Non-lamellar Structure and Negative Charges of Lipopolysaccharides Required for Efficient Folding of Outer Membrane Protein PhoE of Escherichia coli
1999; Elsevier BV; Volume: 274; Issue: 8 Linguagem: Inglês
10.1074/jbc.274.8.5114
ISSN1083-351X
AutoresHans de Cock, Klaus Brandenburg, Andre Wiese, Otto Holst, Ulrich Seydel,
Tópico(s)Escherichia coli research studies
ResumoLipopolysaccharides (LPS) are amphiphilic molecules in the outer leaflet of the bacterial outer membrane. Recently, an early role for LPS in the folding of outer membrane porin PhoE was demonstrated in vitro. In order to elucidate the molecular mechanism of LPS-protein interactions, folding of PhoE protein was studied with a large set of well characterized LPS chemotypes. We demonstrate that negative charges in the inner core region contribute to the high efficiency of folding of PhoE protein. In addition, the supramolecular structure of the LPS aggregate seems to be important. LPS with a lipid A part that prefers a lamellar or a direct micellar structure and a high state of order of its acyl chains is much less efficient to support folding as compared with LPS with lipid A that prefers a non-lamellar structure and a low acyl chain order. Thesein vitro data indicate that extensive interactions between the core and lipid A region of LPS with the protein are required to support protein folding. The LPS-PhoE binding might be promoted by the presence of hydroxy fatty acids in the lipid A moiety of LPS. Lipopolysaccharides (LPS) are amphiphilic molecules in the outer leaflet of the bacterial outer membrane. Recently, an early role for LPS in the folding of outer membrane porin PhoE was demonstrated in vitro. In order to elucidate the molecular mechanism of LPS-protein interactions, folding of PhoE protein was studied with a large set of well characterized LPS chemotypes. We demonstrate that negative charges in the inner core region contribute to the high efficiency of folding of PhoE protein. In addition, the supramolecular structure of the LPS aggregate seems to be important. LPS with a lipid A part that prefers a lamellar or a direct micellar structure and a high state of order of its acyl chains is much less efficient to support folding as compared with LPS with lipid A that prefers a non-lamellar structure and a low acyl chain order. Thesein vitro data indicate that extensive interactions between the core and lipid A region of LPS with the protein are required to support protein folding. The LPS-PhoE binding might be promoted by the presence of hydroxy fatty acids in the lipid A moiety of LPS. Bacterial outer membrane proteins are synthesized as precursor proteins in the cytoplasm. After their translocation across the inner membrane via the Sec machinery (1Wickner W. Driessen A.J.M. Hartl F.-U. Annu. Rev. Biochem. 1991; 60: 101-124Crossref PubMed Scopus (338) Google Scholar) and processing to mature protein, they are assembled into the outer membrane (OM). 1The abbreviations OMouter membraneLPSlipopolysaccharideDOPC1,2-dioleoyl-sn-glycero-3-phosphocholine 1The abbreviations OMouter membraneLPSlipopolysaccharideDOPC1,2-dioleoyl-sn-glycero-3-phosphocholinePore proteins, such as the PhoE protein, are assembled as a trimer in the OM. How membrane proteins fold and assemble into a specific membrane is largely unknown. Kinetic and equilibrium folding studies of mostly soluble, hydrophilic proteins have indicated the existence of at least three main stages in protein folding, the formation of secondary structure, the folding pattern, and detailed tertiary structure (2Ptitsyn O.B. Protein Eng. 1994; 7: 593-596Crossref PubMed Scopus (101) Google Scholar). In vivo, the folding process is mediated by molecular chaperones