Filming protein and water motion within a membrane: We focus on uncovering the fundamental principles governing transmembrane proteins, assemblies that serve as the “gatekeepers” of the cell and are physiologically vital for all forms of life, critically positioned in the hydrophobic membrane surrounding cells. The particular model system studied, the seven-helical Proteorhodopsin (PR), is a light-activated proton pump thought to harvest solar energy for very abundant marine bacteria. The approach taken is to develop site-specific magnetic resonance tools to capture the influential properties of hydration (by Overhauser dynamic nuclear polarization, ODNP) and protein dynamics (by electron paramagnetic resonance, EPR), and relate them to transmembrane protein function, which for the photoactive PR can be quantified using optical absorption measurements.
We observe the mechanics of PR activation, uniquely from the point of view of surface water dynamics. As important as conformational dynamics are for a comprehensive understanding of protein function, few experimental measurements of such are available, especially for large complexes such as membrane proteins. In addition, little is known about the role of water in these largely hydrophobic systems, though there is evidence that water as a solvent drives the dynamic behavior of better-characterized globular proteins and thereby enables their function. Our study of the function-relevant E-F loop segment of PR revealed that there is a distinct rearrangement of surface hydration water upon light activation, which is also greatly modulated by the surrounding surfactant environment (e.g. detergent micelle vs. lipid bilayer). The altered hydration landscape is accompanied by altered kinetics of light-induced motion, suggesting that the water and lipid solvents could be inherently linked influences on membrane protein function.
The spectral signatures of activation obtained from this study provide us with a molecular level snapshot of dynamics that allows the further investigation of environmental effects, such as interactions with the surfactant or with other proteins, as happens in a crowded lipid membrane. We have found that oligomerization, or association of multiple PRs within the membrane-mimetic detergent micelle or native cell membranes, has a profound influence on both the surface and internal properties of the channel, manifested by functional differences. Our results have proven to be useful towards the design of PR-surfactant complexes optimized for a given function, for example energy generation in a solar cell material incorporating PR. What we have learned from PR could parallel the case of higher-order receptors implicated in disease, which also experience similar complex interactions within the membrane.
Knowing the topology and location of protein segments at water-membrane interfaces is critical for rationalizing their functions, but their characterization is challenging under physiological conditions. Our group is pioneering a novel spectroscopic approach to the structure study of membrane-associating proteins by employing the hydration dynamics gradient found across the phospholipid bilayer as an intrinsic ruler for determining the topology, immersion depth, and orientation of protein segments in lipid membranes, particularly at water-membrane interfaces. We pursue this through the site-specific quantification of translational diffusion of hydration water using an emerging tool, 1H Overhauser dynamic nuclear polarization (ODNP)-amplified NMR relaxometry. To name one example, ODNP confirms that the membrane-bound region of ?-synuclein (?S), an amyloid protein known to insert an amphipathic ?-helix into negatively charged phospholipid membranes, forms an extended ?-helix parallel to the membrane surface. We further extend the current knowledge by showing that residues 90-96 of bound ?S, which is a transition segment that links the ?-helix and the C-terminus, adopt a larger loop than an idealized ?-helix. The unstructured C-terminus gradually threads through the surface hydration layers of lipid membranes, with the beginning portion residing within 5-15 Å above the phosphate level, and only the very end of C-terminus surveying bulk water. Remarkably, the intrinsic hydration dynamics gradient along the bilayer normal extends to 20-30 Å above the phosphate level, as demonstrated with another peripheral membrane protein, annexin B12. ODNP offers the opportunity to reveal previously unresolvable structure and location of protein segments well above the lipid phosphate level, whose structure and dynamics critically contribute to the functional versatility of membrane proteins.
- Chi-Yuan Cheng (Collaboration with Prof. Ralf Langen at USC, Dr. M. Ambroso, Dr. J Varkey)
A particular focus of our group’s effort is to develop a set of novel experimental approaches for enhancing the sensitivity and selectivity of magnetic resonance detection by two orders of magnitude compared to existing NMR and EPR experiments. These techniques are employed to pursue detailed study of early protein aggregation mechanisms with high sensitivity and under in situ, solution, conditions. Our target includes time-resolved visualization of molecular interfaces during protein aggregation, in the presence of lipid membranes and/or as a function of chemical signals. These studies are made possible with selective (nitroxide) spin labeling of interfacial protein sites and the sensitive detection of even weak and transient molecular interactions, through the measurements of interfacial hydration water dynamics that is most sensitively modulated upon molecular approach within distant 1 nanometer of the spin labeled sites, as well as through the enhanced measurement of electron spin-spin distances and dynamics. We pursue these new measurements with Overhauser DNP (ODNP) technique developed in our lab and with unprecedented pulse shaping capability to significantly enhance pulsed EPR performance. Long term goals include the transient probing of early protein conformational changes preceding aggregation, the characterization of the soluble oligomer’s size and structure, and the transient probing of changes in membrane permeability.
Overhauser DNP (ODNP) is an emerging and novel magnetic resonance technique that has been used in several of our studies to perform highly localized measurements of translational diffusivity on molecular and lipid membrane surfaces. We find that the local hydration dynamics on the surface and interface of lipid membranes are intimately modulated by the same factors that are known to modulate their key properties, such as Hofmeister cations and anions, various osmolytes, cholesterol, and surface-active polymers, such as poloxamers. As these surface-active species dramatically modulate surface water dynamics, i.e. the compressibility of the surface hydration later, they directly tune the approachability of biomacromolecules to lipid membrane surfaces. Interestingly, crowding agents that may dramatically increase the bulk water viscosity, such as sucrose, glycerol or ficoll, do not retard the lipid membrane surface dynamics accordingly. Specifically, both a >10-fold increase in bulk viscosity and the restriction of water diffusion by confinement on a multiple-nm length-scale, do not change the local translational diffusion coefficient of the membrane surface water by much, namely less than 2.5-fold. We believe that the surface topology and chemistry at the ≤ 1 nm scale, rather than the characteristics of the bulk fluid, nearly exclusively determine the surface hydration dynamics of aqueous macromolecular solutes. These studies impact the unraveling of lipid membrane biophysics, as well as the mechanistic understanding of inter-protein and protein-lipid membrane interaction, a long-term goal of our lab.