SPECTROSCOPY OF SINGLE PROTEINS AND BIOLOGICAL ASSEMBLIES: EQUILIBRIUM DYNAMICS

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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.