Biophysical Aspects of Cotranslational Protein Folding

智力上的功绩。纯蛋白质在溶液中的折叠，从化学或热产生的未折叠状态开始，已经被深入研究了近50年。然而，这些未折叠状态很少出现在活细胞中，因此我们仍然对蛋白质在自然环境中如何折叠以实现其活性构象知之甚少。所有活细胞中的蛋白质都是由核糖体合成的，核糖体是在所有活着的有机体中发现的复杂的分子机器。在活细胞中，许多因素，包括被称为“伴侣蛋白”的辅助蛋白，以及核糖体本身，都可以深刻地影响蛋白质一旦形成后如何正确折叠以及是否正确折叠。了解最少的是折叠的最早阶段，即蛋白质第一次从制造蛋白质的核糖体中出现时。这个项目探索了与核糖体结合的新生蛋白质和释放的核糖体之间的结构和动态差异，并建议比较在存在和不存在伴侣蛋白质(“折叠辅助者”)的情况下导致全长蛋白质链从核糖体释放(“蛋白质诞生”)的时间过程。还将对模型新生蛋白质链进行协同计算和实验研究，以评估核糖体表面在空间上限制新生蛋白质和促进折叠的能力。将追求以下研究目标：#1：平衡分析新生的单域蛋白在核糖体释放前后从核糖体隧道中出现时的构象和动力学。这些研究将通过H/D交换和MALDI质谱学研究新生蛋白质的溶剂暴露程度，从而确定新生蛋白质最稳定和最有结构的区域。#2：实时从核糖体释放新合成的单域蛋白质，以跟随蛋白质3D结构的形成。#3：由多结构域蛋白质衍生的不完整和全长新生链条的平衡和动力学分析。对于简单的多域模型蛋白HMP，将重复在特定目标#1和2中描述的实验。此外，实时翻译研究将探索mRNA沉默突变对蛋白质构象的影响。#4：核糖体结合和核糖体释放的新生蛋白质的计算分析。将开展协同计算和实验研究，以评估核糖体表面对新生蛋白质的构象和空间限制的影响。更广泛的影响。该项目包括追求以下更广泛的影响：#1：招募代表性不足的学生参加研究活动。拟议的研究将涉及指导和培训研究生和本科生，特别是妇女和代表人数不足的少数群体的学生。本科生将定期参加PI小组的高级研究。该协会还将作为讲师参加生物物理学会为少数民族学生举办的夏季生物物理学课程，并将在SACNAS会议上担任导师、招聘者和评委，促进奇卡诺人和拉美裔美国人参与科学。#2：开发关于蛋白质折叠和细胞相关成分对蛋白质折叠和稳定性的影响的新颖讲座演示。PI将开发一些课堂演示，目的是用直接的视觉读数来说明蛋白质折叠的可逆性，它的pH依赖性，以及渗透分子对蛋白质稳定性的影响。#3：在米德尔顿高中举办关于蛋白质折叠的讲座、视频和演示。该项目将与米德尔顿高中分子生物学和生物技术教师Kathryn Eilert合作完成。

Intellectual Merit. The folding of pure proteins in solution, starting from chemically or thermally generated unfolded states, has been studied intensively for almost 50 years. However, these unfolded states rarely occur in living cells and therefore we still know very little about how proteins actually fold in their native environments to achieve their active conformations. Proteins in all living cells are synthesized by ribosomes, complex molecular machines found in all living organisms. In living cells, a number of factors, including helper proteins called "chaperones," crowding by thousands of other molecules, as well as the ribosome itself, can profoundly affect how and whether a protein folds correctly once it is made. Least understood are the earliest stages of folding, when the protein first emerges from the ribosome where it is made.This project explores the structural and dynamic differences between ribosome-bound and ribosome-released nascent proteins, and proposes to compare the timecourse leading to release of full-length protein chains from the ribosome ("protein birth"), in the presence and absence of chaperone proteins ("folding helpers"). Synergistic computational and experimental investigations will also be carried out on model nascent protein chains to assess the ability the ribosome's surface to spatially confine the nascent protein and promote folding. The following research objectives will be pursued:#1: Equilibrium analysis of the conformation and dynamics of nascent single-domain proteins as they emerge from the ribosomal tunnel, before and after their release by the ribosome. These investigations will identify the most stable and most structured regions of nascent proteins by studying their degree of solvent exposure via H/D exchange followed by MALDI mass spectrometry. #2: Kinetic measurements of the release of newly synthesized, single-domain proteins from the ribosome to follow the formation of protein 3D structure in real time.#3: Equilibrium and kinetic analysis of incomplete and full-length nascent chains derived by multidomain proteins. The experiments described in specific aims #1 and 2 will be repeated for the simple multi-domain model protein Hmp. In addition, real-time translation studies will probe the effect of mRNA silent mutations on protein conformation.#4: Computational analysis of ribosome-bound and ribosome-released nascent proteins. Synergistic computational and experimental studies will be carried out to assess the effect of the ribosome's surface on the nascent protein's conformation and spatial confinement. BROADER IMPACTS. The project includes the pursuit of the following broader impacts:#1: Recruitment of underrepresented students to participate in research activities. The proposed studies will involve the mentoring and training of graduate and undergraduate students, especially women and students belonging to underrepresented minorities. Undergraduate students will routinely participate to advanced research in the PI's group. The PI will also participate as a lecturer to the Summer Course in Biophysics for Minority Students organized by the Biophysical Society and will serve as a mentor, recruiter and judge at the SACNAS conference, fostering participation of Chicanos and Latino Americans in science.#2: Development of novel lecture demonstrations on protein folding and the effect of cell-relevant components on protein folding and stability. The PI will develop a number of classroom demos aimed at illustrating, with direct visual readouts, the reversibility of protein folding, its pH dependence, and the effect of osmolytes on protein stability. #3: Presentation of lectures, videos and demonstrations on protein folding at the Middleton High School. This project will be done in collaboration with Kathryn Eilert, Teacher of Molecular Biology and Biotechnology at the Middleton (WI) High School.