Lipid membrane mimetics and oligomerization tune functional properties of proteorhodopsin.

The functional properties of proteorhodopsin (PR) have been found to be strongly modulated by oligomeric distributions and lipid membrane mimetics. This study aims to distinguish and explain their effects by investigating how oligomer formation impacts PR's function of proton transport in lipid-based membrane mimetic environments. We find that PR forms stable hexamers and pentamers in both E. coli membranes and synthetic liposomes. Compared with the monomers, the photocycle kinetics of PR oligomers is ∼2 and ∼4.5 times slower for transitions between the K and M and the M and N photointermediates, respectively, indicating that oligomerization significantly slows PR's rate of proton transport in liposomes. In contrast, the apparent pKa of the key proton acceptor residue D97 (pKaD97) of liposome-embedded PR persists at 6.2-6.6, regardless of cross-protomer modulation of D97, suggesting that the liposome environment helps maintain PR's functional activity at neutral pH. By comparison, when extracted directly from E. coli membranes into styrene-maleic acid lipid particles, the pKaD97 of monomer-enriched E50Q PR drastically increases to 8.9, implying that there is a very low active PR population at neutral pH to engage in PR's photocycle. These findings demonstrate that oligomerization impacts PR's photocycle kinetics, while lipid-based membrane mimetics strongly affect PR's active population via different mechanisms.

Reconstitution of Bam Complex-Mediated Assembly of a Trimeric Porin into Proteoliposomes.

Many integral membrane proteins form oligomeric complexes, but the assembly of these structures is poorly understood. Here, we show that the assembly of OmpC, a trimeric porin that resides in the Escherichia coli outer membrane (OM), can be reconstituted in vitro. Although we observed the insertion of both urea-denatured and in vitro-synthesized OmpC into pure lipid vesicles at physiological pH, the protein assembled only into dead-end dimers. In contrast, in vitro-synthesized OmpC was inserted into proteoliposomes that contained the barrel assembly machinery (Bam) complex, a conserved heterooligomer that catalyzes protein integration into the bacterial OM, and folded into heat-stable trimers by passing through a short-lived dimeric intermediate. Interestingly, complete OmpC assembly was also dependent on the addition of lipopolysaccharide (LPS), a glycolipid located exclusively in the OM. Our results strongly suggest that trimeric porins form through a stepwise process that requires the integration of the protein into the OM in an assembly-competent state. Furthermore, our results provide surprising evidence that interaction with LPS is required not only for trimerization but also for the productive insertion of individual subunits into the lipid bilayer. IMPORTANCE Porins are a widespread family of homotrimers that represent a substantial fraction of the total protein located in the OM of many Proteobacteria. These proteins facilitate the nonspecific diffusion of small molecules across the outer membrane and strongly influence the susceptibility of bacteria to clinically used antibiotics. The assembly of porins and the mechanism by which they are integrated into the outer membrane, however, are poorly understood. Here, we show that assembly can be completely reconstituted in vitro and requires only phospholipid vesicles containing the Bam complex, a molecular chaperone, and LPS. Furthermore, by showing that LPS binding is required for membrane insertion, our results demonstrate that a native lipid promotes a specific stage of porin biogenesis.

Membrane protein production and formulation for drug discovery.

Integral membrane proteins (MPs) are important drug targets across most fields of medicine, but historically have posed a major challenge for drug discovery due to difficulties in producing them in functional forms. We review the state of the art in drug discovery strategies using recombinant multipass MPs, and outline methods to successfully express, stabilize, and formulate them for small-molecule and monoclonal antibody therapeutics development. Advances in structure-based drug design and high-throughput screening are allowing access to previously intractable targets such as ion channels and transporters, propelling the field towards the development of highly specific therapies targeting desired conformations.

Therapeutic Antibodies Targeting Potassium Ion Channels.

