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• W2018131055 abstract "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 β-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. 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 β-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. The number of high resolution membrane protein structures increases each year (1White S.H. Protein Sci. 2004; 13: 1948-1949Crossref PubMed Scopus (236) Google Scholar). These structures provide a static picture of the membrane protein native state but reveal nothing about the dynamic folding process that formed them. Despite these advances in structural biology, the question remains: how does a polypeptide chain encode a structure that folds into a biological membrane? Proteins adopt a variety of folds, yet those that span a biological membrane exhibit only one of two general architectures, either the α-helix or the β-barrel. These transmembrane (TM) 2The abbreviations used are: TMtransmembraneOMPouter membrane proteinPGphosphatidylglycerolPEphosphatidylethanolaminePCphosphatidylcholineLUVlarge unilamellar vesicleSUVsmall unilamellar vesiclediCdiacylglycerol. motifs satisfy the hydrogen bonding requirements of the peptide backbone in the hydrophobic environment of the cellular membrane (2Kleffel B. Garavito R.M. Baumeister W. Rosenbusch J.P. EMBO J. 1985; 4: 1589-1592Crossref PubMed Scopus (111) Google Scholar, 3Engelman D.M. Steitz T.A. Cell. 1981; 23: 411-422Abstract Full Text PDF PubMed Scopus (603) Google Scholar). Interestingly, these motifs are found in different membranes in vivo. Most bilayers contain proteins with an α-helix TM motif, but β-barrel proteins only reside in the outer membranes of chloroplasts (4Schleiff E. Eichacker L.A. Eckart K. Becker T. Mirus O. Stahl T. Soll J. Protein Sci. 2003; 12: 748-759Crossref PubMed Scopus (99) Google Scholar), mitochondria (5Hill K. Model K. Ryan M.T. Dietmeier K. Martin F. Wagner R. Pfanner N. Nature. 1998; 395: 516-521Crossref PubMed Scopus (408) Google Scholar, 6Casadio R. Jacoboni I. Messina A. De Pinto V. FEBS Lett. 2002; 520: 1-7Crossref PubMed Scopus (84) Google Scholar), and Gram-negative bacteria (7Lomize M.A. Lomize A.L. Pogozheva I.D. Mosberg H.I. Bioinformatics (Oxf.). 2006; 22: 623-625Crossref PubMed Scopus (901) Google Scholar). To traverse a membrane, proteins adopt one of only two folds, yet their ability to insert and assume their native structures remains a complex issue that is not well understood. transmembrane outer membrane protein phosphatidylglycerol phosphatidylethanolamine phosphatidylcholine large unilamellar vesicle small unilamellar vesicle diacylglycerol. In vivo, α-helical and β-barrel membrane proteins insert into their respective biological membranes via different molecular machinery. The protein factors that aid bilayer insertion of an α-helix motif have been identified, and an elegant system using in vivo machinery has been developed to study α-helix insertion into membranes (8Hessa T. White S.H. von Heijne G. Science. 2005; 307: 1427Crossref PubMed Scopus (158) Google Scholar, 9Meindl-Beinker N.M. Lundin C. Nilsson I. White S.H. von Heijne G. EMBO Rep. 2006; 7: 1111-1116Crossref PubMed Scopus (56) Google Scholar). For β-barrel proteins, also called outer membrane proteins (OMPs), a few of the in vivo folding factors that facilitate insertion have been proposed only recently and are not yet fully understood (10Bos M.P. Robert V. Tommassen J. Annu. Rev. Microbiol. 2007; 61: 191-214Crossref PubMed Scopus (359) Google Scholar). Although the in vivo OMP folding pathway must yet be completely defined, it has been shown that several OMPs can fold spontaneously into synthetic membranes (11Surrey T. Jahnig F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7457-7461Crossref PubMed Scopus (207) Google Scholar, 12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar, 13Shanmugavadivu B. Apell H.J. Meins T. Zeth K. Kleinschmidt J.H. J. Mol. Biol. 2007; 368: 66-78Crossref PubMed Scopus (69) Google Scholar, 14Pocanschi C.L. Apell H.J. Puntervoll P. Hogh B. Jensen H.B. Welte W. Kleinschmidt J.H. J. Mol. Biol. 2006; 355: 548-561Crossref