The structures and properties of membrane proteins in lipid bilayers are expected to closely resemble those in native cell-membrane environments, although they have been difficult to elucidate. By performing solid-state NMR measurements at very fast (100 kHz) magic-angle spinning rates and at high (23.5 T) magnetic field, severe sensitivity and resolution challenges are overcome, enabling the atomic-level characterization of membrane proteins in lipid environments. This is demonstrated by extensive 1H-based resonance assignments of the fully protonated heptahelical membrane protein proteorhodopsin, and the efficient identification of numerous 1H–1H dipolar interactions, which provide distance constraints, inter-residue proximities, relative orientations of secondary structural elements, and protein–cofactor interactions in the hydrophobic transmembrane regions. These results establish a general approach for high-resolution structural studies of membrane proteins in lipid environments via solid-state NMR.

The plasma membrane is the crucial interface between the cell and its exterior, packed with embedded proteins experiencing simultaneous protein-protein and protein-membrane interactions. A prominent example of cell membrane complexity is the assembly of transmembrane proteins into oligomeric structures, with potential functional consequences that are not well understood. From the study of proteorhodopsin (PR), a prototypical seven-transmembrane light-driven bacterial proton pump, we find evidence that the inter-protein interaction modulated by self-association yields functional changes observable from the protein interior. We also demonstrate that the oligomer is likely a physiologically relevant form of PR, as crosslinking of recombinantly expressed PR reveals an oligomeric population within the E. coli membrane (putatively hexameric). Upon chromatographic isolation of oligomeric and monomeric PR in surfactant micelles, the oligomer exhibits distinctly different optical absorption properties from monomeric PR, as reflected in a prominent decrease in the pKa of the primary proton acceptor residue (D97) and slowing of the light-driven conformational change. These functional effects are predominantly determined by specific PR-PR contacts over nonspecific surfactant interactions. Interestingly, varying the surfactant type alters the population of oligomeric states as well as the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance (EPR) distance measurements. Nevertheless, the dynamic surfactant environment retains the key function-tuning property exerted by oligomeric contacts. A potentially general design principle for transmembrane protein function tuning emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition.

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "b-barrel" architecture, but the assembly of these proteins is poorly understood. The spontaneous assembly of OM proteins (OMPs) into pure lipid vesicles has been studied extensively, but often requires nonphysiological conditions and time scales and is strongly influenced by properties of the lipid bilayer including surface charge, thickness, and fluidity. Furthermore, the membrane insertion of OMPs in vivo is catalyzed by a heterooligomer called the b-barrel assembly machinery (Bam) complex. To determine the role of lipids in the assembly of OMPs under more physiological conditions, we exploited an assay in which the Bam complex mediates their insertion into membrane vesicles. After reconstituting the Bam complex into vesicles that contain a variety of different synthetic lipids, we found that two model OMPs, EspP and OmpA, folded efficiently regardless of the lipid composition. Most notably, both proteins folded into membranes composed of a gel phase lipid that mimics the rigid bacterial OM. Interestingly, we found that EspP, OmpA and another model protein (OmpG) folded at significantly different rates and that an a-helix embedded inside the EspP b-barrel accelerates folding. Our results show that the Bam complex largely overcomes effects that lipids exert on OMP assembly and suggest that specific interactions between the Bam complex and an OMP influence its rate of folding. __________________________ Gram-negative bacteria, a class that includes many pathogenic and emerging antibiotic-resistant organisms, are bound by a double cell membrane. Most of the proteins that reside in the outer membrane (OM) 2 , which serves as a robust barrier, have a distinctive "b-barrel" architecture. A bbarrel is essentially a b-sheet rolled into a closed cylinder that has a hydrophobic exterior and a hydrophilic interior and that is held together by hydrogen bonds between the first and last bstrands. The b-barrel scaffold is found exclusively in the OM of bacteria and organelles of bacterial origin. OM proteins (OMPs) mediate many different functions vital for bacterial survival and range considerably in size from 8-26 b-strands (1, 2). Some OMPs form oligomers (3), while others contain a segment that is embedded inside the bbarrel or a separately folded domain that is displayed on either the periplasmic or extracellular side of the OM (1).OMPs must first traverse the inner membrane (IM) and the periplasmic space, which is devoid of ATP (4), before they attain their final structure in the OM. After OMPs are transported across the IM in an unfolded conformation through the Sec translocon, a variety of periplasmic chaperones including SurA, Skp, and DegP prevent their aggregation in the periplasm (5-7). Integration into the OM is then catalyzed by the heterooligomeric β-barrel assembly machinery (Bam) complex (8,9). In E. coli, the Bam complex is composed of BamA, a b-barrel protein that contains five http://www.jbc.org/cgi

Proteins on film: The Overhauser dynamic nuclear polarization method resolves hydration dynamics to an unprecedented level of detail for a transmembrane protein surface. The heterogeneous hydration landscape of proteorhodopsin rearranges upon photoactivation (see picture), thus providing an insight into how water contributes to protein function even for biological systems embedded in a hydrophobic membrane.