Eight-stranded Β-barrel Research Articles

Structure of the outer membrane porin OmpW from the pervasive pathogen Klebsiella pneumoniae.

Conjugation is the process by which plasmids, including those that carry antibiotic-resistance genes, are mobilized from one bacterium (the donor) to another (the recipient). The conjugation efficiency of IncF-like plasmids relies on the formation of mating-pair stabilization via intimate interactions between outer membrane proteins on the donor (a plasmid-encoded TraN isoform) and recipient bacteria. Conjugation of the R100-1 plasmid into Escherichia coli and Klebsiella pneumoniae (KP) recipients relies on pairing between the plasmid-encoded TraNα in the donor and OmpW in the recipient. Here, the crystal structure of K. pneumoniae OmpW (OmpWKP) is reported at 3.2 Å resolution. OmpWKP forms an eight-stranded β-barrel flanked by extracellular loops. The structures of E. coli OmpW (OmpWEC) and OmpWKP show high conservation despite sequence variability in the extracellular loops.

Yeast mitochondria can process de novo designed β-barrel proteins.

Mitochondrial outer membrane β-barrel proteins are encoded in the nucleus, translated in the cytosol and then targeted to and imported into the respective organelles. Detailed studies have uncovered the mechanisms involved in the import of these proteins and identified the targeting signals and the cytosolic factors that govern their proper biogenesis. Recently, de novo designed eight-stranded β-barrel proteins (Tmb2.3 and Tmb2.17) were shown to fold and assemble into lipid membranes. To better understand the general aspects of the biogenesis of β-barrel proteins, we investigated the fate of these artificial proteins upon their expression in yeast cells. We demonstrate that although these proteins are de novo designed and are not related to bona fide mitochondrial β-barrel proteins, they were targeted to mitochondria and integrated into the organelle outer membrane. We further studied whether this integration requires components of the yeast mitochondrial import machinery like Tom20, Tom70, Tob55/Sam50 and Mas37/Sam37. Whereas it seems that none of the import receptors was required for the biogenesis of the artificial β-barrel proteins, we observed a strong dependency on the TOB/SAM complex. Collectively, our findings demonstrate that the mitochondrial outer membrane is the preferential location in yeast cells for any membrane-embedded β-barrel protein.

Structural and interaction analysis of human lipocalin-type prostaglandin D synthase with the poorly water-soluble drug NBQX.

Lipocalin-type prostaglandin D synthase (L-PGDS) is a secretory lipid-transporter protein that was shown to bind a wide variety of hydrophobic ligands in vitro. Exploiting this function, we previously examined the feasibility of using L-PGDS as a novel delivery vehicle for poorly water-soluble drugs. However, the mechanism by which human L-PGDS binds to poorly water-soluble drugs is unclear. In this study, we determined the solution structure of human L-PGDS and investigated the mechanism of L-PGDS binding to 6-nitro-7-sulfamoyl-benzo[f]quinoxalin-2,3-dione (NBQX), an α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor antagonist. NMR experiments showed that human L-PGDS has an eight-stranded antiparallel β-barrel structure that forms a central cavity, a short 310 -helix, and two α-helices. Titration with NBQX was monitored using 1 H-15 N HSQC spectroscopy. At higher NBQX concentrations, some cross-peaks of the protein exhibited fast-exchanging shifts with a curvature, indicating at least two binding sites. These residues were located in the upper portion of the cavity. Singular value decomposition analysis revealed that human L-PGDS has two NBQX binding sites. Large chemical shift changes were observed in the H2-helix and A-, B-, C-, D-, H-, and I-strands and H2-helix upon NBQX binding. Calorimetric experiments revealed that human L-PGDS binds two NBQX molecules with dissociation constants of 46.7 μM for primary binding and 185.0 μM for secondary binding. Molecular docking simulations indicated that these NBQX binding sites are located within the β-barrel. These results provide new insights into the interaction between poorly water-soluble drugs and human L-PGDS as a drug carrier.

