Suppressed or recovered intensities analysis in site-directed (13)C NMR: assessment of low-frequency fluctuations in bacteriorhodopsin and D85N mutants revisited.

The first proton transfer of bacteriorhodopsin (bR) occurs from the protonated Schiff base to the anionic Asp 85 at the central part of the protein in the L to M states. Low-frequency dynamics accompanied by this process can be revealed by suppressed or recovered intensities (SRI) analysis of site-directed (13)C solid-state NMR spectra of 2D crystalline preparations. First of all, we examined a relationship of fluctuation frequencies available from [1-(13)C]Val- and [3-(13)C]Ala-labeled preparations, by taking the effective correlation time of internal methyl rotations into account.

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.

Dynamic structure of pharaonis phoborhodopsin (sensory rhodopsin II) and complex with a cognate truncated transducer as revealed by site-directed 13C solid-state NMR.

We have recorded (13)C nuclear magnetic resonance (NMR) spectra of [3-(13)C]Ala, [1-(13)C]Val-labeled pharaonis phoborhodopsin (ppR or sensory rhodopsin II) incorporated into egg PC (phosphatidylcholine) bilayer, by means of site-directed high-resolution solid-state NMR techniques. Seven (13)C NMR signals from transmembrane alpha-helices were resolved for [3-(13)C]Ala-ppR at almost the same positions as those of bacteriorhodopsin (bR), except for the suppressed peaks in the loop regions in spite of the presence of at least three Ala residues. In contrast, (13)C NMR signals from the loops were visible from [1-(13)C]Val-ppR but their peak positions of the transmembrane alpha-helices are not always the same between ppR and bR.

Residue-specific millisecond to microsecond fluctuations in bacteriorhodopsin induced by disrupted or disorganized two-dimensional crystalline lattice, through modified lipid-helix and helix-helix interactions, as revealed by 13C NMR.

We have recorded 13C NMR spectra of [3-13C]-, [1-13C]Ala-, and [1-13C]Val-labeled bacteriorhodopsin (bR), W80L and W12L mutants and bacterio-opsin (bO) from retinal-deficient E1001 strain, in order to examine the possibility of their millisecond to microsecond local fluctuations with correlation time in the order of 10(-4) to 10(-5) s, induced or prevented by disruption or assembly of two-dimensional (2D) crystalline lattice, respectively, at ambient temperature. The presence of disrupted or disorganized 2D lattice for W12L, W80L and bO from E1001 strain was readily visualized by increased relative proportions of surrounding lipids per protein, together with their broadened 13C NMR signals of transmembrane alpha-helices and loops in [3-13C]Ala-labeled proteins, with reference to those of wild-type. In contrast, 13C CP-MAS NMR spectra of [1-13C]Ala- and Val-labeled these mutants were almost completely suppressed, owing to the presence of fluctuations with time scale of 10(-4) s interfered with magic angle spinning.