The long term goals of this project are to understand the molecular basis for specific functions in cell membranes. The proposed work focuses two relatively accessible systems from the archae Halobacterium halobium: the purple membrane and the gas vacuole membrane. The purple membrane contains a single protein, bacteriorhodopsin, that functions as a light-driven proton pump. It is thus a convenient system for a detailed examination of an ion transport pathway and the processing of energy for gating. In addition, because bacteriorhodopsin is structurally related to a large class of retinal-based light transducers, including our own visual pigments, it provides an opportunity to elucidate the means by which opsins tune the retinal spectrum and control retinal isomerization. The gas vacuole proteins (one major and one minor) form the lipid-free, pressure sustaining membrane of a compartment that, once formed, passively excludes water. This system thus presents basic issues of protein structure in exaggerated form: the organization of specific polar components to form a pressure resistant structure on the one hand and the organization of non-specific hydrophobic components to exclude water on the other. To study the molecular mechanisms responsible for the effectiveness of these systems in a non-perturbing fashion, we will use recently developed solid state NMR techniques, as well as more routine solid state NMR spectroscopy, some FTIR spectroscopy and some electron microscopy. The proteins will be isotopically labeled at sites of interest. Slow exchange of deuterium from water to specific sites in the protein will be followed by FTIR and NMR spectroscopy. Faster movement will be monitored by the effects of delayed cross polarization in ID 15N NMR spectra and the development of cross peaks in 2D 1H and 3H spectra. Structural changes in the bacteriorhodopsin photocycle intermediates, particularly the controversial L photointermediate, will be characterized by internuclear distance measurements in multiply labeled samples, using the rotational resonance (R2), radiofrequency driven recoupling (RFDR), rotational-echo double-resonance (REDOR) and frequency selective dipolar recoupling (FDR) techniques. Homonuclear distance measurements will also be used to measure bond lengths in the retinal backbone, in order to assess bond orders and rotational barriers influencing isomerization.
Our specific aims are to use these techniques to contribute to our understanding of the proton transport pathway, the gating mechanism, the spectral tuning mechanism, and the control of chromophore isomerization in bacteriorhodopsin, and to the folding and interfacial properties of gas vacuole proteins.
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