High resolution, solid state NMR experiments will be used to investigate three membrane proteins - rhodopsin(RHOD), bacteriorhodopsin(BR), and halorhodopsin(HR). RHOD will be prepared with a variety of 13C labelled retinals and several aspects of the retinal configuration will be studied. The BR investigations will involve 13C labelled retinals and 13C, 15N, and 17O labelled amino acids. The 15N-1H bond distance in the Schiff base will be measured with DIPSHIFT experiments. The configuration of retinal in photointermediates, like M412, will be determined and the involvement of specific amino acids in the BR proton wire will be investigated by looking for deprotonation effects in 13C and 17O spectra. Resonances will be assigned with a new approach involving 15N and doubly 13C labelled amino acids together with heteronuclear chemical shift correlation and spin diffusion experiments (SDE). SDE will also be used to establish the proximity of retinal to particular amino acids by studying BR in which both retinal and the sidechains are labelled. The dynamic structure of BR will be studied with 13C and 2H NMR and the structure of the protein will be investigated by placing BR in a lipid matrix where it will execute fast rotational diffusion. From the rotationally averaged powder patterns the orientations of sidechains with respect to the diffusion axis can be determined. The anion binding site of HR will be studied as will the possibility of isotopically labelling the protein.
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