that are proteins that will act primarily by preventing misfolding and aggregation of partially folded and unassembled protein subunits (3Ellis R.J. Hemmingsen S.M. Trends Biochem. Sci. 1989; 14: 339-342Abstract Full Text PDF PubMed Scopus (368) Google Scholar). In contrast to soluble proteins, membrane proteins will expose much more hydrophobic surfaces, exposed to the lipid phase, whereas the hydrophilic areas will be buried during folding. It can therefore be anticipated that membrane protein folding and assembly in vivo will require a membrane environment and molecular chaperones at or in this membrane. Indeed, refolding of bacterial outer membrane proteins can be accomplished in the presence of detergent and phospholipids (4Eisele J.L. Rosenbusch J.P. J. Biol. Chem. 1990; 265: 10217-10220Abstract Full Text PDF PubMed Google Scholar, 5Surrey T. Jähnig F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7457-7461Crossref PubMed Scopus (204) Google Scholar, 6Surrey T. Jähnig F. J. Biol. Chem. 1995; 270: 28199-28203Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Surrey T. Schmid A. Jähnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (122) Google Scholar). However, the kinetics of refolding of these membrane proteins were slow and, in the case of the trimeric porin OmpF, the yield was also low. Interestingly, phosphatidylethanolamine has recently been demonstrated to act as a non-protein molecular chaperone in the assembly of a bacterial cytoplasmic membrane transporter (8Bogdanov M. Sun J. Kaback H.R. Dowhan W. J. Biol. Chem. 1996; 271: 11615-11618Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Thus, certain lipids might also be required as molecular chaperone in the folding and assembly of bacterial outer membrane proteins. outer membrane lipopolysaccharide 1,2-dioleoyl-sn-glycero-3-phosphocholine outer membrane lipopolysaccharide 1,2-dioleoyl-sn-glycero-3-phosphocholine Various in vivo and in vitro studies have implicated an important role for LPS in the folding and assembly of bacterial outer membrane proteins (9Koplow J. Goldfine H. J. Bacteriol. 1974; 117: 527-543Crossref PubMed Google Scholar, 10Ames G.F.-L. Spudich E.N. Nikaido H. J. Bacteriol. 1974; 117: 406-416Crossref PubMed Google Scholar, 11Tommassen J. Lugtenberg B. J. Bacteriol. 1981; 147: 118-123Crossref PubMed Google Scholar, 12Ried G. Hindennach I. Henning U. J. Bacteriol. 1990; 172: 6048-6053Crossref PubMed Scopus (82) Google Scholar, 13Sen K. Nikaido H. J. Bacteriol. 1991; 173: 926-928Crossref PubMed Google Scholar, 14de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5573Crossref PubMed Scopus (65) Google Scholar). LPS are the major amphiphilic components in the outer leaflet of the bacterial OM (15Lugtenberg B. van Alphen L. Biochim. Biophys. Acta. 1983; 737: 51-115Crossref PubMed Scopus (482) Google Scholar,16Nikaido H. Vaara M. Microbiol. Rev. 1985; 49: 1-32Crossref PubMed Google Scholar). After synthesis in the inner membrane, LPS are translocated in an as yet unknown manner to the OM. Inhibition of fatty acid synthesis with the antibiotic cerulenin interferes with the assembly of pore proteins into the outer membrane (12Ried G. Hindennach I. Henning U. J. Bacteriol. 1990; 172: 6048-6053Crossref PubMed Scopus (82) Google Scholar, 17Bocquet-Pagès C. Lazdunski C. Lazdunski A. Eur. J. Biochem. 1981; 118: 105-111Crossref PubMed Scopus (25) Google Scholar, 18Pagès C. Lazdunski C. Lazdunski A. Eur. J. Biochem. 1982; 122: 381-386Crossref PubMed Scopus (16) Google Scholar, 19Bolla J.