Proton-Detected Solid-State NMR Reveals Intramembrane Polar Networks in a Seven-Helical Transmembrane Protein Proteorhodopsin

Abstract

We used high-resolution proton-detected multidimensional NMR to study the solvent-exposed parts of a seven-helical integral membrane proton pump, proteorhodopsin (PR). PR samples were prepared by growing the apoprotein on fully deuterated medium and reintroducing protons to solvent-accessible sites through exchange with protonated buffer. This preparation leads to NMR spectra with proton resolution down to ca. 0.2 ppm at fast spinning (28 kHz) in a protein back-exchanged at a level of 40%. Novel three-dimensional proton-detected chemical shift correlation spectroscopy allowed for the identification and resonance assignment of the solvent-exposed parts of the protein. Most of the observed residues are located at the membrane interface, but there are notable exceptions, particularly in helix G, where most of the residues are susceptible to H/D exchange. This helix contains Schiff base-forming Lys231, and many conserved polar residues in the extracellular half, such as Asn220, Tyr223, Asn224, Asp227, and Asn230. We proposed earlier that high mobility of the F-G loop may transiently expose a hydrophilic cavity in the extracellular half of the protein, similar to the one found in xanthorhodopsin. Solvent accessibility of helix G is in line with this hypothesis, implying that such a cavity may be a part of the proton-conducting pathway lined by this helix.

Structure of fully protonated proteins by proton-detected magic-angle spinning NMR

“…2C also compares the amide spectrum of AP205CP to that of a perdeuterated sample purified in H 2 O. Although most amide protons exchange during purification, as indicated by the observation of a cross-peak in both spectra, several weak or missing signals for the perdeuterated sample indicate incomplete exchange, a phenomenon observed for a variety of proteins (24,59). For AP205CP, amide groups engaged in hydrogen bonds in the middle of the two α-helices, in the innermost β-strands, or at the dimer interface, are inaccessible to proton exchange.…”

“…This opens the way to rapid sequential assignment of backbone resonances (24)(25)(26)(27), as well as to the unambiguous measurement of detailed structural and dynamical parameters (28)(29)(30)(31)(32). A further increase in the MAS frequency to 100 kHz allows resonance assignment (20), a structure determination of a model protein (16), and interaction studies (15) with as little as 0.5 mg of sample.…”

Dynamics in the solid-state: perspectives for the investigation of amyloid aggregates, membrane proteins and soluble protein complexes

“…The resolution in DMPC/DMPA reconstituted proteorhodopsin (Ward et al 2011) is lower in comparison to bR in native purple membranes (Linser et al 2011). bR is found to be a trimer in the lipid bilayer (Luecke et al 1999;Sapra et al 2006).…”

“…Values for the 1 H and 15 N T 2 decay time from spin-echo experiments, as well as the calculated and experimental line width are summarized in Table 3. Other deuterated protein preparations in the solid-state such as the 20 kDa membrane protein DsbB reconstituted in E. coli lipids ) and the DMPC/DMPA reconstituted proteorhodopsin (Ward et al 2011) yield a 1 H, 15 N spectral resolution which is comparable to the OmpG preparation.…”

“…It is thus conceivable that interdigitation of protomers from different oligomers can affect rotational diffusion in the lipid bilayer. Ladizhansky and co-worker employed DMPC/DMPA to reconstitute proteorhodopsin (Ward et al 2011). Choice of lipids and crystallization conditions will have an effect on the architecture of the oligomeric state of proteorhodopsin, and it remains to be seen what the exact reason for the observed differences in spectral quality is.…”