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

Abstract

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.

Solid-state NMR and protein dynamics

“…During the last few years an expanded series of papers was published [110][111][112][113][114][115][116][117][118][119][120] in which the authors studied dynamic features of bacteriorhodopsin and other membrane proteins by means of site-directed 13 C solid-state NMR spectroscopy. The term 'site directed' means that in the proteins only one carbon of only one type of aminoacid (in most cases, Val and Ala) was labeled.…”

Dynamic aspect of bacteriorhodopsin as a typical membrane protein as revealed by site-directed solid-state 13C NMR

“…In contrast to the abovementioned observations for the 13 C NMR spectra of [1-13 C]Ala-bR, we recently showed that 13 C NMR signals are well visible from 13 C NMR spectral region of loops from [1-13 C]Val-bR: Val 69 from the B-C loop (172.9 ppm), 101 and 199 from the C-D and F-G loops, respectively (171.9 ppm), 103 from the D-E loop (172.0 ppm) (see the caption of Fig. 5A) [29,30]. As a result, the peak-intensities of the above-mentioned three signals for [1-13 C]Val-bR at the loop region [30] were appreciably suppressed by accelerated spin-spin relaxation rate in the presence of Mn 2+ ion (Fig.…”

“…This is mainly because there is no Trp residue in the loop regions nor involvement in specific sequence Trp-Pro which would result in upfield displacement of peaks, if any. Obviously, the peak-intensities of the two a-helical peaks at 177.0 and 175.0 ppm are appreciably suppressed by the accelerated relaxation rate caused by the proximity of Trp residues close to bound Mn 2+ ions located at the surface areas, although the assigned Trp peaks are limited to small numbers, at moment, such as Trp 182 and 189 assigned to the peaks of 175.8 and 175.0 ppm, respectively, utilizing site-directed mutants W182F and W189F mutants [29]. This is expected because Trp residues are mainly located at the extracellular surface as shown in Fig.…”

“…13 C NMR peaks of Val 130 and 101 are superimposed upon the peaks of Val 49 and Val 199, respectively[29].…”

Structural and dynamic studies of proteins by high-resolution solid-state NMR

“…Rather than exploiting sample orientation, MAS experiments average orientation-dependent interactions as an alternative approach to achieve narrow resonances, thereby increasing resolution and sensitivity in the spectrum. Several groups are currently investigating selectively labeled membrane peptides and proteins by high-resolution MAS SSNMR [83][84][85][86][87][88][89][90][91][92][93][94][95][96], or investigate on general features of these proteins [97][98][99][100][101]. The ability of SSNMR to report on structural features of fully labeled ligands interacting with integral membrane proteins was demonstrated for several systems.…”