The crystal structure of DynF from the dynemicin-biosynthesis pathway of Micromonospora chersina.

Dynemicin is an enediyne natural product from Micromonospora chersina ATCC53710. Access to the biosynthetic gene cluster of dynemicin has enabled the in vitro study of gene products within the cluster to decipher their roles in assembling this unique molecule. This paper reports the crystal structure of DynF, the gene product of one of the genes within the biosynthetic gene cluster of dynemicin. DynF is revealed to be a dimeric eight-stranded β-barrel structure with palmitic acid bound within a cavity. The presence of palmitic acid suggests that DynF may be involved in binding the precursor polyene heptaene, which is central to the synthesis of the ten-membered ring of the enediyne core.

Structure-based prediction of the IgE epitopes of the major dog allergen Can f 1.

Allergy to dogs has become increasingly prominent worldwide. Seven dog allergens have been identified, including Canis familiaris allergen 1-7 (Can f 1-7). Although Can f 1 is a major dog allergen sensitized to 50-75% of dog-allergic subjects, its IgE epitopes have not been identified. The structural analysis of an allergen is important to identify conformational epitopes. In this study, we generated a recombinant Can f 1 protein and determined its crystal structure using X-ray crystallography. Can f 1 had a typical lipocalin fold, which is composed of an eight-stranded β-barrel and α-helix, and has high similarity to Can f 2, Can f 4, and Can f 6 in overall structure. However, the localizations of surface charges on these proteins were quite different. Based on sequence alignment and tertiary structure, we predicted five critical residues (His86, Glu98, Arg111, Glu138, and Arg152) for the IgE epitopes. The relevance of these residues to IgE reactivity was assessed by generating Can f 1 mutants with these residues substituted for alanine. Although the effects of the mutation on IgE binding depended on the sera of dog-allergic patients, H86A and R152A mutants showed reduced IgE reactivity compared with wild-type Can f 1. These results suggest that Can f 1 residues His86 and Arg152 are candidates for the IgE conformational epitope.

Conformational changes of loops highlight a potential binding site in Rhodococcus equi VapB.

Virulence-associated proteins (Vaps) contribute to the virulence of the pathogen Rhodococcus equi, but their mode of action has remained elusive. All Vaps share a conserved core of about 105 amino acids that folds into a compact eight-stranded antiparallel β-barrel with a unique topology. At the top of the barrel, four loops connect the eight β-strands. Previous Vap structures did not show concave surfaces that might serve as a ligand-binding site. Here, the structure of VapB in a new crystal form was determined at 1.71 Å resolution. The asymmetric unit contains two molecules. In one of them, the loop regions at the top of the barrel adopt a different conformation from other Vap structures. An outward movement of the loops results in the formation of a hydrophobic cavity that might act as a ligand-binding site. This lends further support to the hypothesis that the structural similarity between Vaps and avidins suggests a potential binding function for Vaps.

Crystal structure of the C-terminal domain of envelope protein VP37 from white spot syndrome virus reveals sulphate binding sites responsible for heparin binding.

White spot syndrome virus (WSSV) is the most virulent pathogen causing high mortality and economic loss in shrimp aquaculture and various crustaceans. Therefore, the understanding of molecular mechanisms of WSSV infection is important to develop effective therapeutics to control the spread of this viral disease. In a previous study, we found that VP37 could bind with shrimp haemocytes through the interaction between its C-terminal domain and heparin-like molecules on the shrimp cells, and this interaction can also be inhibited by sulphated galactan. In this study, we present the crystal structure of C-terminal domain of VP37 from WSSV at a resolution of 2.51 Å. The crystal structure contains an eight-stranded β-barrel fold with an antiparallel arrangement and reveals a trimeric assembly. Moreover, there are two sulphate binding sites found in the position corresponding to R213 and K257. In order to determine whether these sulphate binding sites are involved in binding of VP37 to heparin, mutagenesis was performed to replace these residues with alanine (R213A and K257A), and the Surface Plasmon Resonance (SPR) system was used to study the interaction of each mutated VP37 with heparin. The results showed that mutants R213A and K257A exhibited a significant loss in heparin binding activity. These findings indicated that the sites of R213 and K257 on the C-terminal domain of envelope protein VP37 are essential for binding to sulphate molecules of heparin. This study provides further insight into the structure of C-terminal domain of VP37 and it is anticipated that the structure of VP37 might be used as a guideline for development of antivirus agent targeting on the VP37 protein.

