We report data on the structural dynamics of the neuropeptide Y (NPY) G-protein-coupled receptor (GPCR) type 1 (Y1R), a typical representative of class A peptide ligand GPCRs, using a combination of solid-state NMR and molecular dynamics (MD) simulation. First, the equilibrium dynamics of Y1R were studied using 15N-NMR and quantitative determination of 1H-13C order parameters through the measurement of dipolar couplings in separated-local-field NMR experiments. Order parameters reporting the amplitudes of the molecular motions of the C-H bond vectors of Y1R in DMPC membranes are 0.57 for the Cα sites and lower in the side chains (0.37 for the CH2 and 0.18 for the CH3 groups). Different NMR excitation schemes identify relatively rigid and also dynamic segments of the molecule. In monounsaturated membranes composed of longer lipid chains, Y1R is more rigid, attributed to a higher hydrophobic thickness of the lipid membrane. The presence of an antagonist or NPY has little influence on the amplitude of motions, whereas the addition of agonist and arrestin led to a pronounced rigidization. To investigate Y1R dynamics with site resolution, we conducted extensive all-atom MD simulations of the apo and antagonist-bound state. In each state, three replicas with a length of 20 μs (with one exception, where the trajectory length was 10 μs) were conducted. In these simulations, order parameters of each residue were determined and showed high values in the transmembrane helices, whereas the loops and termini exhibit much lower order. The extracellular helix segments undergo larger amplitude motions than their intracellular counterparts, whereas the opposite is observed for the loops, Helix 8, and termini. Only minor differences in order were observed between the apo and antagonist-bound state, whereas the time scale of the motions is shorter for the apo state. Although these relatively fast motions occurring with correlation times of ns up to a few µs have no direct relevance for receptor activation, it is believed that they represent the prerequisite for larger conformational transitions in proteins.
THE NUMBER of antibiotic agents is constantly increasing. At present there are available, at least for clinical investigation, the following antibiotics: penicillin; streptomycin; dihydrostreptomycin; neomycin; polymyxin B, or aerosporin; tyrothricin; bacitracin; aureomycin, and chloramphenicol U. S. P. (D-[—]-threo-2-dichloroacetamido-1-para-nitrophenyl-1,3-propanediol), also known as chloromycetin.® Many other new antibiotics are still not ready for clinical investigation but are under study at the present time. It appears that more antibiotics will rapidly become available in the future. Many factors keep the problem of antibiotic therapy from being a simple one. A few years ago it was easy to decide which antibiotic to use in a particular case on the basis of whether the infecting organism was gram-positive or gram-negative. Now, however, one must choose between several antibiotics for gram-positive organisms, several for gram-negative organisms and still others for viruses and rickettsias. One must now decide which antibiotic will be the most effective,
Many proteins involved in signal transduction are equipped with covalently attached lipid chains providing a hydrophobic anchor targeting these molecules to membranes. Despite the considerable biological significance of this membrane binding mechanism for 5−10% of all cellular proteins, to date very little is known about structural and dynamical features of lipidated membrane binding domains. Here we report the first comprehensive study of the molecular dynamics of the C-terminus of membrane-associated full-length lipidated Ras protein determined by solid-state NMR. Fully functional lipid-modified N-Ras protein was obtained by chemical-biological synthesis ligating the expressed water soluble N-terminus with a chemically synthesized 2H or 13C labeled lipidated heptapeptide. Dynamical parameters for the lipid chain modification at Cys 181 were determined from static 2H NMR order parameter and relaxation measurements. Order parameters describing the amplitude of motion in the protein backbone and the side chain were determined from site-specific measurements of 1H-13C dipolar couplings for all seven amino acids in the membrane anchor of Ras. Finally, the correlation times of motion were determined from temperature dependent relaxation time measurements and analyzed using a modified Lipari Szabo approach. Overall, the C-terminus of Ras shows a versatile dynamics with segmental fluctuations and axially symmetric overall motions on the membrane surface. In particular, the lipid chain modifications are highly flexible in the membrane.
