Secondary and tertiary structure formation of the beta-barrel membrane protein OmpA is synchronized and depends on membrane thickness

The mechanism of membrane insertion and folding of a beta-barrel membrane protein has been studied using the outer membrane protein A (OmpA) as an example. OmpA forms an eight-stranded beta-barrel that functions as a structural protein and perhaps as an ion channel in the outer membrane of Escherichia coli. OmpA folds spontaneously from a urea-denatured state into lipid bilayers of small unilamellar vesicles. We have used fluorescence spectroscopy, circular dichroism spectroscopy, and gel electrophoresis to investigate basic mechanistic principles of structure formation in OmpA. Folding kinetics followed a second-order rate law and is strongly depended on the hydrophobic thickness of the lipid bilayer. When OmpA was refolded into model membranes of dilaurylphosphatidylcholine, fluorescence kinetics were characterized by a rate constant that was about fivefold higher than the rate constants of formation of secondary and tertiary structure, which were determined by circular dichroism spectroscopy and gel electrophoresis, respectively. The formation of beta-sheet secondary structure and closure of the beta-barrel of OmpA were correlated with the same rate constant and coupled to the insertion of the protein into the lipid bilayer. OmpA, and presumably other beta-barrel membrane proteins therefore do not follow a mechanism according to the two-stage model that has been proposed for the folding of alpha-helical bundle membrane proteins. These different folding mechanisms are likely a consequence of the very different intramolecular hydrogen bonding and hydrophobicity patterns in these two classes of membrane proteins.     

耐冷希瓦氏菌外膜蛋白的体外折叠研究

目的:旨在建立耐低温革兰氏阴性菌外膜蛋白体外折叠体系,为膜蛋白合成耐低温机制提供理论基础。方法:以包涵体的形式在大肠杆菌中过量表达了来源于耐低温希瓦氏菌的OmpA同源外膜蛋白Omp74的全蛋白质和N端跨膜结构域,纯化包涵体后,用高浓度尿素或强阴离子表面活性剂溶液溶解包涵体,以非离子表面活性剂为折叠介质,建立该外膜蛋白的体外折叠体系,同时以大肠杆菌的OmpA作为对照进行了比较研究。结果:与OmpA相比,Omp74体外折叠受温度影响较小,低浓度的阴离子表面活性剂能促Omp74的折叠,但对OmpA的折叠没有影响;C端结构域抑制Omp74在表面活性剂中的折叠;Omp74在0.5%的月桂酰基麦芽糖苷(DDM)和0.4%的十二烷基肌氨酸钠的混合溶液中能达到接近100%的折叠效率。     

The major outer membrane protein of Fusobacterium nucleatum (FomA) folds and inserts into lipid bilayers via parallel folding pathways

Membrane protein insertion and folding was studied for the major outer membrane protein of Fusobacterium nucleatum (FomA), which is a voltage-dependent general diffusion porin. The transmembrane domain of FomA forms a beta-barrel that is predicted to consist of 14 beta-strands. Here, unfolded FomA is shown to insert and fold spontaneously and quantitatively into phospholipid bilayers upon dilution of the denaturant urea, which was shown previously only for outer membrane protein A (OmpA) of Escherichia coli. Folding of FomA is demonstrated by circular dichroism and fluorescence spectroscopy, by SDS-polyacrylamide gel electrophoresis, and by single-channel recordings. Refolded FomA had a single-channel conductance of 1.1 nS at 1 M KCl, in agreement with the conductance of FomA isolated from membranes in native form. In contrast to OmpA, which forms a smaller eight-stranded beta-barrel domain, folding kinetics of the larger FomA were slower and provided evidence for parallel folding pathways of FomA into lipid bilayers. Two pathways were observed independent of membrane thickness with two different lipid bilayers, which were either composed of dicapryl phosphatidylcholine or dioleoyl phosphatidylcholine. This is the first observation of parallel membrane insertion and folding pathways of a beta-barrel membrane protein from an unfolded state in urea into lipid bilayers. The kinetics of both folding pathways depended on the chain length of the lipid and on temperature with estimated activation energies of 19 kJ/mol (dicapryl phosphatidylcholine) and 70 kJ/mol (dioleoyl phosphatidylcholine) for the faster pathways.     