De novo design of transmembrane β barrels.

Transmembrane β-barrel proteins (TMBs) are of great interest for single-molecule analytical technologies because they can spontaneously fold and insert into membranes and form stable pores, but the range of pore properties that can be achieved by repurposing natural TMBs is limited. We leverage the power of de novo computational design coupled with a "hypothesis, design, and test" approach to determine TMB design principles, notably, the importance of negative design to slow β-sheet assembly. We design new eight-stranded TMBs, with no homology to known TMBs, that insert and fold reversibly into synthetic lipid membranes and have nuclear magnetic resonance and x-ray crystal structures very similar to the computational models. These advances should enable the custom design of pores for a wide range of applications.

Building a barrel

Protein Design Computational design offers the possibility of making proteins with customized structures and functions. The range of accessible protein scaffolds has expanded with the design of increasingly complex cytoplasmic proteins and, recently, helical membrane proteins. Vorobieva et al. describe the successful computational design of eight-stranded transmembrane β-barrel proteins (TMBs). Using an iterative approach, they show the importance of negative design to prevent off-target structures and gain insight into the sequence determinants of TMB folding. Twenty-three designs satisfied biochemical screens for a TMB structure, and two structures were experimentally validated by nuclear magnetic resonance spectroscopy or x-ray crystallography. This is a step toward the custom design of pores for applications such as single-molecule sequencing. Science , this issue p. [eabc8182][1] [1]: /lookup/doi/10.1126/science.abc8182

Evidence that immunization with TP0751, a bipartite Treponema pallidum lipoprotein with an intrinsically disordered region and lipocalin fold, fails to protect in the rabbit model of experimental syphilis.

Deconvolution of syphilis pathogenesis and selection of candidate syphilis vaccinogens requires detailed knowledge of the molecular architecture of the Treponema pallidum outer membrane (OM). The T. pallidum OM contains a low density of integral OM proteins, while the spirochete’s many lipoprotein immunogens are periplasmic. TP0751, a lipoprotein with a lipocalin fold, is reportedly a surface-exposed protease/adhesin and protective antigen. The rapid expansion of calycin/lipocalin structures in the RCSB PDB database prompted a comprehensive reassessment of TP0751. Small angle X-ray scattering analysis of full-length protein revealed a bipartite topology consisting of an N-terminal, intrinsically disordered region (IDR) and the previously characterized C-terminal lipocalin domain. A DALI server query using the lipocalin domain yielded 97 hits, 52 belonging to the calycin superfamily, including 15 bacterial lipocalins, but no Gram-negative surface proteins. Surprisingly, Tpp17 (TP0435) was identified as a structural ortholog of TP0751. In silico docking predicted that TP0751 can bind diverse ligands along the rim of its eight-stranded β-barrel; high affinity binding of one predicted ligand, heme, to the lipocalin domain was demonstrated. qRT-PCR and immunoblotting revealed very low expression of TP0751 compared to other T. pallidum lipoproteins. Immunoblot analysis of immune rabbit serum failed to detect TP0751 antibodies, while only one of five patients with secondary syphilis mounted a discernible TP0751-specific antibody response. In opsonophagocytosis assays, neither TP0751 nor Tpp17 antibodies promoted uptake of T. pallidum by rabbit peritoneal macrophages. Rabbits immunized with intact, full-length TP0751 showed no protection against local or disseminated infection following intradermal challenge with T. pallidum. Our data argue that, like other lipoprotein lipocalins in dual-membrane bacteria, TP0751 is periplasmic and binds small molecules, and we propose that its IDR facilitates ligand binding by and offloading from the lipocalin domain. The inability of TP0751 to elicit opsonic or protective antibodies is consistent with a subsurface location.

