The objective of this Project is to characterize crystallographically the changes in protein conformation that occur at distinct stages in the cycle of proton pumping in bacteriorhodopsin. This goal will be accomplished by using high resolution electron diffraction data to compute three- dimensional difference Fourier maps for a number of the different intermediate states in the photocycle of this protein. Recently completed work has produced two-dimensional (projection) difference Fourier maps for two of the intermediate states, designated as M1 and M2. These 2-D projection maps demonstrate that it is indeed possible to see small changes in conformation by the method proposed here. The 2-D projection studies of M1 and M2 will therefore be extended to give high resolution, three-dimensional difference maps by collecting similar data at high tilt angles. The expected outcome is that it will be possible to relate changes in position and tilt angle of transmembrane helices and/or changes in position of critical side-chain groups to the molecular biophysics of the proton-pumping event. After completion of the 3-D, high resolution difference Fourier analysis of the M-state intermediates, work will begin on the L-state (which precedes M) and perhaps on the N-state (which follows M). The structural understanding that will emerge from this work is expected to give us a much improved knowledge of the physical process by which certain membrane proteins are able to actively transport charged ions across cell membranes, thereby creating gradients in ion concentration and membrane potential that, in turn, are vital to metabolism, neuro- and muscular-function, and proper homeostasis.
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| Sazzed, Salim; Song, Junha; Kovacs, Julio A et al. (2018) Tracing Actin Filament Bundles in Three-Dimensional Electron Tomography Density Maps of Hair Cell Stereocilia. Molecules 23: |
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| Howes, Stuart C; Geyer, Elisabeth A; LaFrance, Benjamin et al. (2018) Structural and functional differences between porcine brain and budding yeast microtubules. Cell Cycle 17:278-287 |
| Glaeser, Robert M (2018) PROTEINS, INTERFACES, AND CRYO-EM GRIDS. Curr Opin Colloid Interface Sci 34:1-8 |
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