PubMed Scopus (59) Google Scholar, 15Huysmans G.H. Radford S.E. Brockwell D.J. Baldwin S.A. J. Mol. Biol. 2007; 373: 529-540Crossref PubMed Scopus (51) Google Scholar). The ability of OMPs to spontaneously insert into synthetic vesicles has two important implications. First, there is no additional energy input required for the folding reaction to occur. Therefore, the native state of the protein is an equilibrium structure and thermodynamic measurements of stability can be made in vitro that have relevance in the biological setting. Second, a system composed of synthetic vesicles has significantly fewer variables than the heterogeneous milieu of biological membranes. Furthermore, synthetic vesicles can be modified systematically to dissect the molecular details of how bilayer properties influence OMP folding. Thus, in the absence of a clearly defined in vivo folding pathway, in vitro folding studies can identify both the protein and the bilayer characteristics that promote folding and insertion of membrane proteins. Unlike water, the relatively homogeneous solvent for soluble proteins, the biological membrane can vary in its lipid composition, overall charge, and gross morphology. The most valuable in vitro membrane protein folding system would therefore mimic the in vivo lipid conditions that a protein encounters. Proteins in the outer membrane of Escherichia coli reside in an asymmetric bilayer in which the outer leaflet is made of the glycolipid lipopolysaccharide and the inner leaflet is composed of phospholipids with either the phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) head group (16Osborn M.J. Gander J.E. Parisi E. Carson J. J. Biol. Chem. 1972; 247: 3962-3972Abstract Full Text PDF PubMed Google Scholar, 17Kamio Y. Nikaido H. Biochemistry. 1976; 15: 2561-2570Crossref PubMed Scopus (253) Google Scholar). This asymmetry can be recreated in vitro with planar bilayers, but such bilayers do not remain stable on the time scale of OMP folding experiments (18Seydel U. Schroder G. Brandenburg K. J. Membr. Biol. 1989; 109: 95-103Crossref PubMed Scopus (33) Google Scholar) and therefore are not amenable to folding studies. Instead, vesicles composed entirely of phospholipids have been used to study OMP folding (11Surrey T. Jahnig F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7457-7461Crossref PubMed Scopus (207) Google Scholar, 12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar, 13Shanmugavadivu B. Apell H.J. Meins T. Zeth K. Kleinschmidt J.H. J. Mol. Biol. 2007; 368: 66-78Crossref PubMed Scopus (69) Google Scholar, 14Pocanschi C.L. Apell H.J. Puntervoll P. Hogh B. Jensen H.B. Welte W. Kleinschmidt J.H. J. Mol. Biol. 2006; 355: 548-561Crossref PubMed Scopus (59) Google Scholar, 15Huysmans G.H. Radford S.E. Brockwell D.J. Baldwin S.A. J. Mol. Biol. 2007; 373: 529-540Crossref PubMed Scopus (51) Google Scholar). Despite the asymmetry of outer membranes, it is likely that phospholipids are the most appropriate model for folding studies, because OMPs first encounter the inner leaflet (composed entirely of phospholipids) as they fold into the outer membrane in vivo (10Bos M.P. Robert V. Tommassen J. Annu. Rev. Microbiol. 2007; 61: 191-214Crossref PubMed Scopus (359) Google Scholar). The most extensively studied β-barrel folding model in phospholipid vesicles is outer membrane protein A (OmpA). Studies of OmpA have revealed how insertion occurs (19Kleinschmidt J.H. Tamm L.K. Biochemistry. 1999; 38: 4996-5005Crossref PubMed Scopus (95) Google Scholar, 20Kleinschmidt J.H. Tamm L.K. J. Mol. Biol. 2002; 324: 319-330Crossref PubMed Scopus (143) Google Scholar, 21Surrey T. Jahnig F. J. Biol. Chem. 1995; 270: 28199-28203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 22Kleinschmidt J.H. Tamm L.K. Biochemistry. 1996; 35: 12993-13000Crossref PubMed Scopus (150) Google Scholar, 23Kleinschmidt J.H. den Blaauwen T. Driessen A.J. Tamm L.K. Biochemistry. 