Surface and dynamic structures of bacteriorhodopsin in a 2D crystal, a distorted or disrupted lattice, as revealed by site-directed solid-state 13C NMR.

The 3D structure of bacteriorhodopsin (bR) obtained by X-ray diffraction or cryo-electron microscope studies is not always sufficient for a picture at ambient temperature where dynamic behavior is exhibited. For this reason, a site-directed solid-state 13C NMR study of fully hydrated bR from purple membrane (PM), or a distorted or disrupted lattice, is very valuable in order to gain insight into the dynamic picture. This includes the surface structure, at the physiologically important ambient temperature. Almost all of the 13C NMR signals are available from [3-13C]Ala or [1-13C]Val-labeled bR from PM, although the 13C NMR signals from the surface areas, including loops and transmembrane alpha-helices near the surface (8.7 angstroms depth), are suppressed for preparations labeled with [1-13C]Gly, Ala, Leu, Phe, Tyr, etc. due to a failure of the attempted peak-narrowing by making use of the interfered frequency of the frequency of fluctuation motions with the frequency of magic angle spinning. In particular, the C-terminal residues, 226-235, are present as the C-terminal alpha-helix which is held together with the nearby loops to form a surface complex, although the remaining C-terminal residues undergo isotropic motion even in a 2D crystalline lattice (PM) under physiological conditions. Surprisingly, the 13C NMR signals could be further suppressed even from [3-13C]Ala- or [1-13C]Val-bR, due to the acquired fluctuation motions with correlation times in the order of 10(-4) to 10(-5) s, when the 2D lattice structure is instantaneously distorted or completely disrupted, either in photo-intermediate, removed retinal or when embedded in the lipid bilayers.

Site-directed 13C solid-state NMR studies on membrane proteins: strategy and goals toward revealing conformation and dynamics as illustrated for bacteriorhodopsin labeled with [1-13C]amino acid residues.

We have so far demonstrated that well-resolved and site-specifically assigned (13)C peaks as recorded by site-directed NMR study on (13)C-labeled membrane proteins can serve as a convenient probe to reveal their local conformation and dynamics. We attempted here to clarify the extent to which (13)C NMR spectra of (13)C-labeled fully hydrated bacteriorhodopsin (bR) as a typical membrane protein are visible or well resolved in the presence of inherent fluctuation motions with frequency of 10(2)-10(8) Hz, especially at the membrane surfaces. Accordingly, we estimated the relative proportion of (13)C NMR signals from the surface areas with and without peak suppression by the accelerated transverse relaxation effect by surface-bound Mn(2+) ions, which could be effective for residues within 8.7 angstroms of the membrane surface. It turned out that the experimental findings are consistent with the predicted amount of amino acid residues under consideration located within 8.7 angstroms of the surface for [1-(13)C]Val- and Ile-labeled bR and also [3-(13)C]Ala-bR. In contrast, (13)C NMR peaks from such surfaces area are almost completely or partially suppressed for [1-(13)C]Gly-, Ala-, Leu-, Phe- and Trp-labeled bR, as a result of plausible interference of the fluctuation frequency with frequency of magic angle spinning (10(4) Hz). We further assigned several (13)C NMR signals of [1-(13)C] Val-, Trp- and Ile-labeled bR on the basis of a variety of site-directed mutants with reference to those of the wild type. Further, we recorded the (13)C NMR of bR in lipid bilayers to search for the optimal conditions to be able to obtain signals with the highest peak intensities and spectral resolution. Backbone dynamics turn out to be essential for recording (13)C NMR spectra so as to escape from motional frequencies of the order of 10(4)-10(5) Hz, either in the direction of accelerated fluctuation or slowed motions in the direction of forming the 2D array.