Mediation Of Aromatic Residues On Lipid Bilayers And 7TM Receptors Activation

Membrane proteins are main components of the cell membrane, representing about 20-30% of the proteomes of most organisms, and serving a multitude of cellular functions, such as photosynthesis, respiration, neural signaling, immune response, nutrient absorption, and serving more than 50% of drug targets. Clearly, knowledge of a membrane protein structure will enable us to insight into its structure-function relationship, dynamic conformation, and further for rational drug design.Over the past years, nuclear magnetic resonance (NMR) technique has made tremendous progress showing its capability of determining a protein structure and studying protein dynamic conformation on a variety of time scales at atomic resolution. Since there is no special requirements and restrictions on the sample form and molecular weight, solid-state NMR (ssNMR) is a suitable method for the study of membrane protein structural biology. Combining a variety of labeling strategies and multidimensional correlation experiments, ssNMR can be used to determine three-dimensional structure of membrane proteins, and to study protein dynamic conformation, ligand binding and dynamic activation mechanism in the native membrane lipid environment, and has its unique advantage over the other techniques.Mediation mechanism of the aromatic residues on cellular membranes in hydrophilic peptides and peripheral proteins has been an issue for a long time, deep and systematic studies have been carried out in this thesis first to probe location and dynamic conformation of tryptophan and other aromatic amino acids within phospholipid membranes by using high-resolution 1H NMR. Our results show that rather than having a preference in the membrane bilayers, location and penetration of the basic and aromatic residues depend on the sequence and structural context of the peptide in which the residues are located in the membrane bilayers.In order to study the function of Tyrosine 185(Tyrl 85) and Tyrosine 57(Tyr57) in photoreaction mechanism of the seven trans-membrane receptor bacteriorhodopsin (bR), we have focused on couplings of Tyr185 and Tyr57 with protonated Schiff base with respect to the thermal isomerization of retinal chromophore at the bR ground state, and stabilization of the pentagonal hydrogen-bonded network containing Asp85-Asp212-Arg82 on the extracellular side of the protonated Schiff base during photoreaction. Heteronuclear polarization transfer through a single spin-pair, combined with homonuclear correlation expetimens, UV spectroscopy, molecular dynamic simulations, lighted-induced transient absorption change measurements and mutagenesis, has been established as a powerful tool for providing insight into the conformation and coupling for Tyr185 and Tyr57 with retinal chromophore and the Asp85-Asp212-Arg82 hydrogen-bonded network at the ground state and activite states.Choosing an appropriate sample environment is very important to correctly interpret a membrane protein structure and function. In this study, dynamic regulation of bR function under alkaline pH conditions has been investigated by magic-angle spinning solid-state NMR through chemical shifts, torsion angle and C-H bond length measurements on reconstituted bR purple membrane samples. Combined with UV spectroscopy, light-induced transient absorption changes and molecular dynamic simulations, possible perturbation mechanism from the retinal binding pocket to the overall structure have been discussed. Our results show that the conformational change of the local residues at the binding pocket may lead to the increase of partial flexible of bRcis, and further shifts the cis-trans thermal equilibrium to a more bRtrans populated state. Deprotonation of the alkaline residues on the helices C, D, F and G may result in the reformation of the H-bond network on both proton release and uptake channels and further the cause of elongation and attenuation of the M and N states, and vanish of the O state in the bR photocycle.This work is not only of great significance to a deeper understanding of bR structure, function of key residues and proton pumping mechanism, but is also of great significance to insight into structure-function relationship of other retinal protein family members. Since bR possesses a highly sequence similarity and a similar activation mechanism with G Protein Coupled Receptors (GPCRs), which are drug targets of a variety of diseases, the outcome of our studies could also guide the research on structure, function and relate diseases of any other rhodopsin-like GPCR, and provide some basic experimental data on the structure based GPCR drug design.