Monoclonal antibodies combine specificity and high affinity binding with excellent pharmacokinetic properties and are rapidly being developed for a wide range of drug targets including clinically important potassium ion channels. Nonetheless, while therapeutic antibodies come with great promise, K+ channels represent particularly difficult targets for biologics development for a variety of reasons that include their dynamic structures and relatively small extracellular loops, their high degree of sequence conservation (leading to immune tolerance), and their generally low-level expression in vivo. The process is made all the more difficult when large numbers of antibody candidates must be screened for a given target, or when lead candidates fail to cross-react with orthologous channels in animal disease models due to their highly selective binding properties. While the number of antibodies targeting potassium channels in preclinical or clinical development is still modest, significant advances in the areas of protein expression and antibody screening are converging to open the field to an avalanche of new drugs. Here, the opportunities and constraints associated with the discovery of antibodies against K+ channels are discussed, with an emphasis on novel technologies that are opening the field to exciting new possibilities for biologics development.

Bam complex-mediated assembly of bacterial outer membrane proteins synthesized in an in vitro translation system.

Bacterial outer membrane proteins (OMPs) contain a unique "ß barrel" segment that is inserted into the membrane by the barrel assembly machinery (Bam) complex by an unknown mechanism. OMP assembly has been reconstituted in vitro, but assembly reactions have involved the use of urea-denatured protein purified from inclusion bodies. Here we show that the E. coli Bam complex catalyzes the efficient assembly of OMPs synthesized de novo in a coupled in vitro transcription/translation system. Interestingly, the in vitro translated forms of the OMPs we analyzed were assembled more rapidly and were effectively engaged by fewer periplasmic chaperones than their urea-denatured counterparts. Taken together, our results strongly suggest that the mode of production influences the conformational states sampled by OMPs and thereby affects their recognition by both chaperones and the Bam complex. Besides providing insights into OMP biogenesis, our work describes a novel, streamlined method to reconstitute OMP assembly in vitro.

Chaperone OsmY facilitates the biogenesis of a major family of autotransporters.

OsmY is a widely conserved but poorly understood 20 kDa periplasmic protein. Using a folding biosensor, we previously obtained evidence that OsmY has molecular chaperone activity. To discover natural OsmY substrates, we screened for proteins that are destabilized and thus present at lower steady-state levels in an osmY-null strain. The abundance of an outer membrane protein called antigen 43 was substantially decreased and its ß-barrel domain was undetectable in the outer membrane of an osmY-null strain. Antigen 43 is a member of the diffuse adherence family of autotransporters. Like strains that are defective in antigen 43 production, osmY-null mutants failed to undergo cellular autoaggregation. In vitro, OsmY assisted in the refolding of the antigen 43 ß-barrel domain and protected it from added protease. Finally, an osmY-null strain that expressed two members of the diffuse adherence family of autotransporters that are distantly related to antigen 43, EhaA and TibA, contained reduced levels of the proteins and failed to undergo cellular autoaggregation. Taken together, our results indicate that OsmY is involved in the biogenesis of a major subset of autotransporters, a group of proteins that play key roles in bacterial pathogenesis.

Identification of a novel post-insertion step in the assembly of a bacterial outer membrane protein.

Although the barrel assembly machinery (Bam) complex has been shown to facilitate the insertion of ß barrel proteins into the bacterial outer membrane (OM), the stage at which ß barrels fold is unknown. Here, we describe insights into ß barrel assembly that emerged from an analysis of a member of the autotransporter family of OM proteins (EspP) in Escherichia coli. EspP contains an extracellular 'passenger' domain that is translocated across the OM and then released from the covalently linked ß barrel domain in an intra-barrel cleavage reaction. We found that the mutation of an unusual lipid-exposed lysine residue impairs a previously unidentified late folding step that follows both the membrane insertion of the ß barrel domain and the secretion of the passenger domain but that precedes proteolytic maturation. Our results demonstrate that ß barrel assembly can be completed at a post-insertion stage and raise the possibility that interactions with membrane lipids can promote folding in vivo. Furthermore, by showing that the passenger domain is secreted before the ß barrel domain is fully assembled, our results also provide evidence against the long-standing hypothesis that autotransporters are autonomous protein secretion systems.

Functionally Active Membrane Proteins Incorporated in Mesostructured Silica Films.