Non-native fold of the putative VPS39 zinc finger domain.

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Non-native fold of the putative VPS39 zinc finger domain

Background: The multi-subunit homotypic fusion and vacuole protein sorting (HOPS) membrane-tethering complex is involved in regulating the fusion of late endosomes and autophagosomes with lysosomes in eukaryotes. The C-terminal regions of several HOPS components have been shown to be required for correct complex assembly, including the C-terminal really interesting new gene (RING) zinc finger domains of HOPS components VPS18 and VPS41. We sought to structurally characterise the putative C-terminal zinc finger domain of VPS39, which we hypothesised may be important for binding of VPS39 to cellular partners or to other HOPS components. Methods: We recombinantly expressed, purified and solved the crystal structure of the proposed zinc-binding region of VPS39. Results: In the structure, this region forms an anti-parallel β-hairpin that is incorporated into a homotetrameric eight-stranded β-barrel. However, the fold is stabilised by coordination of zinc ions by residues from the purification tag and an intramolecular disulphide bond between two predicted zinc ligands. Conclusions: We solved the structure of the VPS39 C-terminal domain adopting a non-native fold. Our work highlights the risk of non-native folds when purifying small zinc-containing domains with hexahistidine tags. However, the non-native structure we observe may have implications for rational protein design.

Structural Characterization of Cuta- and Tusavirus: Insight into Protoparvoviruses Capsid Morphology

Several members of the Protoparvovirus genus, capable of infecting humans, have been recently discovered, including cutavirus (CuV) and tusavirus (TuV). To begin the characterization of these viruses, we have used cryo-electron microscopy and image reconstruction to determine their capsid structures to ~2.9 Å resolution, and glycan array and cell-based assays to identify glycans utilized for cellular entry. Structural comparisons show that the CuV and TuV capsids share common features with other parvoviruses, including an eight-stranded anti-parallel β-barrel, depressions at the icosahedral 2-fold and surrounding the 5-fold axes, and a channel at the 5-fold axes. However, the viruses exhibit significant topological differences in their viral protein surface loops. These result in three separated 3-fold protrusions, similar to the bufaviruses also infecting humans, suggesting a host-driven structure evolution. The surface loops contain residues involved in receptor binding, cellular trafficking, and antigenic reactivity in other parvoviruses. In addition, terminal sialic acid was identified as the glycan potentially utilized by both CuV and TuV for cellular entry, with TuV showing additional recognition of poly-sialic acid and sialylated Lewis X (sLeXLeXLeX) motifs reported to be upregulated in neurotropic and cancer cells, respectively. These structures provide a platform for annotating the cellular interactions of these human pathogens.

Crystal structures of the GH18 domain of the bifunctional peroxiredoxin-chitinase CotE from Clostridium difficile.

CotE is a coat protein that is present in the spores of Clostridium difficile, an obligate anaerobic bacterium and a pathogen that is a leading cause of antibiotic-associated diarrhoea in hospital patients. Spores serve as the agents of disease transmission, and CotE has been implicated in their attachment to the gut epithelium and subsequent colonization of the host. CotE consists of an N-terminal peroxiredoxin domain and a C-terminal chitinase domain. Here, a C-terminal fragment of CotE comprising residues 349-712 has been crystallized and its structure has been determined to reveal a core eight-stranded β-barrel fold with a neighbouring subdomain containing a five-stranded β-sheet. A prominent groove running across the top of the barrel is lined by residues that are conserved in family 18 glycosyl hydrolases and which participate in catalysis. Electron density identified in the groove defines the pentapeptide Gly-Pro-Ala-Met-Lys derived from the N-terminus of the protein following proteolytic cleavage to remove an affinity-purification tag. These observations suggest the possibility of designing peptidomimetics to block C. difficile transmission.

