Session J46: Invited Session: Physics of Proteins: Integrating Computation with Experiment

Protein Folding Transition Paths: Single Molecule Experiments, Theory and Simulations

The transition-path is the tiny fraction of an equilibrium, single-molecule trajectory when the transition over a free-energy barrier occurs between two states. In the case of protein folding, the distribution of transition paths contains all of the mechanistic information on how a protein folds and unfolds. Transition path distributions can now be predicted for fast folding proteins by all-atom molecular dynamics simulations and by an Ising-like theoretical model [1,2]. Experimental information on transition paths should provide the most demanding test of both simulations and theoretical models. However, transition-paths for barrier crossings have never been observed experimentally for any molecular system in solution. Because it is a single molecule property, even determining the average transition-path time is challenging. In this presentation, I will discuss how we use measurements of Foerster resonance energy transfer in single molecule fluorescence experiments and a photon-by-photon analysis to measure average transition path times for proteins of different topology and folding rate coefficients using the Gopich/Szabo maximum likelihood method [3.4]. These results, which are surprisingly interesting, are just the first, but important, steps toward measuring intra-molecular distances during individual transition paths. [1] Best, R.B.; Hummer, G.; Eaton. W.A. Native contacts determine protein folding mechanisms in atomistic simulations.'' Proc. Natl. Acad. Sci. USA 2013, 110, 17874; [2] Henry, E.R.; Best, R.B.; Eaton, W.A. Comparing a simple theoretical model for protein folding with all-atom molecular dynamics simulations. Proc. Natl. Acad. Sci. USA 2013,110, 17880; [3] Chung, H.S.; McHale, K.; Louis, J.M.; Eaton, W.A. Single-molecule fluorescence experiments determine protein folding transition path times. Science 2012, 335, 981; [4] Chung, H.S.; Eaton, W.A.Single molecule fluorescence probes dynamics of barrier crossing. Nature 2013, 502, 685.   

Exploring the active site structure of photoreceptor proteins by Raman optical activity

Understanding protein function at the atomic level is a major challenge in a field of biophysics and requires the combined efforts of structural and functional methods. We use photoreceptor proteins as a model system to understand in atomic detail how a chromophore and a protein interact to sense light and send a biological signal. A potential technique for investigating molecular structures is Raman optical activity (ROA), which is a spectroscopic method with a high sensitivity to the structural details of chiral molecules. However, its application to photoreceptor proteins has not been reported. Thus we have constructed ROA spectrometer using near-infrared (NIR) laser excitation at 785 nm. The NIR excitation enables us to measure ROA spectra for a variety of biological samples, including photoreceptor proteins, without fluorescence from the samples. In the present study, we have applied the NIR-ROA to bacteriorhodopsin (BR) and photoactive yellow protein (PYP). BR is a light-driven proton pump and contains a protonated Schiff base of retinal as a chromophore. PYP is a blue light receptor, and this protein has the 4-hydroxycinnamyl chromophore, which is covalently linked to Cys69 through a thiolester bond. We have successfully obtained the ROA spectra of the chromophore within a protein environment. Furthermore, calculations of the ROA spectra utilizing density functional theory provide detailed structural information, such as data on out-of-plane distortions of the chromophore. The structural information obtained from the ROA spectra includes the positions of hydrogen atoms, which are usually not detected in the crystal structures of biological samples.   

integrating Solid State NMR and Computations in Membrane Protein Science

Helical membrane protein structures are influenced by their native environment. Therefore the characterization of their structure in an environment that models as closely as possible their native environment is critical for achieving not only structural but functional understanding of these proteins. Solid state NMR spectroscopy in liquid crystalline lipid bilayers provides an excellent tool for such characterizations. Two classes of restraints can be obtained - absolute restraints that constrain the structure to a laboratory frame of reference when using uniformly oriented samples (approximately 1$^{\circ}$ of mosaic spread) and relative restraints that restrain one part of the structure with respect to another part such as torsional and distance restraints. Here, I will discuss unique restraints derived from uniformly oriented samples and the characterization of initial structures utilizing both restraint types, followed by restrained molecular dynamics refinement in the same lipid bilayer environment as that used for the experimental restraint collection. Protein examples will be taken from Influenza virus and Mycobacterium tuberculosis. When available comparisons of structures to those obtained using different membrane mimetic environments will be shown and the causes for structural distortions explained based on an understanding of membrane biophysics and its sophisticated influence on membrane proteins.   

Discovering novel ligands for understanding molecular mechanism of bacteria chemotaxis

In order to understand the molecular mechanism of bacteria chemotaxis, we used a combined experimental and computational approach to discover novel chemoeffector molecules and compare their binding features, as well as the conformational changes they produce. We first used molecular docking to computationally screen a large chemical library and tested binding strengths of the top-ranking molecules for the E. coli chemoreceptor Tar. Chemotactic properties of the binding molecules were then studied using a specially designed microfluidic device. Novel attractant and antagonist molecules were identified that bind directly with the E. coli chemoreceptor Tar. Molecular dynamics simulations showed that attractant and antagonist binding result in distinct conformational changes in Tar. Differences of antagonist and attractant binding suggest that molecules lacking triggering interaction with the receptor behave as antagonist. For Tar, the triggering interaction is mediated by the hydrogen bonds formed between a donor group in the attractant and the main-chain carbonyls in the fourth helix of Tar. This ?bind-and-trigger? mechanism of receptor signaling is verified experimentally by converting an antagonist into an attractant when introducing an NH group into the antagonist compound. Similar conformational changes were also observed in the E. coli Tsr system.