Single Molecule Analysis of Protein Folding Energy Landscapes

该项目包含一个综合的研究和教育计划，涉及指导、教育和培训本科生和研究生在生物物理研究方面的工作。该项目的目标是了解细胞色素c （cyt c）折叠的分子复杂性，为表征cyt c的能量景观提供至关重要的数据。这些测量将测试与理解蛋白质折叠相关的两个基本假设：1)氨基酸序列的螺旋倾向的程度和分布在确定常见cyt c折叠中间体的稳定性，种群和异质性方面发挥重要作用；2) Cyt - c分子在折叠能量格局上主要表现为交换扩展和紧凑构象，其相对数量取决于变性条件。实验方法将采用单分子（SM）水平的折叠光学研究，这将允许分析多肽集合中单个成员的构象和动力学。拟议的研究将涉及时间集成和时间分辨的SM荧光共振能量转移测量，从而突变到蛋白质表面的残基将允许获得蛋白质结构的动力学。Cyt c具有许多关键优势：1)共价连接的血红素基团在能量转移实验中提供了一个内在受体，从而简化了样品制备，并最大限度地减少了衍生化对蛋白质折叠的干扰。2) Cyts c是高可溶性、稳定和健壮的蛋白质。3) Cyts c折叠简单（即由双态模型近似），这很重要，因为随着对集合成员的探测，复杂性将出现。4) Cyt c折叠在宏观水平上有很好的表征，但在单分子水平上还没有表征。

This project contains an integrated research and educational plan involving the mentoring, education, and training of undergraduate and graduate students in biophysical research. The goal of the project is to understand the molecular complexity of cytochrome c (cyt c) folding, providing data vital for characterization of energy landscapes for cyt c. The measurements will test two fundamental hypotheses relevant to understanding protein folding: 1) The extent and distribution of helical propensities of the amino acid sequences play important roles in determining the stability, population, and heterogeneity of the common cyt c folding intermediate; 2) Cyt c molecules primarily sample interchanging extended and compact conformations on the folding energy landscape, the relative populations of which depend on denaturation conditions. The experimental approach will be to employ optical studies of folding on the single-molecule (SM) level, which will allow analysis of conformations and dynamics of individual members of an ensemble of polypeptides. Proposed studies will involve both time-integrated and time resolved SM fluorescence resonance energy transfer measurements, whereby mutations to residues on the protein surface will allow for access to dynamics of protein structure. Cyt c offers a number of key advantages: 1) The covalently attached heme group provides an intrinsic acceptor in energy transfer experiments, thus simplifying sample preparation and minimizing perturbation of protein folding by derivatization. 2) Cyts c are highly soluble, stable, and robust proteins. 3) Cyts c fold simply (i.e. approximated by a two-state model), which is important, as complexity will arise as members of the ensemble are probed. 4) Cyt c folding is well characterized on the macroscopic level, but not yet characterized on the single-molecule level.