-M. Lazdunski C. Pagès J.M. EMBO J. 1988; 7: 3595-3599Crossref PubMed Scopus (51) Google Scholar). Thus, there appears to be a direct relationship between de novo synthesis of lipids, LPS and/or phospholipids, and correct folding and assembly of pore proteins in vivo. Recently, an early role for LPS in folding of PhoE protein was demonstrated in vitro (14de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5573Crossref PubMed Scopus (65) Google Scholar). Both, LPS and divalent cations were shown to be involved in the formation of an early intermediate, i.e. a folded monomer of PhoE protein. The subsequent assembly of monomers into trimers requires an additional incubation with outer membranes and Triton X-100 (0.08% w/v). The kinetics of the folding of PhoE with LPS is much higher (20de Cock H. van Blokland S. Tommassen J. J. Biol. Chem. 1996; 271: 12885-12890Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) as compared with the kinetics observed in refolding studies with OmpA and OmpF in the presence of phospholipids (5Surrey T. Jähnig F. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7457-7461Crossref PubMed Scopus (204) Google Scholar, 6Surrey T. Jähnig F. J. Biol. Chem. 1995; 270: 28199-28203Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 7Surrey T. Schmid A. Jähnig F. Biochemistry. 1996; 35: 2283-2288Crossref PubMed Scopus (122) Google Scholar). These in vitro results implicate a special role for LPS in outer membrane protein folding. In order to understand how LPS can support outer membrane protein folding, it is important to elucidate the molecular mechanism of LPS-protein interactions. Both hydrophilic and hydrophobic interactions, involving the negatively charged phosphates in the core and at the C-1 and C-4′ positions of the lipid A backbone and the fatty acids, respectively, might be directly involved in LPS-protein interactions. In addition, the state of order of the acyl chains (described by the order parameter S37) and the three-dimensional supramolecular structures of LPS might directly influence the capacity to support protein folding (for an overview see Refs. 21Seydel U. Brandenburg K. Morrison D.C. Ryan Y. Bacterial Endotoxic Lipopolysaccharides. 1. CRC Press, Inc., Boca Raton, FL1992: 225-250Google Scholar and 22Seydel U. Wiese A. Schromm A.B. Brandenburg K. Morrison D.C. Brade H. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1998Google Scholar). The relationship between the molecular shape of the lipid molecule and the structural polymorphism of its aggregates is well documented for phospholipids (23Israelachvili J.N. Marcelja S. Horm R.G. Q. Rev. Biophys. 1980; 13: 121-200Crossref PubMed Scopus (1133) Google Scholar). The same principles also apply for LPS (21Seydel U. Brandenburg K. Morrison D.C. Ryan Y. Bacterial Endotoxic Lipopolysaccharides. 1. CRC Press, Inc., Boca Raton, FL1992: 225-250Google Scholar, 22Seydel U. Wiese A. Schromm A.B. Brandenburg K. Morrison D.C. Brade H. Opal S. Vogel S. Endotoxin in Health and Disease. Marcel Dekker, Inc., New York1998Google Scholar). Thus, the supramolecular structure of LPS aggregates can either be lamellar (L, M) or non-lamellar (cubic, Q or HII). Factors like degree of saturation of acyl chains, temperature, head group size and ionization (pH, divalent cation concentration), and water content can influence the type of aggregate structures (lamellar and various non-lamellar phases). We have used a large set of different LPS molecules, of which the chemical and biophysical properties were previously characterized, in the developed in vitro folding system to investigate which properties of LPS are involved in folding of PhoE protein into its folded monomeric, native-like state. Isolation of S135 cell extract from Escherichia coli strain MC4100 (24Casadaban M.J. J. Mol. Biol. 1976; 104: 541-555Crossref PubMed Scopus (1298) Google Scholar) and the in vitro transcription and translation reactions were performed as described previously (25De Vrije T. Tommassen J. de Kruijff B. Biochim. Biophys. Acta. 1987; 900: 63-72Crossref PubMed Scopus (111) Google Scholar). Plasmid pJP370 (26de Cock H. Hekstra D. Tommassen T. Biochimie (Paris). 