The advent of single molecule fluorescence microscopy has allowed experimental molecular biophysics and biochemistry to transcend traditional ensemble measurements, where the behavior of individual proteins could not be precisely sampled. The recent explosion in popularity of new super-resolution and super-localization techniques coupled with technical advances in optical designs and fast highly sensitive cameras with single photon sensitivity and millisecond time resolution have made it possible to track key motions, reactions, and interactions of individual proteins with high temporal resolution and spatial resolution well beyond the diffraction limit. Within the purview of membrane proteins and ligand gated ion channels (LGICs), these outstanding advances in single molecule microscopy allow for the direct observation of discrete biochemical states and their fluctuation dynamics. Such observations are fundamentally important for understanding molecular-level mechanisms governing these systems. Examples reviewed here include the effects of allostery on the stoichiometry of ligand binding in the presence of fluorescent ligands; the observation of subdomain partitioning of membrane proteins due to microenvironment effects; and the use of single particle tracking experiments to elucidate characteristics of membrane protein diffusion and the direct measurement of thermodynamic properties, which govern the free energy landscape of protein dimerization. The review of such characteristic topics represents a snapshot of efforts to push the boundaries of fluorescence microscopy of membrane proteins to the absolute limit.

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Protein conformation change depending not only on the values of temperature, denaturant concentration but also on the values of solvent pH. The difference of the pH-denaturation from the thermal or urea denaturation is that hydrogen atoms (un)bind exclusively to R, K, Y, C, H, D, E amino acids. Thus the pH effect on the protein conformation is selective so that the physico-chemical machinery for the biological function of a protein frequently has its origin due to the solvent pH. Although several previous approaches were suggested to elucidate the (un)protonation behavior of a protein conformation, those were mainly oriented on evaluating pKa values of titratable residues in a given static protein conformation. The theoretical and calculation framework for describing the effect of solvent pH to the thermodynamic and kinetic properties of proteins under the equilibrium fluctuation is indispensable for the fundamental understanding of important biological phenomena of proteins. Here we present a development of the pH-dependent free energy function of proteins incorporating its equilibrium fluctuations based on the concept of statistical physics. The validity of our approach is justified by reproducing the experimental pKa values of titratable residues in several proteins. We also present the analytical and calculation framework for describing the pHdependent thermodynamics and folding kinetics of proteins by the exact calculation. The effects of pH not only on the free energy landscape but also on the folding characters of several proteins are discussed.

then tested the GLIC-ELIC chimera with crotonic acid and picrotoxin. Crotonic acid inhibits GLIC with an IC50 of 110mM; our data indicate it binds to the extracellular domain. Picrotoxin (IC50¼2.6mM) blocks the GLIC pore(4); it likely cannot access the ELIC pore(5), but may bind to the extracellular domain (IC50¼96mM;6). These compounds were less potent than expected in the chimera (IC50>300mM). Overall the data suggest that domain specific effects may not be accurately reproduced in complex chimeras with intercommunicating domains, such as an orthosteric binding site and a pore in ligand-gated ion channels.