SPECTROSCOPY OF SINGLE PROTEINS AND BIOLOGICAL ASSEMBLIES: EQUILIBRIUM DYNAMICS

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。与典型体实验中的系综平均相比，单分子光谱在研究分子性质分布方面具有很大的优势。我们已经开发了各种装置，可以与实验室中可用的所有激励源相结合，并提供频率和时间分辨检测以及成像功能。我们的主要研究方向是生理条件下蛋白质动力学和蛋白质折叠的研究以及单细胞内分子过程的研究。我们将继续研究各种固定技术，进一步研究凝胶，溶胶-凝胶，海藻糖玻璃，反胶束，γ -环糊精，沸石笼和胶束在水环境中固定单个蛋白质和酶。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（螺旋线圈转变）、酶反应和钙离子信号（钙调素）的方案。此外，还通过监测构象变化以及温度、pH、盐和离子在空腔中扩散的影响的实验，研究了水在包封中的作用、空腔中含水量的变化、蛋白质水的包封以及蛋白质与周围环境的相互作用。我们最近研究的目的是定量评估具有不同曲率、胆固醇、洗涤剂和头基团的各种脂类的膜中结合和解离的异质性、扩散和螺旋聚集的动态效应。本实验通过单分子荧光寿命和特异性标记和环境敏感探针的FRET实验，实现了糖蛋白- a （GpA）和其他TM蛋白跨膜（TM）部分结构波动和分布的可视化。另一个目的是通过环境敏感探针的单分子荧光相关实验，寻求定量描述钙调素钙信号传导机制的异质性。针对肌红蛋白和细胞色素c在膜、囊泡和脂质双分子层中的构象态分布和自由能面的实验正在进行中。探测荧光寿命分布、FRET和环境敏感响应的单分子方法，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，这些都是无法从大量实验中获得的。

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.