Folding kinetics of the outer membrane proteins OmpA and FomA into phospholipid bilayers

The folding mechanism of outer membrane proteins (OMPs) of Gram-negative bacteria into lipid bilayers has been studied using OmpA of E. coli and FomA of F. nucleatum as examples. Both, OmpA and FomA are soluble in unfolded form in urea and insert and fold into phospholipid bilayers upon strong dilution of the denaturant urea. OmpA is a structural protein and forms a small ion channel, composed of an 8-stranded transmembrane beta-barrel domain. FomA is a voltage-dependent porin, predicted to form a 14 stranded beta-barrel. Both OMPs fold into a range of model membranes of very different phospholipid compositions. Three membrane-bound folding intermediates of OmpA were discovered in folding studies with dioleoylphosphatidylcholine bilayers that demonstrated a highly synchronized mechanism of secondary and tertiary structure formation of beta-barrel membrane proteins. A study on FomA folding into lipid bilayers indicated the presence of parallel folding pathways for OMPs with larger transmembrane beta-barrels.     

Beta-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro

Little is known about the dynamic process of membrane protein folding, and few models exist to explore it. In this study we doubled the number of Escherichia coli outer membrane proteins (OMPs) for which folding into lipid bilayers has been systematically investigated. We cloned, expressed, and folded nine OMPs: outer membrane protein X (OmpX), OmpW, OmpA, the crcA gene product (PagP), OmpT, outer membrane phospholipase A (OmpLa), the fadl gene product (FadL), the yaet gene product (Omp85), and OmpF. These proteins fold into the same bilayer in vivo and share a transmembrane beta-barrel motif but vary in sequence and barrel size. We quantified the ability of these OMPs to fold into a matrix of bilayer environments. Several trends emerged from these experiments: higher pH values, thinner bilayers, and increased bilayer curvature promote folding of all OMPs. Increasing the incubation temperature promoted folding of several OMPs but inhibited folding of others. We discovered that OMPs do not have the same ability to fold into any single bilayer environment. This suggests that although environmental factors influence folding, OMPs also have intrinsic qualities that profoundly modulate their folding. To rationalize the differences in folding efficiency, we performed kinetic and thermal denaturation experiments, the results of which demonstrated that OMPs employ different strategies to achieve the observed folding efficiency.     

Mixed macromolecular crowding accelerates the refolding of rabbit muscle creatine kinase: implications for protein folding in physiological environments

The effects of four single macromolecular crowding agents, Ficoll 70, dextran 70, polyethylene glycol (PEG) 2000, and calf thymus DNA (CT DNA), and three mixed crowding agents containing both CT DNA and polysaccharide (or PEG 2000) on the refolding of guanidine hydrochloride-denatured rabbit muscle creatine kinase (MM-CK) have been examined by activity assay. When the total concentration of the mixed crowding agent is 100 g/l, in which the weight ratio of CT DNA to Ficoll 70 is 1:9, the refolding yield of MM-CK after refolding for 3 h under these conditions increases 23% compared with that in the presence of 10 g/l CT DNA, 18% compared with 100 g/l Ficoll 70, and 19% compared with that in the absence of crowding agents. A remarkable increase in the refolding yield of MM-CK by a mixed crowding agent containing CT DNA and dextran 70 (or PEG 2000) is also observed. Further folding kinetics analyses show that these three mixed crowding agents remarkably accelerate the refolding of MM-CK, compared with single crowding agents. Aggregation of MM-CK in the presence of any of the three mixed crowding agents is less serious than that in the presence of a single crowding agent at the same concentration but more serious than that in the absence of crowding agents. Both the refolding yield and the refolding rate of MM-CK in mixtures of these agents are increased relative to the individual agents by themselves, indicating that mixed macromolecular crowding agents are more favorable to MM-CK folding and can be used to reflect the physiological environment more accurately than single crowding agents.     