1990; 72: 177-182Crossref PubMed Scopus (25) Google Scholar) was used to direct the synthesis of the mature form of PhoE protein that was radioactively labeled due to the incorporation of [35S]methionine during protein synthesis. Folding of PhoE protein was initiated by addition of purified LPS and 0.015% Triton X-100 after inhibition of protein synthesis with puromycin and was essentially performed as described previously (14de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5573Crossref PubMed Scopus (65) Google Scholar). In short, 20 μl of a mixture of LPS with Triton X-100 (0.0338%, w/v) in buffer L (50 mm triethanolamine acetate, pH 7.5, 250 mmsucrose, 1 mm dithiothreitol) was mixed with 25 μl of a translation mixture and incubated for 30 min at 37 °C. Samples were treated with trypsin (45 μg/ml) for 15 min at 37 °C, phenylmethylsulfonyl fluoride (1 mm) was added, and the samples were transferred to ice. Prior to electrophoresis, sample buffer containing 2% SDS was added to the protein samples which were divided into two equal portions. One portion was incubated for 10 min at room temperature (lane a in Figs. Figure 2, Figure 3, Figure 4) and the other portion at 100 °C (lane c in Figs. Figure 2, Figure 3, Figure 4). In addition, 5 μl of the total translation mixture (lacking LPS, Triton X-100) was incubated for 10 min at 100 °C in sample buffer (Figs. Figure 2, Figure 3,lanes TL) and was used for determining the total amount of full-length PhoE present in 25 μl of translation mixture. SDS-polyacrylamide gels (27Lugtenberg B. Meijers J. Peters R. van der Hoek P. van Alphen L. FEBS Lett. 1975; 58: 254-258Crossref PubMed Scopus (961) Google Scholar) were run at 20 mA in a temperature-controlled room at 4 °C to prevent denaturation of the various folded forms of the PhoE protein during electrophoresis. The folded monomer (Figs. Figure 2, Figure 3, Figure 4, lanes a and designated m*) often runs as a smear originating from a species with a molecular mass of approximately 31 kDa up to the position of the denatured PhoE form (m; 38 kDa). Gels were incubated with Amplify, dried, and exposed to film (Fuji) at −70 °C. Data were quantified with a PhosphorImager (Molecular Dynamics). The amount of trypsin-resistant PhoE is calculated from the amount of denatured PhoE (m) present in lanes designatedc (Figs. Figure 2, Figure 3, Figure 4) as percentage of the total amount of full-length PhoE present in 25 μl of translation mixture.Figure 3LPS-dependent protein folding. Folding of in vitro synthesized PhoE protein was performed with LPS of chemotype Re (A) in its natural salt form (−) or in the salt form as indicated. B, folding of PhoE protein with LPS derived from S. minnesota (S. min), C. violaceum (C. viol), R. capsulatus (Rb. caps), and P. denitrificans(P. den), all of chemotypes S. Protein samples were analyzed as described in the legend of Fig. 1. The positions of denatured PhoE (m) and the folded monomer of PhoE (m*) are indicated. TL, total translation products containing chloramphenicol acetyltransferase (Cat) and mature PhoE (mPhoE).View Large Image Figure ViewerDownload (PPT)Figure 2LPS-dependent protein folding. Folding of in vitro synthesized PhoE protein was performed with LPS of various chemotypes from S. minnesota. Samples, containing the folded proteins, were loaded onto an SDS-polyacrylamide gel after incubation for 10 min in sample buffer at room temperature (lanes indicated witha) or 100 °C (lanes indicated withc). The positions