Research Overview

Halophilic archae 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: their structure is primarily beta-sheet and they are not dissolved by detergents. 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 and bond torsion angles.

Water is often a functionally important part of the active sites of proteins. In order to understand how it can play more than a structural role, it is necessary to take its polarizability and its amphiproticity into account. Of course, this is best done by quantum mechanics. However, most systems of interest are sufficiently large that it is useful to have models that are less computer intensive. On the other hand, classical models of the water molecule generally comprise a fixed array of partial charges that is incapable of polarization or ionization. Our approach is to start with a Lewis-inspired model of water and add quantum features to the interactions between the submolecular particles. 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. Using this model we are simulating water in various simple environments, as a prelude to simulations of water inside proteins.

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 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.