Research Overview

Many micro-organisms produce retinal containing membrane proteins similar to the mammalian visual pigments. These rhodopsins include energy transducers that use light to drive ion transport, as well as signal transducers that use light to stimulate phototaxis. The rhodopsins are thus light-driven analogues of the chemically-driven energy transducers (membrane ATPases) and signal transducers (hormone receptors) found in mammalian cells. The first rhodopsin to be discovered in a unicellular organism (known therefore as bacteriorhodopsin) is also the most abundant. It undergoes a photocycle in which an ion is released on one side of the membrane and replaced from the other side of the membrane. Thus, light is used to create an electrochemical potential gradient across the membrane that the cell can use to drive other processes. To study bacteriorhodopsin in the native membrane, we employ solid-state NMR methods that achieve the high resolution of solution spectra while preserving the three-dimensional information of powder spectra. The results are interpreted empirically using data from model compounds and theoretically via quantum mechanical calculations. Specifically, we are probing the features of the photocycle intermediates that enforce unidirectional ion transport. Recent results point to the release of photon-induced torsion in the chromophore as responsible for the switch in hydrogen bond connections that prevents ion backflow. Other recent results suggest that bacteriorhodopsin may be an inwardly-driven hydroxyl pump rather than an outwardly-driven proton pump.

Gas vesicles present a particularly intriguing protein folding problem. Their thin shells must withstand considerable pressure while presenting a highly hydrophobic interior surface to prevent the condensation of permeating water vapor. But it is difficult to imagine how such a shell is created by the self-association of many small (7 kDa) subunits. Gas vesicles also have significant similarities with amyloid fibers: they are not dissolved by detergents and beta-sheet is a prominent, conserved feature. Yet, the host organisms can recycle the subunits, indicating that they are able to reversibly disassemble the gas vesicles. The potential for controlling amyloid pathologies might be enhanced by understanding how aquatic organisms manage their gas vesicle subunits. To determine the structure of gas vesicles, we employ recently developed multi-dimensional solid-state NMR methods. Resonances are assigned by homo- and hetero-nuclear correlations, and structural constraints are obtained by measuring dipolar interactions that reflect internuclear distances.

Proton transfer is important in many chemical and biochemical processes, including those that support energy transduction in organisms and in fuel cells. At this time the dominant tools for simulating these reactions either rely directly on quantum mechanical calculations or make heavy use of them to parametrize potential energy surfaces. In contrast, we have pursued an approach that side-steps wave-function calculations by treating electron pairs as point particles. Key to this approach is absorbing the quantum mechanical effects in the potentials among electron pairs and between electron pairs and nuclei. With these pseudopotentials, the particles are able to produce stable neutral, protonated and deprotonated water monomers and water clusters, all with bond angles and bond lengths in close agreement with experiment. In addition, experimental energies for water dissociation, proton transfer and hydrogen bonding are well reproduced. Extension to other protic systems and more general reaction force fields is underway.

Scenarios for the origins of life begin with the abiotic formation of biologically significant molecules from simple inorganic precursors. Much of our current understanding of this prebiotic chemistry relates to small molecules. However, polymers are also thought to have played significant roles. For example, HCN, which is thought to have been abundant in the prebiotic environment, spontaneously forms polymers that have been the focus of much speculation. Using the unique probes afforded by solid state NMR, we have been able to identify three different types of HCN polymers, all of which are unlike any of the structures that have been previously proposed. Sugars, another large class of prebiotically abundant molecules, also form polymers readily. Again, solid state NMR is providing the first insights into the structures of these macromolecules.

Cytoskeletal proteins have been the subject of intense experimental scrutiny. Of particular interest is the molecular basis for the spatial organization of cytoskeletal fibers in cells. Typically it is assumed that protein polymers should be randomly disposed in solution and that the non-random organization of cytoskeletal fibers must be due to the effects of various accessory binding proteins. However, the cytosol is a very crowded and therefore highly non-ideal solution. Under these circumstances, the theory of liquid crystals tells us that elongated particles may spontaneously align, coalesce into bundles, and form gels. We adapt these theories to heterogeneous systems with self-assembling fibers to characterize the various mechanisms by which cells can control the spatial arrangement of cytoskeletal elements. We find that, under physiological conditions, long filaments are not only predicted to form bundles spontaneously, but these bundles will be spontaneously segregated according to the diameters and flexibilities of the filaments. The function of cross-linking by "bundling proteins" therefore appears to be only to fine tune the bundles (e.g., as to polarity and registration). The theory also predicts that the cell can prevent crowding-induced bundling by using its capping proteins to reduce the lengths of the filaments. And it can frustrate bundling and form a gel by using other accessory proteins that cross-link filaments in orthogonal configurations.