of denatured mature PhoE (m) and the folded monomer of PhoE (m*) are indicated.TL, total translation products containing the cytosolic protein chloramphenicol acetyltransferase (Cat) and mature PhoE (mPhoE).View Large Image Figure ViewerDownload (PPT) Bacteria from enterobacterial strains as well as the non-enterobacterial speciesParacoccus denitrificans and Rhodobacter capsulatus were cultured as described (28Mayer H. Krauss J.H. Yokota A. Weckesser J. Friedman H. Klein T.W. Nakano M. Nowotny A. Endotoxin. Plenum Publishing Corp., New York1990: 45-70Google Scholar, 29Krauss J.H. Seydel U. Weckesser J. Mayer H. Eur. J. Biochem. 1989; 180: 519-526Crossref PubMed Scopus (77) Google Scholar). The non-enterobacterial strain Chromobacterium violaceum was obtained from the Institute for Fermentation, Osaka, Japan (IFO number 12614), and was cultivated in polypeptone growth media. LPS was extracted from phenol-killed bacteria, and rough mutant strains were extracted according to a modified PCP (PCP I, phenol/chloroform/petrol ether, 2:5:8 volume %) procedure (30Galanos C. Lüderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1363) Google Scholar) and for the wild type strain ofSalmonella minnesota by a phenol/water extraction (31Westphal O. Jann K. Whistler R.L. Methods in Carbohydrate Chemistry. 5. Academic Press, New York1965: 83-91Google Scholar). LPS in the water phase was enzyme-treated with proteinase K, RNase, and DNase (purchased from Sigma, Deisenhofen, Germany, and Boehringer, Mannheim, Germany) and further purified by the PCP procedure (PCP II, phenol/chloroform/petrol ether, 5:5:8 volume %). LPS were lyophilized and usually used in their natural salt form. In some cases, LPS Re samples were converted to various defined salt forms (Na+, Li+, Mg2+, Ba2+, and Ca2+) by extensive dialysis for 48 h against the corresponding salts. The preparations of de-O-acylated LPS, de-O-acylated and de-phosphorylated LPS, de-acylated LPS and of lipid A from LPS of E. coli and K. pneumoniaeR20 were performed as described (33Müller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1994; 224: 751-760Crossref PubMed Scopus (48) Google Scholar, 34Holst O. Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 1993; 214: 695-701Crossref PubMed Scopus (46) Google Scholar). The structures of the modified LPS form of J5 are schematically indicated in Fig.1 B. The chemical structures of the various lipid A and LPS from S. minnesota strains are given in Ref. 32Holst O. Morrison D.C. Brade H. Opal S. Vogel S. Endotoxins in Health and Disease. Marcel Dekker, Inc., New York1998Google Scholar (and schematically in Fig.1 A), those of E. coli J-5 in Refs. 33Müller-Loennies S. Holst O. Brade H. Eur. J. Biochem. 1994; 224: 751-760Crossref PubMed Scopus (48) Google Scholar and 34Holst O. Müller-Loennies S. Lindner B. Brade H. Eur. J. Biochem. 1993; 214: 695-701Crossref PubMed Scopus (46) Google Scholar, those of Klebsiella pneumoniae in Ref. 35Süsskind M. Brade L. Brade H. Holst O. J. Biol. Chem. 1998; 273: 7006-7017Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, and those of the non-enterobacterial LPS and lipid A in Refs. 28Mayer H. Krauss J.H. Yokota A. Weckesser J. Friedman H. Klein T.W. Nakano M. Nowotny A. Endotoxin. Plenum Publishing Corp., New York1990: 45-70Google Scholar, 29Krauss J.H. Seydel U. Weckesser J. Mayer H. Eur. J. Biochem. 1989; 180: 519-526Crossref PubMed Scopus (77) Google Scholar, and 36Rietschel E.Th. Brade L. Lindner B. Zähringer U. Morrison O.C. Ryan Y.L. Bacterial Endotoxic Lipopolysaccharides, Molecular Biochemistry and Cellular Biology. 