A versatile synthetic protocol is reported that allows high concentrations of functionally active membrane proteins to be incorporated in mesostructured silica materials. Judicious selections of solvent, surfactant, silica precursor species, and synthesis conditions enable membrane proteins to be stabilized in solution and during subsequent coassembly into silica-surfactant composites with nano- and mesoscale order. This was demonstrated by using a combination of nonionic ( n-dodecyl-ß-d-maltoside or Pluronic P123), lipid-like (1,2-diheptanoyl- s n-glycero-3-phosphocholine), and perfluoro-octanoate surfactants under mild acidic conditions to coassemble the light-responsive transmembrane protein proteorhodopsin at concentrations up to 15 wt % into the hydrophobic regions of worm-like mesostructured silica materials in films. Small-angle X-ray scattering, electron paramagnetic resonance spectroscopy, and transient UV-visible spectroscopy analyses established that proteorhodopsin molecules in mesostructured silica films exhibited native-like function, as well as enhanced thermal stability compared to surfactant or lipid environments. The light absorbance properties and light-activated conformational changes of proteorhodopsin guests in mesostructured silica films are consistent with those associated with the native H+-pumping mechanism of these biomolecules. The synthetic protocol is expected to be general, as demonstrated also for the incorporation of functionally active cytochrome c, a peripheral membrane protein enzyme involved in electron transport, into mesostructured silica-cationic surfactant films.

The Bam complex catalyzes efficient insertion of bacterial outer membrane proteins into membrane vesicles of variable lipid composition.

Most proteins that reside in the bacterial outer membrane (OM) have a distinctive "ß-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 non-physiological 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 ß-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 α-helix embedded inside the EspP ß-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.

Proton-Based Structural Analysis of a Heptahelical Transmembrane Protein in Lipid Bilayers.

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.

Functional consequences of the oligomeric assembly of proteorhodopsin.

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 Escherichia 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 and the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance 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 emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition.

Structure and function of G protein-coupled receptor oligomers: implications for drug discovery.

G protein-coupled receptor (GPCR) oligomers are promising targets for the design of new highly selective therapeutics. GPCRs have historically been attractive drug targets for their role in nearly all cellular processes, and their localization at the cell surface makes them easily accessible to most small molecule therapeutics. However, GPCRs have traditionally been considered a monomeric entity, a notion that greatly oversimplifies their function. As evidence accumulates that GPCRs tune function through oligomer formation and protein-protein interactions, we see a greater demand for structural information about these oligomers to facilitate oligomer-specific drug design. These efforts are slowed by difficulties inherent to studying membrane proteins, such as low expression yield, in vitro stability and activity. Such obstacles are amplified for the study of specific oligomers, as there are limited tools to directly isolate and characterize these receptor complexes. Thus, there is a need to develop new interdisciplinary approaches, combining biochemical and biophysical techniques, to address these challenges and elucidate structural details about the oligomer and ligand binding interfaces. In this review, we provide an overview of mechanistic models that have been proposed to underlie the function of GPCR oligomers, and perspectives on emerging techniques to characterize GPCR oligomers for structure-based drug design.

Determining the oligomeric structure of proteorhodopsin by Gd3+ -based pulsed dipolar spectroscopy of multiple distances.

The structural organization of the functionally relevant, hexameric oligomer of green-absorbing proteorhodopsin (G-PR) was obtained from double electron-electron resonance (DEER) spectroscopy utilizing conventional nitroxide spin labels and recently developed Gd3+ -based spin labels. G-PR with nitroxide or Gd3+ labels was prepared using cysteine mutations at residues Trp58 and Thr177. By combining reliable measurements of multiple interprotein distances in the G-PR hexamer with computer modeling, we obtained a structural model that agrees with the recent crystal structure of the homologous blue-absorbing PR (B-PR) hexamer. These DEER results provide specific distance information in a membrane-mimetic environment and across loop regions that are unresolved in the crystal structure. In addition, the X-band DEER measurements using nitroxide spin labels suffered from multispin effects that, at times, compromised the detection of next-nearest neighbor distances. Performing measurements at high magnetic fields with Gd3+ spin labels increased the sensitivity considerably and alleviated the difficulties caused by multispin interactions.