Nuclear magnetic resonance spectroscopy is a powerful and unique tool for the study of matter in its various states, including solids, liquids, liquid crystals, and biological materials. With solid-state 2H NMR spectroscopy, we obtain experimental data pertinent to both structure and dynamics. The role of lipid bilayer membranes is crucial. First, the membrane lipids provide a semipermeable barrier that modulates the passage of molecules into and out of the cell. Second, the membrane provides an interface and matrix for proteins that support their structural and functional properties. Both of these roles of the membrane are intrinsically related and are in large part determined by lipid structure and dynamics. Nuclear magnetic resonance spectroscopy is one of the premier tools for studying the phospholipid membrane.
From an application standpoint, nuclear spin couplings and relaxation measurements allow characterization of structural and dynamic adaptations of membrane lipids on multiple time and length scales. A major emphasis involves developing solid-state NMR techniques and relaxation theory for the study of molecular solids and liquid crystals. We are presently utilizing 2H NMR to investigate influences of the bilayer thickness, polyunsaturation, polar head groups, and the incorporation of detergents, cholesterol, and proteins such as rhodopsin on the elastic properties of phospholipid membranes.
Structure and Dynamics of Proteins Studied by Multidimensional NMR Spectroscopy
Additional work involves extending and applying the above approaches to proteins, to obtain information on their molecular dynamics over a wide range of time scales. As a rule, the relaxation times of proteins depend on the mean-squared amplitudes of the motions of the various functional groups and the associated correlation times. Hence one can deduce information regarding the average structure as well as dynamical properties of the polypeptide backbone and amino acid side chains. Using multidimensional NMR spectroscopy in conjunction with modern NMR relaxation methods, one can thus understand how protein structural and dynamical properties are related to chemical reactivity, and to biological function.
Solid-State NMR Spectroscopy of Rhodopsin and Molecular Modeling of G Protein-Coupled Receptors
Another major area of research involves the use of solid-state NMR techniques to investigate membrane proteins which are receptors for light, hormones, and neurotransmitters. Cell membrane receptors are pharmacologically important and constitute about 45% of all molecular drug targets of current therapies. The initial analysis of the human genome has located more than 500 genes involving the rhodopsin-like superfamily of G protein-coupled receptors(GPCRs).
Here we use rhodopsin and its cognate G protein transducin as a model to illuminate general features of GPCR and G protein activation. Our strategy involves deuterium labeling of retinylidene ligand of rhodopsin and C terminal peptide of transducin in conjunction with 2H NMR spectroscopy.
In this manner, one can obtain information at atomic level resolution regarding the structure and dynamics of the membrane-associated peptides and proteins that cannot be prepared for X-ray diffraction structural studies.
The study of retinal ligand has shown us that motions are different for different methyl group at different carbon position of retinal, can conclude that the C9 methyl group, with lowest activation energy barrier, is the hot pot during the light activation of rhodopsin. Current we study the C terminal peptide of transducin, which has to bind to active rhodopsin in order to activate transducin.
The study of this peptide can help us understand the general activation mechanism of G proteins. And let us know if there is coupling between the local motions of ligand and peptide and the global motions of proteins. This work will be interesting to the molecular dynamics (MD) scientists, since the study of local motion can help define the force field of the protein motions. Future work involves an extension to other integral membrane proteins and receptors.
Exciting studies with membrane-lipid-protein interactions
Rhodopsin is a membrane protein which is responsible for vision in the dim light. Membrane lipids play a key role in the rhodopsin function and activation. It is known that rhodopsin activation leads to structural changes in the protein establishing equilibrium between inactive Metarhodopsin-I (Meta-I) and activated Metarhodopsin-II (Meta-II) states. The canonical thinking is that rhodopsin activation is a simple on-off switch with Meta-I being the inactive state and Meta-II being the active state.
In our study, using UV-visible and FTIR spectroscopy for the protein reconstituted in the membrane lipids, we have shown for the first time that rhodopsin activation is not a simple on-off switch but there is an ensemble of functional substrates explained by the ensemble activation mechanism (EAM).
The results of EAM is in agreement with the flexible surface model (FSM) which describes the elastic coupling of the integral membrane lipids through a balance of curvature and hydrophobic forces in lipid-protein interaction. Furthermore, the thermodynamic analysis shows that the rhodopsin activation is analogous to the protein unfolding reaction where the increase in entropy compensates for unfavorable enthalpy changes on rhodopsin activation. The study further gives an understanding of activation of other rhodopsin-like GPCRs in natural environment system.