Structural and Functional Role of the Surface-Exposed Loops of Ail During Complement-Mediated Evasion by Y. pestis

Yersinia pestis, the causative agent of plague, promotes its proliferation in the host by expressing various bacterial proteins that inhibit the host innate immune system. One of these is attachment invasion locus (Ail), which is an eight-stranded β-barrel outer membrane protein with four surface exposed extracellular loops. Ail is essential for Y. pestis pathogenesis and proliferation as it recruits host serum proteins to inhibit the complement-dependent bacteriolysis and inflammatory response, and regulates host cell attachment and delivery of Yersinia outer proteins to host tissue. Recently, we demonstrated that Y. pestis can also recruit vitronectin (Vn) from the human serum in Ail-dependent manner. Vn is a 459-residue multi-domain protein that interacts with various molecules to promote cell adhesion and migration, and inhibits formation of the membrane attack complex, indicating that the interaction between Ail and Vn may be responsible for promoting Y. pestis survival and pathogenesis. Notably, previous works have indicated that serum proteins such as Vn are recruited by Ail via its extracellular loops. Thus, we hypothesized that Ail's extracellular loops may also be responsible for binding to Vn. To characterize this interaction, amino acids in each extracellular loop of Ail were substituted for alanine to individually characterize the loops for Vn binding. The structure and function of each Ail loop mutant was characterized in a native like environment inside membrane (detergent micelle and nanodiscs) by solution state NMR and ELISA binding assays. These studies identify specific extracellular loops of Ail that are important for binding to Vn, which will guide future developments of therapeutics aimed at combating Y. pestis infections.

Periplasmic protein EipA determines envelope stress resistance and virulence in Brucella abortus.

Molecular components of the Brucella abortus cell envelope play a major role in its ability to infect, colonize and survive inside mammalian host cells. In this study, we have defined a role for a conserved gene of unknown function in B. abortus envelope stress resistance and infection. Expression of this gene, which we name eipA, is directly activated by the essential cell cycle regulator, CtrA. eipA encodes a soluble periplasmic protein that adopts an unusual eight-stranded β-barrel fold. Deletion of eipA attenuates replication and survival in macrophage and mouse infection models, and results in sensitivity to treatments that compromise the cell envelope integrity. Transposon disruption of genes required for LPS O-polysaccharide biosynthesis is synthetically lethal with eipA deletion. This genetic connection between O-polysaccharide and eipA is corroborated by our discovery that eipA is essential in Brucella ovis, a naturally rough species that harbors mutations in several genes required for O-polysaccharide production. Conditional depletion of eipA expression in B. ovis results in a cell chaining phenotype, providing evidence that eipA directly or indirectly influences cell division in Brucella. We conclude that EipA is a molecular determinant of Brucella virulence that functions to maintain cell envelope integrity and influences cell division.

An engineered lipocalin that tightly complexes the plant poison colchicine for use as antidote and in bioanalytical applications.

Colchicine is a toxic alkaloid prevalent in autumn crocus (Colchicum autumnale) that binds to tubulin and inhibits polymerization of microtubules. Using combinatorial and rational protein design, we have developed an artificial binding protein based on the human lipocalin 2 that binds colchicine with a dissociation constant of 120 pm, i.e. 10000-fold stronger than tubulin. Crystallographic analysis of the engineered lipocalin, dubbed Colchicalin, revealed major structural changes in the flexible loop region that forms the ligand pocket at the open end of the eight-stranded β-barrel, resulting in a lid-like structure over the deeply buried colchicine. A cis-peptide bond between residues Phe71 and Pro72 in loop #2 constitutes a peculiar feature and allows intimate contact with the tricyclic ligand. Using directed evolution, we achieved an extraordinary dissociation half-life of more than 9 h for the Colchicalin-colchicine complex. Together with the chemical robustness of colchicine and availability of activated derivatives, this also opens applications as a general-purpose affinity reagent, including facile quantification of colchicine in biological samples. Given that engineered lipocalins, also known as Anticalin® proteins, represent a class of clinically validated biopharmaceuticals, Colchicalin may offer a therapeutic antidote to scavenge colchicine and reverse its poisoning effect in situations of acute intoxication.

