Biophysical Aspects of Cotranslational Protein Folding

智力优势。 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发生在活细胞中，因此我们仍然对蛋白质在其天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，核糖体是所有活生物体中都存在的复杂分子机器。在活细胞中，许多因素，包括被称为“伴侣”的辅助蛋白，被成千上万的其他分子拥挤，以及核糖体本身，可以深刻地影响蛋白质一旦被制造出来后如何以及是否正确折叠。最不为人所知的是折叠的最早阶段，即蛋白质首次从核糖体中出现的阶段。本项目探讨了核糖体结合和核糖体释放的新生蛋白质之间的结构和动力学差异，并提出在有和没有伴侣蛋白（“折叠助手”）的情况下，比较导致全长蛋白质链从核糖体中释放（“蛋白质诞生”）的时间过程。协同计算和实验研究也将进行模型新生蛋白质链，以评估核糖体的表面空间限制新生蛋白质和促进折叠的能力。以下研究目标将被追求：#1：新生的单域蛋白质的构象和动力学的平衡分析，因为它们出现在核糖体隧道，之前和之后的核糖体释放。 这些研究将通过研究新生蛋白质的溶剂暴露程度，通过H/D交换，然后进行MALDI质谱，来确定新生蛋白质的最稳定和最结构化的区域。二：新合成的单域蛋白质从核糖体释放的动力学测量，以真实的时间形成蛋白质3D结构。3：多结构域蛋白质衍生的不完整和全长新生链的平衡和动力学分析。 将针对简单的多结构域模型蛋白Hmp重复具体目标#1和2中描述的实验。此外，实时翻译研究将探测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。 将进行协同计算和实验研究，以评估核糖体表面对新生蛋白质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。 拟议的研究将涉及对研究生和本科生，特别是妇女和属于代表性不足的少数群体的学生进行辅导和培训。本科生将定期参加PI组的高级研究。PI还将参加由生物物理学会组织的少数民族学生生物物理学暑期课程的讲师，并将在SACNAS会议上担任导师，招聘人员和法官，促进奇卡诺人和拉丁美洲人参与科学。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讲座演示。 PI将开发一些课堂演示，旨在说明，与直接的视觉读数，蛋白质折叠的可逆性，其pH值的依赖性，以及渗透剂对蛋白质稳定性的影响。#3：在米德尔顿高中的讲座，视频和演示演示蛋白质折叠。 该项目将与米德尔顿（WI）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.