 了解蛋白质的环境如何调节这一景观。 从序列到功能的一个主要障碍是我们缺乏对非天然区域的了解， 风景高能构象对于指导蛋白质的稳定性和折叠是重要的， 该系综的调节在错误折叠、蛋白质信号传导、催化活性和变构中起作用。而 许多序列可以编码相同的结构，它们的功能和动力学可以显着变化-由于 景观的变化。一个序列中的微小变化可以产生从检测不到到 病态的很快我们就能获得数千个人类基因组， 这些数据的变化，这些数据的潜力，影响医学和人类健康将不会得到充分的认识。 这里概述的实验将集中在细胞环境（核糖体， 翻译后修饰和共翻译折叠）影响蛋白质的能量格局以及新的 方法来调查这些景观序列变化的影响。 目标1.细胞成分（核糖体和泛素化）如何调节能量景观？ a.确定泛素化对靶蛋白能量分布的影响。 B.确定核糖体对核糖体结合的新生链的能量景观的影响 （RNC），监测动力学和氢交换。 目的2：共翻译折叠的生物物理学研究 a.通过研究HaloTag的环状排列体的共翻译折叠来确定拓扑结构的作用。 B.使用X射线羟基足迹法（XF/MS）监测复杂环境中的蛋白质折叠。 C.利用单分子探针研究翻译与新生链折叠之间的时间协调 零模波导中的荧光。 目的3：利用ASR研究蛋白质景观的序列决定因子 a.通过研究一个含有动力学稳定和 稳定蛋白质。 B.α-裂解蛋白酶家族的ASR分析。

All proteins sample a diverse array of conformations (folded, unfolded, and excited states) with differing free energies and dynamics depending on the environmental conditions. A protein’s primary 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 and modulated. The goal of this proposal is a quantitative and predictive understanding of the relationship between sequence and the landscape, together with an understanding of how a protein’s 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 – 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 an ability to interpret variation, the potential of these data to impact medicine and human health will not be fully appreciated. The experiments outlined here will focus on how modulations in the cellular environment (the ribosome, post-translational modifications and co-translational folding) affect a protein’s energy landscape as well as new approaches to investigate the effect of sequence variation on these landscapes. Aim 1. How do cellular components (the ribosome and ubiquitination) modulate the energy landscape? a. Determine the effect of ubiquitination on the energy landscape of target proteins. b. Determine the effects of the ribosome on the energy landscape of ribosome-bound nascent chains (RNCs), monitoring kinetics and hydrogen exchange. Aim 2: Biophysical studies of co-translational folding a. Determine the role of topology by studying the co-translational folding of circular permutants of HaloTag. b. Monitor protein folding in complex environments using X-ray hydroxyl radical footprinting (XF/MS). c. Probe the temporal coordination between translation and nascent chain folding using single-molecule fluorescence in zero-mode waveguides. Aim 3: ASR studies to probe the sequence determinants of protein landscapes a. Probing the rate-limiting step in protein folding by investigating a family containing kinetically stable and thermodynamically stable proteins. b. ASR analysis on the alpha-lytic protease family.