Probing Complexity in Protein Structural Landscapes by Fluorescence and Microfluidics

蛋白质已经进化成在多维能量环境中编码的高度复杂性。鉴于它们在细胞中的功能作用，对这些方面的详细了解是至关重要的。改进的实验方法的发展可以打开大门，揭示对这种理解至关重要的以前无法获得的信息。在这个项目中，将结合新的高分辨率荧光显微镜和微流控技术，研究蛋白质系统中的拓扑、动力学和其他结构复杂性的物理学。进展将包括一种新的实验技术的发展，该技术能够快速冷却少量蛋白质溶液，用于高分辨率动力学研究。这些技术与单分子Förster共振能量转移显微镜(SmFRET)相结合，将被应用于蛋白质生物物理学中的一些关键问题，包括蛋白质打结的复杂性、对称性相关效应和非平衡动力学的研究。实验研究还将与理论和计算分析相结合，以进一步提高获得的洞察力水平。这项研究有望开发一套新的先进实验工具，并提供关于蛋白质结构复杂性的重要信息，对细胞和组织中的蛋白质功能具有广泛的影响。该项目的广泛影响预计将在多个方向上进行。学生和博士后研究员将接受新出现的多学科方法及其在分子生物物理学中的应用方面的培训。将继续参与与该项目的科学研究有关的教育工作。研究结果将通过在同行评议的期刊上发表论文和研讨会介绍来传播。还将继续努力扩大参与，包括通过TSRI外联方案。最后，预计拟议工作中产生的新的实验技术和见解将对生物物理学和生物学界具有广泛的实用性和吸引力。研究人员的实验室之间的协同作用，以及他们在尖端单分子生物物理学和微流体研究方面的互补专业领域，将特别有利于更广泛的影响。总体而言，这个多学科项目的目标是提供增强的实验方法，并将在蛋白质复杂性物理学中产生新的基本见解，同时还将在培训、传播和基础设施方面整合更广泛的影响。该项目由MCB的生物分子动力学、结构和功能以及物理部的生命系统物理计划共同支持。

Proteins have evolved to assume a large degree of complexity encoded in a multidimensional energy landscape. Given their functional roles in cells, it is of the utmost importance to achieve a detailed understanding of these aspects. The development of enhanced experimental methods can open the door to revealing previously unavailable information critical to such an understanding. In this project, to study the physics of topological, dynamic and other structural complexity in protein systems, a combination of novel high-resolution fluorescence microscopy and microfluidic technologies will be utilized. Advances will include developments in a new experimental technique enabling rapid cooling of a small volume of protein solution for high resolution kinetic studies. These techniques in combination with single-molecule Förster resonance energy transfer (smFRET) microscopy will be applied to some key problems in protein biophysics, including investigations of complexity in protein knotting, symmetry-related effects, and non-equilibrium dynamics. The experimental research will also be linked with theoretical and computational analysis to further enhance the level of insight gained. The research is expected to lead to the development of a new set of advanced experimental tools and provide important information on protein structural complexity, with broad implications for protein function in cells and organisms.Broader impacts of the project are anticipated in multiple directions. Students and a post-doctoral researcher will be trained in emerging multidisciplinary methods and their application to molecular biophysics. Involvement in educational efforts that are related to the scientific research in the project will be continued. The results of the research will be disseminated through publication of papers in peer-reviewed journals and seminar presentations. Efforts to broaden participation, including through a TSRI outreach program, will also be continued. Finally, it is anticipated that the new experimental techniques and insights generated in the proposed work will have wide utility and appeal to the Biophysics and Biology communities. The synergy between the labs of the investigators, with their complementary areas of expertise in cutting-edge single-molecule biophysics and microfluidics research, will be particularly beneficial for broader impacts. Overall, this multidisciplinary project is aimed at providing enhanced experimental methods and will lead to new fundamental insights in the physics of protein complexity, while also integrating broader impacts in training, dissemination, and infrastructure.This project is being supported jointly by Biomolecular Dynamics, Structure and Function in MCB, and the Physics of Living Systems Program in the Physics Division.