1999; 38: 5006-5016Crossref PubMed Scopus (124) Google Scholar) and have measured the stability of the native structure in different lipid environments (24Hong H. Szabo G. Tamm L.K. Nat. Chem. Biol. 2006; 2: 627-635Crossref PubMed Scopus (103) Google Scholar, 25Hong H. Tamm L.K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4065-4070Crossref PubMed Scopus (198) Google Scholar, 26Tamm L.K. Hong H. Liang B. Biochim. Biophys. Acta. 2004; 1666: 250-263Crossref PubMed Scopus (234) Google Scholar, 27Hong H. Park S. Jimenez R.H. Rinehart D. Tamm L.K. J. Am. Chem. Soc. 2007; 129: 8320-8327Crossref PubMed Scopus (129) Google Scholar). The principles garnered from these studies allow conclusions to be drawn for how OmpA behaves, but they cannot reliably be applied to all OMPs until the behaviors of other proteins are observed in the same environment. Moreover, folding studies have been performed on other OMPs (12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar, 13Shanmugavadivu B. Apell H.J. Meins T. Zeth K. Kleinschmidt J.H. J. Mol. Biol. 2007; 368: 66-78Crossref PubMed Scopus (69) Google Scholar, 14Pocanschi C.L. Apell H.J. Puntervoll P. Hogh B. Jensen H.B. Welte W. Kleinschmidt J.H. J. Mol. Biol. 2006; 355: 548-561Crossref PubMed Scopus (59) Google Scholar, 15Huysmans G.H. Radford S.E. Brockwell D.J. Baldwin S.A. J. Mol. Biol. 2007; 373: 529-540Crossref PubMed Scopus (51) Google Scholar), but no comprehensive folding screen exists that facilitates comparison between proteins. To directly compare the folding propensities of membrane proteins, we probed the folding conditions of nine different β-barrel OMPs: 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. The OMPs we chose all reside in the outer membrane of E. coli. Despite inhabiting the same bilayer environment in vivo and sharing a common TM motif, the primary sequences of these nine OMPs could not be aligned altogether or in pairwise BLAST queries (data not shown). Furthermore, the structure of each OMP varies from the next (Fig. 1). These model OMPs have barrel sizes ranging from eight β-strands (OmpX, OmpW, OmpA, and PagP) to 16 β-strands (OmpF). Their extramembrane structures also vary. OmpA and Omp85 have periplasmic domains as large as their TM domains, whereas FadL and OmpT have significant amounts of structure extending from their barrels toward the extracellular side of the biological membrane. The study of these nine OMPs constitutes the largest set of OMPs evaluated in tandem to date. In this work, we have established OmpW, OmpT, OmpLa, FadL, and Omp85 as novel models for folding studies. For purposes of direct comparison, we included OMPs that have previously been shown to fold into phospholipid bilayers in vitro: OmpA (11Surrey T. Jahnig F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7457-7461Crossref PubMed Scopus (207) Google Scholar), OmpF (12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar), OmpX (28Mahalakshmi R. Franzin C.M. Choi J. Marassi F.M. Biochim. Biophys. Acta. 2007; 1768: 3216-3224Crossref PubMed Scopus (26) Google Scholar), and PagP (15Huysmans G.H. Radford S.E. Brockwell D.J. Baldwin S.A. J. Mol. Biol. 2007; 373: 529-540Crossref PubMed Scopus (51) Google Scholar). Our data set doubles the number of E. coli OMPs for which folding has been systematically examined in lipid bilayers. Further, because these OMPs are sequentially and structurally diverse yet fold into the same environment in vivo, our results allowed the deduction of broad rules that define folding similarities and differences. Vesicle Preparation—Lipids dissolved in chloroform (Avanti Polar Lipids) were dried to a thin film in glass vials under a gentle stream of nitrogen gas. Lipid films were evacuated for at least 3 h to remove residual solvent and stored at -20 °C until use. Lipid films were reconstituted in buffer containing 2 mm EDTA (Fluka) and 10 mm borate (Sigma), pH 10. Vesicles used in pH studies were brought up in the same concentration of appropriate buffers at various pH values. To make small unilamellar vesicles (SUVs), lipids reconstituted in buffer were sonicated on ice for 50 min with a 50% duty cycle with a Branson Sonifier as described previously (25Hong H. Tamm L.K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4065-4070Crossref PubMed Scopus (198) Google Scholar). Large unilamellar vesicles (LUVs) were made by extruding reconstituted lipids 11 times through a 0.1 μm filter using a mini-extruder (Avanti Polar Lipids). Cloning and Expression of OMPs—Primers were designed to encompass the mature forms of the OMPs and add NdeI (5′) and BamHI (3′) sites. Primers are listed in supplemental Table S1. OMP genes were amplified using ExTaq polymerase (Takara) from an overnight growth of E. coli K12 MG1655 (29Blattner F.R. Plunkett 3rd, G. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6053) Google Scholar). The PCR products were cut with restriction enzymes and ligated into a pET11a vector. The resulting plasmids were transformed into a laboratory supply of electrocompetent DH5α cells. The sequences were confirmed by double-stranded DNA sequencing using the T7 promoter and T7 terminator primers for all clones. Additional primers were designed and used for Omp85 to cover the length of the insert. The expression products were confirmed by matrix-assisted laser desorption ionization (MALDI) mass spectrometry at the Johns Hopkins Medical Institute. Plasmids were transformed into BL21(DE3) Star™ cells (Invitrogen). Transformed cells were grown in 500 ml of LB medium to an optical density of 0.6 at 600 nm before expression was induced by the addition of 1 mm β-d-1-thiogalactopyrano-side. Cells continued to incubate for 3-4 h at 37 °C and were harvested by centrifugation (5500 rpm, 30 min, 4 °C). Pellets were resuspended in 25 ml of lysis buffer (50 mm Tris, pH 8, 40 mm EDTA, 125 μl of 20 mg ml-1 lysozyme) and incubated for 30 min on ice before being sonicated three times for 1 min at a 50% duty cycle. Brij-35 (Sigma) was added to a final concentration of 0.1%. Inclusion bodies were centrifuged (6000 rpm, 30 min, 4 °C) and washed twice by resuspension in 25 ml of wash buffer (10 mm Tris, pH 8, 2 mm EDTA) followed by centrifugation (6000 rpm, 30 min, 4 °C). Cells were resuspended a third time in wash buffer and aliquoted into 3-ml fractions before a final centrifugation (6000 rpm, 30 min, 4 °C). Supernatant was discarded, and inclusion body pellets were stored at -20 °C. OMP Folding—Inclusion body pellets were dissolved in 8 m urea (ultra pure grade, Amresco), 2 mm EDTA, and 10 mm borate, pH 10, to a final concentration of 100 μm protein. Protein concentration was determined by measuring the absorbance at 280 nm. The extinction coefficients for each OMP were determined using Sequence Analysis software (Informagen), with the exception of OmpLa, for which we used a previously published value (90,444 m-1 cm-1) (30Dekker N. Merck K. Tommassen J. Verheij H.M. Eur. J. Biochem. 1995; 232: 214-219Crossref PubMed Scopus (88) Google Scholar). OMPs were refolded by rapid dilution into 3.2 mm synthetic lipid or 2.3 mg ml-1 lipid extract in folding buffer (1 m urea, 2 mm EDTA, 10 mm borate, pH 10) to a final concentration of 4 μm protein. Folding temperatures were controlled by incubating reactions in a PTC-200 Peltier thermal cycler (MJ Research). Overnight reactions were incubated for 15 h. SDS-PAGE—Folding reactions were quenched by adding 5× SDS gel-loading buffer (31Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989: B.25Google Scholar) to a final dilution of 1× SDS gel-loading buffer. Samples were stored at -20 °C, if not immediately loaded onto a gel. 15 μl of sample (or only 5 μl of Omp85 sample because of its high molecular weight) were then loaded on a precast gel (Bio-Rad) without boiling. OmpX, OmpW, and PagP required gels with 4-20% acrylamide continuous gradient to resolve the folded and unfolded populations, but all other OMPs could be resolved on 10% acrylamide gels. After electrophoresis, gels were stained with Coomassie Blue and scanned digitally. Densitometry was performed using ImageJ software. 