OmpA: Gating and dynamics via molecular dynamics simulations

Outer membrane proteins (OMPs) of Gram-negative bacteria have a variety of functions including passive transport, active transport, catalysis, pathogenesis and signal transduction. Whilst the structures of ∼ 25 OMPs are currently known, there is relatively little known about their dynamics in different environments. The outer membrane protein, OmpA from Escherichia coli has been studied extensively in different environments both experimentally and computationally, and thus provides an ideal test case for the study of the dynamics and environmental interactions of outer membrane proteins. We review molecular dynamics simulations of OmpA and its homologues in a variety of different environments and discuss possible mechanisms of pore gating. The transmembrane domain of E. coli OmpA shows subtle differences in dynamics and interactions between a detergent micelle and a lipid bilayer environment. Simulations of the crystallographic unit cell reveal a micelle-like network of detergent molecules interacting with the protein monomers. Simulation and modelling studies emphasise the role of an electrostatic-switch mechanism in the pore-gating mechanism. Simulation studies have been extended to comparative models of OmpA homologues from Pseudomonas aeruginosa (OprF) and Pasteurella multocida (PmOmpA), the latter model including the periplasmic C-terminal domain.     

细菌外膜蛋白参与革兰氏阴性细菌对异丙醇耐受的调节

细菌外膜蛋白与细菌对异丙醇耐受关系密切,但迄今为止尚未见相关研究.本文首先采用基于双向电泳(two dimensional electrophoresis,2-DE)的蛋白质组学技术,研究E.coli K-12 BW25113在有无异丙醇条件下外膜蛋白表达的差异.结果发现,外膜蛋白LamB、FadL和OmpC以及OmpT、Tsx、OmpA和OmpF在异丙醇应激条件下表达量分别上调和下调.然后通过基因敲除、补救和高表达等功能基因组学的方法,探讨这些功能外膜蛋白在异丙醇应激耐受中所起的作用,发现LamB、OmpA和OmpC在E.coli K-12 BW25113对异丙醇耐受过程中起到更重要的作用.最后,对EnvZ/OmpR双组分信号转导系统在对异丙醇耐受中的作用进行了研究,证实EnvZ/OmpR双组分信号转导系统确实参与细菌对异丙醇的耐受.因此,外膜蛋白的改变和EnvZ/OmpR双组分信号转导系统的调节是革兰氏阴性细菌对异丙醇耐受的一种重要机制。     

Protein folding by the effects of macromolecular crowding

Unfolded states of ribonuclease A were used to investigate the effects of macromolecular crowding on macromolecular compactness and protein folding. The extent of protein folding and compactness were measured by circular dichroism spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In addition, the effects of macromolecular crowding on inert macromolecule compactness were investigated by fluorescence correlation spectroscopy using Fluorescence-labeled PEG as a test macromolecule. The size of Fluorescence-labeled PEG decreased remarkably with an increase in the concentration of PEG 20000 or Ficoll 70. These results show that macromolecules are favored compact conformations in the presence of a high concentration of macromolecules and indicate the importance of a crowded environment for the folding and stabilization of globular proteins. Furthermore, the magnitude of the effects on macromolecular crowding by the different sizes of background molecules was investigated. RNase A and Fluorescence-labeled PEG did not become compact, and had folded conformation by the addition of PEG 200. The effect of the chemical potential on the compaction of a test molecule in relation to the relative sizes of the test and background molecules is also discussed.     

The trimeric periplasmic chaperone Skp of Escherichia coli forms 1:1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions

The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various OMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NalP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltage-dependent anion-selective channel, human isoform 1 (hVDAC1) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDAC1 nor for soluble bovine serum albumin. The Skp trimer formed 1:1 complexes, OMP·Skp₃, with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP·Skp₃ complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp₃ with YaeT displayed the smallest dissociation constants, complexes with NalP the largest. OMP binding to Skp₃ was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and stability of OMP·Skp₃ complexes. Light-scattering and circular dichroism experiments demonstrated that Skp₃ remained a stable trimer from pH 3 to pH 11. In the OmpA·Skp₃ complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA·Skp₃ complexes at low stoichiometries. Acrylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpA were located closer to the surface than in binary OmpA·Skp₃ complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS.     

