Suchithranga M.D.C. Perera

Investigation of Rhodopsin Dynamics in Its Signaling State by Solid-State Deuterium NMR Spectroscopy

Site-directed deuterium NMR spectroscopy is a valuable tool to study the structural dynamics of biomolecules in cases where solution NMR is inapplicable. Solid-state (2)H NMR spectral studies of aligned membrane samples of rhodopsin with selectively labeled retinal provide information on structural changes of the chromophore in different protein states. Moreover (2)H NMR relaxation time measurements allow one to study the dynamics of the ligand during the transition from the inactive to the active state. Here we describe the methodological aspects of solid-state (2)H NMR spectroscopy for functional studies of rhodopsin, with an emphasis on the dynamics of the retinal cofactor. We provide complete protocols for the preparation of NMR samples of rhodopsin with 11-cis-retinal selectively deuterated at the methyl groups in aligned membranes. In addition we review optimized conditions for trapping the rhodopsin photointermediates; and we address the challenging problem of trapping the signaling state of rhodopsin in aligned membrane films.

Quasi-elastic Neutron Scattering Reveals Ligand-Induced Protein Dynamics of a G-Protein-Coupled Receptor

Light activation of the visual G-protein-coupled receptor (GPCR) rhodopsin leads to significant structural fluctuations of the protein embedded within the membrane yielding the activation of cognate G-protein (transducin), which initiates biological signaling. Here, we report a quasi-elastic neutron scattering study of the activation of rhodopsin as a GPCR prototype. Our results reveal a broadly distributed relaxation of hydrogen atom dynamics of rhodopsin on a picosecond-nanosecond time scale, crucial for protein function, as only observed for globular proteins previously. Interestingly, the results suggest significant differences in the intrinsic protein dynamics of the dark-state rhodopsin versus the ligand-free apoprotein, opsin. These differences can be attributed to the influence of the covalently bound retinal ligand. Furthermore, an idea of the generic free-energy landscape is used to explain the GPCR dynamics of ligand-binding and ligand-free protein conformations, which can be further applied to other GPCR systems.

Powdered G-Protein-Coupled Receptors.

Preparation and storage of functional membrane proteins such as G-protein-coupled receptors (GPCRs) are crucial to the processes of drug delivery and discovery. Here, we describe a method of preparing powdered GPCRs using rhodopsin as the prototype. We purified rhodopsin in CHAPS detergent with low detergent to protein ratio so the bulk of the sample represented protein (ca. 72% w/w). Our new method for generating powders of membrane proteins followed by rehydration paves the way for conducting functional and biophysical experiments. As an illustrative application, powdered rhodopsin was prepared with and without the cofactor 11-cis-retinal to enable partial rehydration of the protein with D2O in a controlled manner. Quasi-elastic neutron scattering studies using both spatial motion and energy landscape models form the basis for crucial insights into structural fluctuations and thermodynamics of GPCR activation.

Membrane Lipid-Protein Interactions

In this review we describe how the properties of cellular membranes govern protein structure and activity. Lipids can modulate protein functional states through general bilayer properties, or by specific binding and acting as allosteric regulators. Hydrophobic matching by solvation of the protein surface entails short-range interactions of the lipids, and cellular water affects bilayer structure through hydrating the lipid polar head groups and protein hydrophilic domains. Biomembranes have important analogies to supercritical fluids leading to raft-like nanostructures with cholesterol. Additional long-range interactions of the lipids and proteins involve the curvature stress field, where a flexible surface model (FSM) describes how collective properties of the lipids affect the conformational energetics of membrane proteins. Curvature elasticity and hydrophobicity of native lipid mixtures play key roles in functional proteolipid couplings and give insights into protein activation mechanisms in cellular membranes.