3Program authored by Wayne Rasband. The linear regions of the densitometry were determined by measuring the density of standards of known protein amounts. The fraction folded is calculated by dividing the intensity of the folded band by the sum of the intensities of both the folded and unfolded bands. Folding Efficiency—We defined folding efficiency as the fraction of folded protein quantified at the overnight (15 h) time point. Folding Kinetics—After folding was initiated, small aliquots of the folding reaction were removed and quenched with 5× SDS-PAGE loading buffer at the following time points: 5, 10, 20, 30, 45, 60, 120, 180, 300, 420, 720, 1200, and 1800 s. The fraction folded for each time point was determined by SDS-PAGE and densitometry as described above. We fit the results of folding kinetics to either a single exponential equation, y=yo+Aexp(-kt)(Eq. 1) or a double exponential equation, y=yo+A1exp(-k1t)+A2exp(-k2t)(Eq. 2) where y is the fraction folded at a given time, t, y0 is the fraction folded as time approaches infinity, k1 and k2 are rate constants, and A1 and A2 are the negative amplitudes associated with each rate constant. The burst phase is calculated as a sum of y0, A1, and A2. The lag phase is defined as the first time point at which fraction folded was equal to or greater than 0.05. The reported values and error bars equal the average and standard deviations, respectively, of three independent kinetic experiments. Thinner Bilayers and Smaller Vesicles Promote Folding Efficiency—To probe the bilayer properties that affect OMP folding, we incubated OMPs overnight with synthetic vesicles composed of a variety of lipids. The overnight time point (15 h) was chosen because it is an experimentally convenient time point for screening a large number of conditions and is intermediate to the incubation times used in previous folding studies (12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar, 14Pocanschi C.L. Apell H.J. Puntervoll P. Hogh B. Jensen H.B. Welte W. Kleinschmidt J.H. J. Mol. Biol. 2006; 355: 548-561Crossref PubMed Scopus (59) Google Scholar, 21Surrey T. Jahnig F. J. Biol. Chem. 1995; 270: 28199-28203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). Although some of the folding reactions were not complete by 15 h, this time point was sensitive to the fastest and most efficient folding events. The buffer we used contained 10 mm borate, pH 10, 2 mm EDTA, and 1 m urea. Previous OMP studies have used residual amounts of urea under folding conditions (11Surrey T. Jahnig F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7457-7461Crossref PubMed Scopus (207) Google Scholar, 12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar, 13Shanmugavadivu B. Apell H.J. Meins T. Zeth K. Kleinschmidt J.H. J. Mol. Biol. 2007; 368: 66-78Crossref PubMed Scopus (69) Google Scholar, 14Pocanschi C.L. Apell H.J. Puntervoll P. Hogh B. Jensen H.B. Welte W. Kleinschmidt J.H. J. Mol. Biol. 2006; 355: 548-561Crossref PubMed Scopus (59) Google Scholar, 21Surrey T. Jahnig F. J. Biol. Chem. 1995; 270: 28199-28203Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), yet OmpA can fold in 4 m urea (25Hong H. Tamm L.K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 4065-4070Crossref PubMed Scopus (198) Google Scholar) and PagP can fold in 7 m urea (15Huysmans G.H. Radford S.E. Brockwell D.J. Baldwin S.A. J. Mol. Biol. 2007; 373: 529-540Crossref PubMed Scopus (51) Google Scholar). Although OMPs can fold into synthetic vesicles at a variety of urea concentrations, we chose a low concentration of urea (1 m) in which to conduct this folding screen. To determine the fraction of protein that folded under a given condition, we used SDS-PAGE. Even before the first OMP structure was solved, it was observed by SDS-PAGE that OMPs share a characteristic called “heat modifiability”; that is, adding SDS to OMPs captures the folded and unfolded populations present in solution. During electrophoresis, these populations migrate to different positions (32Inouye M. Yee M.L. J. Bacteriol. 1973; 113: 304-312Crossref PubMed Google Scholar, 33Nakamura K. Mizushima S. J. Biochem. 1976; 80: 1411-1422Crossref PubMed Scopus (181) Google Scholar). When OMPs are solubilized in SDS and subsequently boiled, they lose their native β-content and migrate to their expected molecular weight on polyacrylamide gels (33Nakamura K. Mizushima S. J. Biochem. 1976; 80: 1411-1422Crossref PubMed Scopus (181) Google Scholar). However, unboiled samples of folded OMPs retain a high content of β-structure (33Nakamura K. Mizushima S. J. Biochem. 