1. CRC Press, Inc., Boca Raton, FL1992: 3-41Google Scholar. The state of order of the hydrocarbon chains of the endotoxins was determined by Fourier-transform spectroscopy by evaluating the symmetric stretching vibration νs(CH2) of the methylene groups. The position of this vibrational band is a sensitive marker of lipid order, and an estimation of the order parameter S at 37 °C, S37, can be performed according to a procedure described earlier (37Brandenburg K. Mayer H. Koch M.H.J. Weckesser J. Rietschel E.T. Seydel U. Eur. J. Biochem. 1993; 218: 555-563Crossref PubMed Scopus (155) Google Scholar), in which S can be calculated from the peak position x s of νs (CH2) by S = −941.8 + 0.7217x s −7.823 10−5 x s2 −2.068 10−8 x s3. It should be noted that S37 defined in this way should be similar but not identical to the order parameter used in NMR spectroscopy (S = 1 for perfectly aligned and 0 for isotropic acyl chains). The supramolecular structures of LPS and lipid A aggregates were deduced from x-ray diffraction patterns, which were obtained by measurements at the European Molecular Biology Laboratory (EMBL) outstation at the Hamburg synchrotron radiation facility HASYLAB kindly performed by M. H. J. Koch using the double-focusing monochromator-mirror camera X33 (38Koch M.H.J. Bordas J. Nucleic Instr. Methods. 1983; 208: 461-469Crossref Scopus (287) Google Scholar). The evaluation of the x-ray spectra was achieved according to procedures described (39Luzzati V. Vargas R. Mariani P. Gulik A. Delacroix H. J. Mol. Biol. 1993; 229: 540-551Crossref PubMed Scopus (183) Google Scholar) and in previous papers (37Brandenburg K. Mayer H. Koch M.H.J. Weckesser J. Rietschel E.T. Seydel U. Eur. J. Biochem. 1993; 218: 555-563Crossref PubMed Scopus (155) Google Scholar, 40Seydel U. Koch M.H.J. Brandenburg K. J. Struct. Biol. 1993; 110: 232-243Crossref PubMed Scopus (73) Google Scholar, 41Brandenburg K. Koch M.J.H. Seydel U. J. Struct. Biol. 1992; 108: 93-106Crossref PubMed Scopus (68) Google Scholar) which allow us to assign the spacing ratios of the main scattering maxima to defined three-dimensional structures. Here, in particular lamellar (L) and non-lamellar inverted cubic (Q) structures are of relevance. From this, the conformation of the individual molecules can be derived, which is cylindrical in the case of L-structures and conical, the cross-section of the hydrophobic moiety is larger than that of the hydrophilic moiety, in the case of inverted Q-structures. For some endotoxin derivatives, de-O-acyl-LPS and de-O-acyl-dephospho-LPS, for which only limited amounts were available, the aggregate structures were predicted according to procedures published earlier (42Brandenburg K. Kusumoto S. Seydel U. Biochim. Biophys. Acta. 1997; 1329: 183-201Crossref PubMed Scopus (98) Google Scholar). Phospholipids were purified from E. coliMC4100 (24Casadaban M.J. J. Mol. Biol. 1976; 104: 541-555Crossref PubMed Scopus (1298) Google Scholar) grown overnight at 30 °C in L broth (43Tommassen J. van Tol H. Lugtenberg B. EMBO J. 1983; 2: 1275-1279Crossref PubMed Scopus (117) Google Scholar) as described (44Bligh G.G. Dyer W.G. Can. J. Biochem. 1959; 37: 911-917Crossref PubMed Scopus (42382) Google Scholar). The phosphor content was determined by the methods of Rouseret al. (45Rouser G. Fleischer S. Yamamoto A. Lipids. 