Folding of outer membrane protein A in the anionic biosurfactant rhamnolipid

Folding and stability of bacterial outer membrane proteins (OMPs) are typically studied in vitro using model systems such as phospholipid vesicles or surfactant. OMP folding requires surfactant concentrations above the critical micelle concentration (cmc) and usually only occurs in neutral or zwitterionic surfactants, but not in anionic or cationic surfactants. Various Gram-negative bacteria produce the anionic biosurfactant rhamnolipid. Here we show that the OMP OmpA can be folded in rhamnolipid at concentrations above the cmc, though the thermal stability is reduced compared to the non-ionic surfactant dodecyl maltoside. We discuss implications for possible interactions between OMPs and biosurfactants in vivo.     

Refolding of an integral membrane protein. OmpA of Escherichia coli

OmpA is an integral membrane protein from the outer membrane of Escherichia coli. Purified, lipopolysaccharide-free OmpA was denatured by boiling in sodium dodecyl sulfate (SDS). Refolding was then induced by replacement of SDS with the nonionic detergent octylglucoside. The structure of both the denatured and refolded protein were investigated by SDS-gel electrophoresis, protease digestion, Raman and fluorescence spectroscopy. Refolded OmpA could be reconstituted into membranes of the synthetic lipid dimyristoylphosphatidylcholine. Thus, lipopolysaccharide is neither necessary for proper folding of OmpA nor for its insertion into lipid membranes. Based on this result, models for sorting of OmpA into the outer membrane of E. coli are discussed.     

Mixed macromolecular crowding accelerates the oxidative refolding of reduced, denatured lysozyme: implications for protein folding in intracellular environments

The oxidative refolding of reduced, denatured hen egg white lysozyme in the presence of a mixed macromolecular crowding agent containing both bovine serum albumin (BSA) and polysaccharide has been studied from a physiological point of view. When the total concentration of the mixed crowding agent is 100 g/liter, in which the weight ratio of BSA to dextran 70 is 1:9, the refolding yield of lysozyme after refolding for 4 h under this condition increases 24% compared with that in the presence of BSA and 16% compared with dextran 70. A remarkable increase in the refolding yield of lysozyme by a mixed crowding agent containing BSA and Ficoll 70 is also observed. Further folding kinetics analyses show that these two mixed crowding agents accelerate the oxidative refolding of lysozyme remarkably, compared with single crowding agents. These results suggest that the stabilization effects of mixed macromolecular crowding agents are stronger than those of single polysaccharide crowding agents such as dextran 70 and Ficoll 70, whereas the excluded volume effects of mixed macromolecular crowding agents are weaker than those of single protein crowding agents such as BSA. Both the refolding yield and the rate of the oxidative refolding of lysozyme in these two mixed crowded solutions with suitable weight ratios are higher than those in single crowded solutions, indicating that mixed macromolecular crowding agents are more favorable to lysozyme folding and can be used to simulate the intracellular environments more accurately than single crowding agents do.     

OmpA can form folded and unfolded oligomers

The monomeric outer membrane protein OmpA from Escherichia coli has long served as a model protein for studying the folding and membrane insertion of β-barrel membrane proteins. Here we report that when OmpA is refolded in limiting amounts of surfactant (close to the cmc), it has a high propensity to form folded and unfolded oligomers. The oligomers exist both in a folded and (partially) unfolded form which both dissociate under denaturing conditions. Oligomerization does not require the involvement of the periplasmic domain and is not strongly affected by ionic strength. The folded dimers can be isolated and show native-like secondary structure; they are resistant to proteolytic attack and do not dissociate in high surfactant concentrations, indicating high kinetic stability once formed. Remarkably, OmpA also forms significant amounts of higher order structures when refolding in the presence of lipid vesicles. We suggest that oligomerization occurs by domain swapping favored by the high local concentration of OmpA molecules congregating on the same micelle or vesicle. In this model, the unfolded oligomer is stabilized by a small number of intermolecular β-strand contacts and subsequently folds to a more stable state where these intermolecular contacts are consolidated in a native-like fashion by contacts between complementary β-strands from different molecules. Our model is supported by the ability of complementary fragments to associate with each other in vitro. Oligomerization is probably avoided in the cell by the presence of cellular chaperones which maintain the protein in a monomeric state.     

