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Environmental factors such as temperature, hydration, and lipid bilayer properties are tightly coupled to the dynamics of membrane proteins. So far, site-resolved data visualizing the protein's response to alterations in these factors are rare, and conclusions had to be drawn from dynamic data averaged over the whole protein structure. In the current study, high-resolution solid-state NMR at high magnetic field was used to investigate their effects on the molecular dynamics of green proteorhodopsin, a bacterial light-driven proton pump. Through-space and through-bond correlation experiments were employed to identify and characterize highly mobile and motionally restricted regions of proteorhodopsin. Our data show that hydration water plays an essential role for enhancing molecular dynamics of residues in tails and interhelical loops, while it is found less important for residues in transmembrane domains or rigid, structured loop segments. In contrast, switching the lipids from the gel to their liquid crystalline phase enhances molecular fluctuations mainly in transmembrane helices on a time scale of 10-6 s, but has little effect on loop and tail residues. Increased mobility is especially observed in helices C, F, and G, but also in the EF loop. Fluctuations in those regions are relevant to structural dynamics during the photocycle of proteorhodopsin. Our data are important for the functional understanding of proteorhodopsin, but also offer an important contribution to the general understanding of site-resolved effects of water and lipid bilayers onto the dynamic properties of membrane proteins. [ABSTRACT FROM AUTHOR]

Solid state NMR spectra from uniformly 13C, 15N enriched bacteriorhodospin (bR) purified from H. salinarium were acquired at 18.8 T using magic angle spinning methods. Isolated resonances of 2D 13C−13C spectra exhibited 0.50–0.55 ppm line-widths. Several amino acid types could be assigned, and at least 12 out of 15 Ile peaks could be resolved clearly and identified based on their characteristic chemical shifts and connectivities. This study confirms that high resolution solid state NMR spectra can be obtained for a 248 amino acid uniformly labeled membrane protein in its native membrane environment and indicates that site-specific assignments are likely to be feasible with heteronuclear multidimensional spectra. [ABSTRACT FROM AUTHOR]

Abstract: Proteins of the proteorhodopsin (PR) family are found abundantly in many marine bacteria in the photic zone of the oceans. They are colour-tuned to their environment. The green absorbing species has been shown to act as a light-driven proton pump and thus could form a potential source of energy. The pK a of the primary proton acceptor is close to the pH of seawater which could also indicate a regulatory role. Here, we review and summarize our own recent findings in the context of known data and present some new results. Proton transfer in vitro by PR is shown by a fluorescence assay which confirms a pH dependent vectoriality. Previously reported low diffracting 2D crystal preparations of PR are assessed for their use for solid-state NMR by two dimensional 13C–13C DARR spectra. 15N–1H HETCOR MAS NMR experiments show bound water in the vicinity of the protonated Schiff base which could play a role in proton transfer. The effect of highly conserved H75 onto the properties of the chromophore has been investigated by single site mutations. They do show a pronounced effect onto the optical absorption maximum and the pK a of the proton acceptor but have only a small effect onto the 15N chemical shifts of the protonated Schiff base. [Copyright &y& Elsevier]