G Grobner

Photoreceptor rhodopsin: structural and conformational study of its chromophore 11-cis retinal in oriented membranes by deuterium solid state NMR

Rhodopsin is the retinal photoreceptor responsible for visual signal transduction. To determine the orientation and conformation of retinal within the binding pocket of this membrane bound receptor, an ab initio solid state 2H NMR approach was used. Bovine rhodopsin containing 11‐cis retinal, specifically deuterated at its methyl groups at the C19 or C20 position, was uniaxially oriented in DMPC bilayers. Integrity of the membranes and quality of alignment were monitored by 31P NMR. Analysis of the obtained 2H NMR spectra provided angles for the individual labelled chemical bond vectors leading to an overall picture for the three dimensional structure of the polyene chain of the chromophore in the protein binding pocket around the Schiff base attachment site.

Structural and orientational information of the membrane embedded M13 coat protein by (13)C-MAS NMR spectroscopy.

Oriented and unoriented M13 coat protein, incorporated into dimyristoyl phosphatidylcholine bilayers, has been studied by (13)C-magic angle spinning nuclear magnetic resonance (MAS NMR) spectroscopy. Rotational resonance experiments provided two distance constraints between Calpha and C&z.dbnd6;O positions of the labelled residues Val-29/Val-30 (0.4+/-0.5nm) and Val-29/Val-31 (0.45+/-0. 5nm) in its hydrophobic domain. The derived dihedral angles (Phi, Psi) for Val-30 revealed a local alpha-helical conformation. (13)C-CP-MAS experiments on uniformly aligned samples (MAOSS experiments) using the (13)C&z.dbnd6;O labelled site of Val-30 allowed the determination of the helix tilt (20 degrees +/-10 degrees ) in the membrane. It is shown that one uniform MAS high-resolution solid state NMR approach can be used to obtain structural and orientational data.

Structural descriptions of ligands in their binding site of integral membrane proteins at near physiological conditions using solid-state NMR

Abstract Using solid-state NMR approaches, it is now possible to define the structure and dynamics of binding for a small, isotopically (2H, 13C, 19F, 15N) labelled ligand, prosthetic group or solute in its binding site of a membrane-bound protein at near physiological conditions in natural membrane fragments or in reconstituted complexes. Studies of oriented membranes permit the orientational bond vectors of labelled groups to be determined to good precision, as shown for retinal in bacteriorhodopsin and bovine rhodopsin. Using novel magic angle spinning NMR methods on membrane dispersions, high-resolution NMR spectra can be obtained. Dipolar couplings can be reintroduced into the spectrum of labelled ligands in their binding sites of membrane-bound proteins to give interatomic distances to high precision (±0.5 Å). Relaxation and cross-polarization data give estimates for the kinetics for on-off rates for binding. In addition, chemical shifts can be measured directly to help provide details of the binding environment for a bound ligand, as shown for analogues of drugs used in peptic ulcer treatment in the gastric ATPase, and for acetylcholine in the acetylcholine receptor.

Use of solid-state 2H NMR for studying protein-lipid interactions at emulsion interfaces

Interactions between myosin and β‐casein with lipids at lipid–water interfaces were studied by solid‐state 2H NMR using dimyristoylphosphatidylcholine with the four hydrogens at α‐ and β‐positions (DMPC‐d4) and the nine protons at the γ‐position substituted by deuterium (DMPC‐d9). Quadrupole splittings and spin–lattice relaxation times were used to describe the amplitude and rate of molecular motion of the choline segment, respectively, in liposomes made of pure labeled dimyristoylphosphatidylcholine or admixed with non‐labeled dimyristoylphosphatidylglycerol (DMPG) in a 1:1 mole ratio. No changes were observed in these NMR parameters for the deuterons when increasing amounts of myosin were added to liposomes exclusively made of DMPC‐d9 or DMPC‐d4. However, when DMPG was present, myosin was found to interact electrostatically with the liposomes, and both the quadrupolar splittings and spin–lattice relaxation times of all head‐group segments were affected, demonstrating that DMPG was necessary in the liposomes for the interaction to occur. The results suggest that positively charged lysine residues located at the tail domain of myosin provided the necessary sites for the lipid–protein interaction, leaving free the head domain for further structural interaction. On the other hand, β‐casein was found to interact both with the charged (with DMPG) and neutral, zwitterionic (DMPC only) liposomes, although this interaction was more pronounced in the charged lipids. In the interaction with charged liposomes, β‐casein was able to affect the lineshape of the NMR spectra from DMPC‐d9 deuterons, even at low protein concentration (lipid/protein mole ratio=30000:1), indicating its ability to locate at emulsion interfaces.