1976; 80: 1411-1422Crossref PubMed Scopus (181) Google Scholar) as well as their activity (12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar, 15Huysmans G.H. Radford S.E. Brockwell D.J. Baldwin S.A. J. Mol. Biol. 2007; 373: 529-540Crossref PubMed Scopus (51) Google Scholar, 30Dekker N. Merck K. Tommassen J. Verheij H.M. Eur. J. 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To determine the folding propensities under the most native-like conditions, we tested whether OMPs could fold into extracts prepared from their native lipids. We purchased two E. coli lipid extracts from Avanti Polar Lipids: total extract (57.5% PE, 15.1% PG, 9.8% cardiolipin, and 17.6% of uncharacterized mass) and polar extract (67.0% PE, 23.2% PG, 9.8% cardiolipin). These E. coli extracts could not be extruded to make LUVs; therefore we made SUVs by sonication. We incubated OMPs with SUVs of native lipids for 15 h, quenched folding by adding SDS-loading buffer, and performed electrophoresis on the unboiled samples. The fraction folded for each protein was determined by densitometry of the scanned gels. Fig. 3 shows that total and polar extracts gave similar results. In both extracts, a small population of only four of the nine proteins (OmpX, OmpA, OmpT, and Omp85) could fold into SUVs of native lipids. We then tested the ability of synthetic lipids to recapitulate the native lipids result. Fig. 3 shows that SUVs composed of synthetic lipids with a composition similar to the native extracts (75% C16C18:1PE, 25% C16C18:1PG) gave comparable results. We therefore conclude that pure preparations of synthetic lipids can reproduce the results obtained using native lipids. Further, unlike the native lipid extracts, the synthetic lipids could be extruded through 100-nm filters to make LUVs, which allowed us to assess the effect of bilayer curvature on OMP folding. With the exception of OmpT, we found that none of the OMPs folded into LUVs of synthetic lipids of native-like composition. Because less than half of the OMPs could fold into extracted lipids or native-like synthetic lipids, we tried bilayers with phosphatidylcholine (PC) head groups. Although E. coli do not contain any PC in their bilayers, PC-containing membranes are used in many aspects of OMP research, including computational simulations (41Khalid S. Bond P.J. Carpenter T. Sansom M.S. Biochim. Biophys. Acta. 2007; (10.101b/j.bbamem.2007.05.024)Google Scholar), ion channel measurements (42Hong H. Patel D.R. Tamm L.K. van den Berg B. J. Biol. Chem. 2006; 281: 7568-7577Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 43Basle A. Iyer R. Delcour A.H. Biochim. Biophys. Acta. 2004; 1664: 100-107Crossref PubMed Scopus (54) Google Scholar), and folding studies (11Surrey T. Jahnig F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7457-7461Crossref PubMed Scopus (207) Google Scholar, 12Surrey T. Schmid A. Jahnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (123) Google Scholar, 13Shanmugavadivu B. Apell H.J. Meins T. Zeth K. Kleinschmidt J.H. J. Mol. Biol. 2007; 368: 66-78Crossref PubMed Scopus (69) Google Scholar, 14Pocanschi C.L. Apell H.J. Puntervoll P. Hogh B. Jensen H.B. Welte W. Kleinschmidt J.H. J. Mol. Biol. 2006; 355: 548-561Crossref PubMed Scopus (59) Google Scholar, 22Kleinschmidt J.H. Tamm L.K. Biochemistry. 1996; 35: 12993-13000Crossref PubMed Scopus (150) Google Scholar). Moreover, the TM β-barrels found in eukaryotes are located in the outer membranes of mitochondria, which are composed primarily of PC lipids, further supporting the notion that membranes composed of synthetic PC lipids are appropriate in vitro substitutes for biological lipid bilayers. Consistent with this idea, the data shown in Figs. 3 and 4 demonstrate that PC lipids promote in vitro folding of all the OMPs in this study. Using PC lipids, we further investigated how acyl chain length, acyl chain saturation, and vesicle size influenced fo" @default.
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• W2018131055 title "β-Barrel Proteins That Reside in the Escherichia coli Outer Membrane in Vivo Demonstrate Varied Folding Behavior in Vitro" @default.
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