SPECTROSCOPY OF SINGLE PROTEINS AND BIOLOGICAL ASSEMBLIES: EQUILIBRIUM DYNAMICS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 与典型整体实验中的系综平均值相比，单分子光谱学在研究分子性质的分布方面具有很大的优势。我们开发了各种设置，可以与实验室中所有可用的激励源相结合，提供频率和时间分辨率检测以及成像能力。我们的主要研究方向是研究生理条件下的蛋白质动力学和蛋白质折叠，以及研究单细胞内的分子过程。 我们将继续研究各种固定化技术，进一步研究凝胶、溶胶凝胶、海藻糖玻璃、反胶束、伽马环糊精、沸石笼子和胶束在水环境中固定单一蛋白质和酶的方法。我们正在获得在这些环境中测量结构波动、蛋白质和模型肽的折叠/展开(螺旋螺旋转变)、酶反应和钙离子信号(钙调蛋白)的协议。此外，通过监测构象变化的实验以及温度、pH、盐和离子在空腔中扩散的影响，还在研究水在包膜中的作用、空腔中水分含量的变化、蛋白质水的包埋以及蛋白质与周围环境的相互作用。 我们最新研究的目的是对具有不同曲率、胆固醇、洗涤剂和头基的各种脂质膜中螺旋聚集的缔合和解离、扩散和动态效应的异质性进行定量评估。这些实验可以通过专门标记的环境敏感探针的单分子荧光寿命和FRET实验来可视化糖蛋白A(GPA)和其他TM蛋白的跨膜(TM)部分的结构波动和分布。 另一个目的是通过环境敏感探针的单分子荧光相关实验，定量描述钙调蛋白钙信号机制的异质性。 针对无肌红蛋白和细胞色素-c在膜、囊泡和脂质双层中的构象状态分布和自由能表面的实验都在进行中。单分子方法探测荧光寿命分布、FRET和环境敏感响应，为解决构象运动、蛋白质折叠和展开特性以及自由能表面的异质性提供了强有力的途径，这些都是从整体实验中无法获得的。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Single molecule spectroscopy offers the great advantages of studying distributions of molecular properties compared to ensemble averages in a typical bulk experiment. We have developed various set ups which can be combined with all excitation sources available in the lab, and which offer frequency- and time-resolved detection as well as imaging capabilities. Our main research direction is the investigation of protein dynamics and protein folding under physiological conditions and the study of molecular processes within single cells. We will continue to investigate various immobilization techniques, further examining gels, sol-gels, trehalose glasses, reverse micelles, gamma-cyclodextrin, zeolite cages and micelles to immobilize single proteins and enzymes in aqueous environments. We are obtaining protocols for measurements of structural fluctuations, folding/unfolding of proteins and model peptides (helix coil transitions), enzyme reactions and calcium ion signaling (calmodulin) in these environments. Also the role of water in encapsulation, the variation of the water content in cavities, the encapsulation of protein water and the interaction of proteins with the surrounding environment through experiments that monitor conformational changes and the effects of temperature, pH, salts and ions diffusing in the cavities are under study. The purpose of our most recent research is to make quantitative assessments of the heterogeneity of association and dissociation, diffusion and dynamic effects of helix aggregation in membranes with various lipids having a range of curvatures, cholesterol, detergents and head groups. The experiments enable the visualization of structural fluctuations and distributions of trans-membrane (TM) sections of Glycophorin-A (GpA) and other TM proteins by single molecule fluorescence lifetime and FRET experiments for specifically labeled and environmentally sensitive probes. Another aim seeks quantitative descriptions of the heterogeneity of the calcium signaling mechanisms of Calmodulin by single molecule fluorescence correlation experiments with environmentally sensitive probes. Experiments aimed at conformational state distributions and free energy surfaces of apomyoglobin and cytochrome-c in membranes, vesicles, and lipid bilayers are all on-going. Single molecule methods that probe fluorescence lifetime distributions, FRET and environment sensitive responses, provide powerful avenues with which to address the heterogeneity of conformational motions, protein folding and unfolding properties and free energy surfaces that are not obtainable from bulk experiments.