Macromolecular crowding induces a molten globule state in the C-terminal domain of histone H1

We studied the secondary structure of the C-terminal domains of the histone H1 subtypes H1 degrees (C-H1 degrees ) and H1t (C-H1t) in the presence of macromolecular crowding agents (Ficoll 70 and PEG 6000) by IR spectroscopy. The carboxyl-terminal domain has little structure in aqueous solution but became extensively folded in the presence of crowding agents. In 30% PEG, C-H1 degrees contained 19% alpha-helix, 28% beta-sheet, 16% turns, and 31% open loops. Similar proportions were observed in 30% Ficoll 70 and for C-H1t in both crowding agents. The proportions of secondary structure motifs were comparable to those of the DNA-bound domain. Kratky plots of the small-angle x-ray scattering showed that in crowding agents the C-terminus had the compaction of a globular state. Progressive dissipation of the secondary structure and a linear increase in partial heat capacity with temperature together with increased binding of ANS indicated that the C-terminus is not cooperatively folded in crowded conditions. Native-like secondary structure and compactness in absence of folding cooperativity indicate that the C-terminus in crowding agents is in a molten globule state. Folding of the C-terminus in crowded conditions may increase the rate of the transition toward the DNA-bound state and facilitate H1 diffusion inside cell nuclei.     

Folding and insertion of the outer membrane protein OmpA is assisted by the chaperone Skp and by lipopolysaccharide

We have studied the folding pathway of a beta-barrel membrane protein using outer membrane protein A (OmpA) of Escherichia coli as an example. The deletion of the gene of periplasmic Skp impairs the assembly of outer membrane proteins of bacteria. We investigated how Skp facilitates the insertion and folding of completely unfolded OmpA into phospholipid membranes and which are the biochemical and biophysical requirements of a possible Skp-assisted folding pathway. In refolding experiments, Skp alone was not sufficient to facilitate membrane insertion and folding of OmpA. In addition, lipopolysaccharide (LPS) was required. OmpA remained unfolded when bound to Skp and LPS in solution. From this complex, OmpA folded spontaneously into lipid bilayers as determined by electrophoretic mobility measurements, fluorescence spectroscopy, and circular dichroism spectroscopy. The folding of OmpA into lipid bilayers was inhibited when one of the periplasmic components, either Skp or LPS, was absent. Membrane insertion and folding of OmpA was most efficient at specific molar ratios of OmpA, Skp, and LPS. Unfolded OmpA in complex with Skp and LPS folded faster into phospholipid bilayers than urea-unfolded OmpA. Together, these results describe a first assisted folding pathway of an integral membrane protein on the example of OmpA.     

Mixed macromolecular crowding inhibits amyloid formation of hen egg white lysozyme

The effects of two single macromolecular crowding agents, Ficoll 70 and bovine serum albumin (BSA), and one mixed macromolecular crowding agent containing both BSA and Ficoll 70, on amyloid formation of hen egg white lysozyme have been examined by thioflavin T binding, Congo red binding, transmission electron microscopy, and activity assay, as a function of crowder concentration and composition. Both the mixed crowding agent and the protein crowding agent BSA at 100 g/l almost completely inhibit amyloid formation of lysozyme and stabilize lysozyme activity on the investigated time scale, but Ficoll 70 at the same concentration neither impedes amyloid formation of lysozyme effectively nor stabilizes lysozyme activity. Further kinetic and isothermal titration calorimetry analyses indicate that a mixture of 5 g/l BSA and 95 g/l Ficoll 70 inhibits amyloid formation of lysozyme and maintains lysozyme activity via mixed macromolecular crowding as well as weak, nonspecific interactions between BSA and nonnative lysozyme. Our data demonstrate that BSA and Ficoll 70 cooperatively contribute to both the inhibitory effect and the stabilization effect of the mixed crowding agent, suggesting that mixed macromolecular crowding inside the cell may play a role in posttranslational quality control mechanism.     