1970; 5: 494-496Crossref PubMed Scopus (2865) Google Scholar). Folding of PhoE with LPS of a deep rough mutant, lacking the complete core region up to the 3-deoxy-d-manno-octulopyranosonic acid residues, was previously shown to be much less efficient as compared with wild-type LPS (14de Cock H. Tommassen J. EMBO J. 1996; 15: 5567-5573Crossref PubMed Scopus (65) Google Scholar). In order to identify which region of the core was important to support protein folding, we made use of a well characterized series of chemotypes of LPS derived from S. minnesota (Table I). The primary chemical structures of wild type LPS and of the LPS mutants down to the deep rough mutant (LPS Re) are largely known (Fig.1 A; 32, 46, and references therein) except for the precise substitution with phosphate groups, in particular for lipopolysaccharides with longer sugar chains (>LPS Rc). In addition to differences in total amount of negative charges, the type of supramolecular structure of the LPS aggregates (Refs. 40Seydel U. Koch M.H.J. Brandenburg K. J. Struct. Biol. 1993; 110: 232-243Crossref PubMed Scopus (73) Google Scholar and41Brandenburg K. Koch M.J.H. Seydel U. J. Struct. Biol. 1992; 108: 93-106Crossref PubMed Scopus (68) Google Scholar; aggregate structure of the lipid A part) and the state of order of the acyl chains at 37 °C (Ref. 46Brandenburg K. Seydel U. Biochim. Biophys. Acta. 1984; 775: 225-238Crossref Scopus (99) Google Scholar; order parameter S37) are indicated (Table I).Table IFolding of in vitro synthesized PhoE protein with LPSStrain and (LPS chemotype)TrypRNegative chargeAggregate structureOrder parameter S37 ("0.03)%S. minnesotaS form53≥5Q0.63R60 (Ra)61≥5Q0.60R345 (Rb1)55≥5Q0.58R5 (Rc/P−)374Q0.55Rz (Rd1/P+)736Q0.43R7 (Rd1/P−)484Q0.44R4 (Rd2)384Q0.42R595 (Re)283–4Q0.39Re Na+ salt263–4Q0.37Re Li+salt303–4ND0.70Re Mg2+ salt13NDL0.43Re Ba2+saltNO≤2L0.86Re Ca2+ saltNO≤2L0.88C. violaceum28≥4L0.26IFO 12612 (S form)Rb. capsulatus11≥4.5L0.2837b4 (S form)P. denitrificans (S form)5≥5L0.45LPS was purified from the indicated strains. Folding was performed at 37 °C with 0.015% Triton X-100 and 138 nmol/ml LPS (Kdo content). No folding was observed in the absence of LPS. The total negative charge, preferred aggregate structure of the lipid A part of LPS, and the order parameter (S37) of the lipid A acyl chains are indicated. The amount of trypsin-resistant PhoE (TrypR) as % of the total amount of full-length PhoE synthesized is an average of two independent experiments. Q, inverted cubic structure. L, lamellar structure, ND, not determined. NO, not observed. Open table in a new tab LPS was purified from the indicated strains. Folding was performed at 37 °C with 0.015% Triton X-100 and 138 nmol/ml LPS (Kdo content). No folding was observed in the absence of LPS. The total negative charge, preferred aggregate structure of the lipid A part of LPS, and the order parameter (S37) of the lipid A acyl chains are indicated. The amount of trypsin-resistant PhoE (TrypR) as % of the total amount of full-length PhoE synthesized is an average of two independent experiments. Q, inverted cubic structure. L, lamellar structure, ND, not determined. NO, not observed. Folding of in vitro synthesized PhoE protein with LPS of chemotype S, Ra, Rb1, and Rd1/P+ were more efficient as compared with the folding efficiencies obtained with LPS of chemotypes Rc/P−, Rd1/P−, Rd2, and Re (Table I and Fig.2). The efficiency of folding is very reproducible and varies usually between 10% of the obtained average folding efficiency (as indicated in Tables I and II; e.g.60 ± 6%). The reduction of the folding efficiencies with this latter group appears to be mainly due to the absence of a negative charge in the inner core region. The negative charge of the phosphate group at the first heptopyranose, designated Hep I, appears to be most critical in this respect, as has been suggested previously (13Sen K. Nikaido H. J. Bacteriol. 