HDX-MS reveals orthosteric and allosteric changes in apolipoprotein-D structural dynamics upon binding of progesterone.

Apolipoprotein-D is a glycosylated tetrameric lipocalin that binds and transports small hydrophobic molecules such as progesterone and arachidonic acid. Like other lipocalins, apolipoprotein-D adopts an eight-stranded β-barrel fold stabilized by two intramolecular disulphide bonds, with an adjacent α-helix. Crystallography studies of recombinant apolipoprotein-D demonstrated no major conformational changes upon progesterone binding. Amide hydrogen-deuterium exchange mass spectrometry (HDX-MS) reports structural changes of proteins in solution by monitoring exchange of amide hydrogens in the protein backbone with deuterium. HDX-MS detects changes in conformation and structural dynamics in response to protein function such as ligand binding that may go undetected in X-ray crystallography, making HDX-MS an invaluable orthogonal technique. Here, we report an HDX-MS protocol for apolipoprotein-D that solved challenges of high protein rigidity and low pepsin cleavage using rigorous quenching conditions and longer deuteration times, yielding 85% sequence coverage and 50% deuterium exchange. The relative fractional deuterium exchange of ligand-free apolipoprotein-D revealed apolipoprotein-D to be a highly structured protein. Progesterone binding was detected by significant reduction in deuterium exchange in eight peptides. Stabilization of apolipoprotein-D dynamics can be interpreted as a combined orthosteric effect in the ligand binding pocket and allosteric effect at the N-terminus and C-terminus. Together, our experiments provide insight into apolipoprotein-D structural dynamics and map the effects of progesterone binding that are relayed to distal parts of the protein. The observed stabilization of apolipoprotein-D dynamics upon progesterone binding demonstrates a common behaviour in the lipocalin family and may have implications for interactions of apolipoprotein-D with receptors or lipoprotein particles. Statement: We reveal for the first time how apolipoprotein-D, which is protective in Alzheimer's disease, becomes more ordered when bound to a molecule of steroid hormone. These results significantly extend the understanding of apolipoprotein-D structure from X-ray crystallography studies by incorporating information on how protein motion changes over time. To achieve these results an improved protocol was developed, suitable for proteins similar to apolipoprotein-D, to elucidate how proteins change flexibility when binding to small molecules.

Quaternary structure of the small amino acid transporter OprG from Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that causes nosocomial infections. The P. aeruginosa outer membrane contains specific porins that enable substrate uptake, with the outer membrane protein OprG facilitating transport of small, uncharged amino acids. However, the pore size of an eight-stranded β-barrel monomer of OprG is too narrow to accommodate even the smallest transported amino acid, glycine, raising the question of how OprG facilitates amino acid uptake. Pro-92 of OprG is critically important for amino acid transport, with a P92A substitution inhibiting transport and the NMR structure of this variant revealing that this substitution produces structural changes in the barrel rim and restricts loop motions. OprG may assemble into oligomers in the outer membrane (OM) whose subunit interfaces could form a transport channel. Here, we explored the contributions of the oligomeric state and the extracellular loops to OprG's function. Using chemical cross-linking to determine the oligomeric structures of both WT and P92A OprG in native outer membranes and atomic force microscopy, and single-molecule fluorescence of the purified proteins reconstituted into lipid bilayers, we found that both protein variants form oligomers, supporting the notion that subunit interfaces in the oligomer could provide a pathway for amino acid transport. Furthermore, performing transport assays with loop-deleted OprG variants, we found that these variants also can transport small amino acids, indicating that the loops are not solely responsible for substrate transport. We propose that OprG functions as an oligomer and that conformational changes in the barrel-loop region might be crucial for its activity.