Macromolecular crowding modulates α-synuclein amyloid fiber growth

The crowdedness of living cells, hundreds of milligrams per milliliter of macromolecules, may affect protein folding, function, and misfolding. Still, such processes are most often studied in dilute solutions in vitro. To assess consequences of the in vivo milieu, we here investigated the effects of macromolecular crowding on the amyloid fiber formation reaction of α-synuclein, the amyloidogenic protein in Parkinson’s disease. For this, we performed spectroscopic experiments probing individual steps of the reaction as a function of the macromolecular crowding agent Ficoll70, which is an inert sucrose-based polymer that provides excluded-volume effects. The experiments were performed at neutral pH at quiescent conditions to avoid artifacts due to shaking and glass beads (typical conditions for α-synuclein), using amyloid fiber seeds to initiate reactions. We find that both primary nucleation and fiber elongation steps during α-synuclein amyloid formation are accelerated by the presence of 140 and 280 mg/mL Ficoll70. Moreover, in the presence of Ficoll70 at neutral pH, secondary nucleation appears favored, resulting in faster overall α-synuclein amyloid formation. In contrast, sucrose, a small-molecule osmolyte and building block of Ficoll70, slowed down α-synuclein amyloid formation. The ability of cell environments to modulate reaction kinetics to a large extent, such as severalfold faster individual steps in α-synuclein amyloid formation, is an important consideration for biochemical reactions in living systems.     

Outer membrane protein A of E. coli folds into detergent micelles, but not in the presence of monomeric detergent.

Outer membrane protein A (OmpA) of Escherichia coli is a beta-barrel membrane protein that unfolds in 8 M urea to a random coil. OmpA refolds upon urea dilution in the presence of certain detergents or lipids. To examine the minimal requirements for secondary and tertiary structure formation in beta-barrel membrane proteins, folding of OmpA was studied as a function of the hydrophobic chain length, the chemical structure of the polar headgroup, and the concentration of a large array of amphiphiles. OmpA folded in the presence of detergents only above a critical minimal chain length of the apolar chain as determined by circular dichroism spectroscopy and a SDS-PAGE assay that measures tertiary structure formation. Details of the chemical structure of the polar headgroup were unimportant for folding. The minimal chain length required for folding correlated with the critical micelle concentration in each detergent series. Therefore, OmpA requires preformed detergent micelles for folding and does not adsorb monomeric detergent to its perimeter after folding. Formation of secondary and tertiary structure is thermodynamically coupled and strictly dependent on the interaction with aggregated amphiphiles.     

Discrimination of outer membrane proteins using machine learning algorithms

Discriminating outer membrane proteins (OMPs) from other folding types of globular and membrane proteins is an important task both for identifying OMPs from genomic sequences and for the successful prediction of their secondary and tertiary structures. In this work, we have analyzed the performance of different methods, based on Bayes rules, logistic functions, neural networks, support vector machines, decision trees, etc. for discriminating OMPs. We found that most of the machine learning techniques discriminate OMPs with similar accuracy. The neural network-based method could discriminate the OMPs from other proteins [globular/transmembrane helical (TMH)] at the fivefold cross-validation accuracy of 91.0% in a dataset of 1,088 proteins. The accuracy of discriminating globular proteins is 88.8% and that of TMH proteins is 93.7%. Further, the neural network method is tested with globular proteins belonging to 30 different folding types and it could successfully exclude 95% of the considered proteins. The proteins with SAM domain such as knottins, rubredoxin, and thioredoxin folds are eliminated with 100% accuracy. These accuracy levels are comparable to or better than other methods in the literature. We suggest that this method could be effectively used to discriminate OMPs and for detecting OMPs in genomic sequences.