1991; 173: 926-928Crossref PubMed Google Scholar). A further reduction in folding efficiency, as observed with LPS of chemotype Re, should be due to a further reduction of the net negative charge present. The variations in the folding efficiencies were not due to changes in the supramolecular structure and changes in the state of order of the acyl chains since the lipid A moiety of all LPS chemotypes prefers a nonlamellar cubic phase (Q) and the order parameter S37 decreased gradually, due to the decrease ofT c from 37 to 30 °C accompanying the reduction of the size of the core structure (21Seydel U. Brandenburg K. Morrison D.C. Ryan Y. Bacterial Endotoxic Lipopolysaccharides. 1. CRC Press, Inc., Boca Raton, FL1992: 225-250Google Scholar, 46Brandenburg K. Seydel U. Biochim. Biophys. Acta. 1984; 775: 225-238Crossref Scopus (99) Google Scholar).Table IIFolding of in vitro synthesized PhoE protein with chemically modified LPSStrain and (LPS chemotype)TrypRNegative chargeAggregate structureOrder parameter S37 ("0.03)%E. coliJ-5 (Rc)76≥5Q0.51De-O-acyl LPS from J-535≥5MaPrediction according to data from Ref. 42.0.37De-O-acyl and dephospho LPS from J-5263MaPrediction according to data from Ref. 42.0.07Lipid A from LPS J-5112Q0.83K. pneumoniaeR20 (R form)867QaPrediction according to data from Ref. 42.0.38De-O-acyl LPS from R20127MaPrediction according to data from Ref. 42.0.23De-O-acyl and dephospho LPS from R20125MaPrediction according to data from Ref. 42.0.27Lipid A from LPS R2082Q/L0.80Purified LPS of E. coli J-5 and K. pneumoniae R20 were chemically modified and re-purified as described under "Materials and Methods" (see also Fig. 1 B). The amount of lipid A of J-5 and of R20 used was 291 nmol/ml and 311 μg/ml, respectively. M, direct micellar structure. For further details, see Table I.a Prediction according to data from Ref. 42Brandenburg K. Kusumoto S. Seydel U. Biochim. Biophys. Acta. 1997; 1329: 183-201Crossref PubMed Scopus (98) Google Scholar. Open table in a new tab Purified LPS of E. coli J-5 and K. pneumoniae R20 were chemically modified and re-purified as described under "Materials and Methods" (see also Fig. 1 B). The amount of lipid A of J-5 and of R20 used was 291 nmol/ml and 311 μg/ml, respectively. M, direct micellar structure. For further details, see Table I. All LPS chemotypes used above were in their natural salt form. The folding efficiencies did not change significantly when the counterions in the LPS of chemotype Re were exchanged by dialysis for Na+ or Li+ (Table I and Fig.3 A). Interestingly, the folding efficiency of Re LPS in the Mg2+ salt form was significantly reduced, whereas those of the Ba2+ and Ca2+ forms had lost the capacity to support protein folding completely (Table I and Fig. 3 A). This reduced capacity to support folding seems to be correlated with a change in the supramolecular structure from a cubic, i.e. non-lamellar, to a lamellar phase and an increase in S37 (41Brandenburg K. Koch M.J.H. Seydel U. J. Struct. Biol. 1992; 108: 93-106Crossref PubMed Scopus (68) Google Scholar). Parallel to the change in aggregate structure, there is also a further reduction of the amount of free negative charges that might influence the capacity to support folding. The exact charge density of the LPS in the divalent cation salt forms as indicated in Table I, however, can only be estimated to be ≤2 from the measurement of the electrophoretic mobility of the lipid aggregates (Zeta potential, kindly performed by B. Lindner, Borstel Research Institute). In order to further substantiate the influence of the supramolecular structure further, LPS purified from C. violaceum, R. capsulatus, and P. denitrificans were used. The LPS from these non-enterobacterial species contain a lipid A structure that is different to the basic chemical structure of enterobacterial lipid A, mainly at the level of fatty acid composition (28Mayer H. Krauss J.H. Yokota A. Weckesser J. Friedman H. Klein T.W. Nakano M. Nowotny A. Endotoxin. Plenum Publis
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