Title: Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy
Abstract: In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region. In the light of occurrence of bacterial strains with multiple resistances against most antibiotics, antimicrobial peptides that interact with the outer layer of Gram-negative bacteria, such as polymyxin (PMX), have recently received increased attention. Here we present a study of the interactions of PMX-B, -E, and -M with lipopolysaccharide (LPS) from a deep rough mutant strain of Escherichia coli. A method for efficient purification of biosynthetically produced LPS using reversed-phase high-performance liquid chromatography in combination with ternary solvent mixtures was developed. LPS was incorporated into a membrane model, dodecylphosphocholine micelles, and its interaction with polymyxins was studied by heteronuclear NMR spectroscopy. Data from chemical shift mapping using isotope-labeled LPS or labeled polymyxin, as well as from isotope-filtered nuclear Overhauser effect spectroscopy experiments, reveal the mode of interaction of LPS with polymyxins. Using molecular dynamics calculations the complex of LPS with PMX-B in the presence of dodecylphosphocholine micelles was modeled using restraints derived from chemical shift mapping data and intermolecular nuclear Overhauser effects. In the modeled complex the macrocycle of PMX is centered around the phosphate group at GlcN-B, and additional contacts from polar side chains are formed to GlcN-A and Kdo-C, whereas hydrophobic side chains penetrate the acyl-chain region. Cellular membranes segregate the interior of cells from their surroundings and therefore are crucial to maintain cells as autonomously functioning systems (1.Gennis R.B. Biomembranes: Molecular Structure and Function. Springer, New York1989Crossref Google Scholar). The chemical constituents of outer membranes from mammalian cells and bacteria are fundamentally different (2.Yeagle P.L. The Membranes of Cells. Academic Press, San Diego, CA1993Google Scholar). The mammalian outer membranes are largely formed by phospholipid bilayers, whereas additional coating structures are present covering these in bacteria. In Gram-positive bacteria a thick peptidoglycan layer is built around the phospholipid bilayer. In Gram-negative bacteria, the peptidoglycan structure is much thinner and coated by an additional phospholipid-containing bilayer, whose outer leaflet is mainly composed of lipopolysaccharides (LPSs) 2The abbreviations used are: LPS, lipopolysaccharide; Kdo, 3-deoxy-d-manno-oct-2-ulosonate; PMX, polymyxin; Dab, diaminobutyric acid; NOE, nuclear Overhauser effect; Re-LPS, LPS from the deep rough mutant D31m4 of E. coli; HPLC, high-performance liquid chromatography; DPC, dodecylphosphocholine; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MES, 4-morpholineethanesulfonic acid; MD, molecular dynamics; TOCSY, total correlation spectroscopy; HM, hydroxymyristoyl; LM, lauroxymyristoyl. (3.Raetz C.R. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith D.C. Ribeiro A.A. Murphy R.C. Ulevitch R.J. Fearns C. Reichart D. Glass C.K. Benner C. Subramaniam S. Harkewicz R. Bowers-Gentry R.C. Buczynski M.W. Cooper J.A. Deems R.A. Dennis E.A. J. Lipid Res. 2006; 47: 1097-1111Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). LPS are high molecular weight, strongly negatively charged molecules, which for smooth LPS can be divided in three regions: the lipid A portion of LPS inserts into the phospholipidic membrane and in many Gram-negative bacteria consists of a di-glucosamine diphosphate with 5 to 7 fatty acid chains extending to one side of the disaccharide. The lipid A is appended to a region (the inner core) of 8-12 variable sugars (including the negatively charged 3-deoxy-d-manno-oct-2-ulosonate (Kdo) units) and 3-8 phosphate residues. To the inner core is covalently associated the O-antigen, an oligosaccharide chain of variable length and chemical composition, depending on the exact type of LPS. Sepsis caused by Gram-negative bacteria is a serious source of mortality in many clinical cases, accounting for ∼200,000 deaths in the U.S. annually (see David (4.David S.A. J. Mol. Recognit. 2001; 14: 370-387Crossref PubMed Scopus (107) Google Scholar) and references therein). The primary trigger for sepsis was identified as LPS, and LPS-neutralizing agents are therefore valuable therapeutics. Antimicrobial peptides against Gram-negative bacteria can interfere with the integrity of this LPS layer. PMX-B is considered as the "gold standard" for LPS-sequestering agents. Polymyxin-B, -M, and -E are characterized by a heptapeptide ring and a fatty acid tail (see Fig. 1). These highly cationic decapeptides contain six diaminobutyric acid (Dab) residues, a macrocylic ring involving residues 4-10, and an acyl chain coupled to the N terminus. Severe toxic side effects have limited their usage to treatments against bacteria resistant against most other antibiotics such as Pseudomonas aeruginosa. The interaction of LPS with various antimicrobial peptides has been the subject of a number of studies (4.David S.A. J. Mol. 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Chem. 1999; 42: 4604-4613Crossref PubMed Scopus (155) Google Scholar, 12.Rana F.R. Blazyk J. FEBS Lett. 1991; 293: 11-15Crossref PubMed Scopus (40) Google Scholar, 13.Rosenfeld Y. Papo N. Shai Y. J. Biol. Chem. 2006; 281: 1636-1643Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 14.Rosenfeld Y. Shai Y. Biochim. Biophys. Acta. 2006; 1758: 1513-1522Crossref PubMed Scopus (254) Google Scholar, 15.Wang H. Head J. Kosma P. Brade H. Muller-Loennies S. Sheikh S. McDonald B. Smith K. Cafarella T. Seaton B. Crouch E. Biochemistry. 2008; 47: 710-720Crossref PubMed Scopus (54) Google Scholar). In some of these, the conformation of the LPS-bound peptides was established using transferred NOE effects, and the complex between LPS and the peptides was established by docking the transferred NOE-derived peptide conformer to LPS (11.Pristovsek P. Kidric J. J. Med. Chem. 1999; 42: 4604-4613Crossref PubMed Scopus (155) Google Scholar), whose coordinates were taken from the crystal-structure of FhuA-bound LPS (16.Ferguson A.D. Hofmann E. Coulton J.W. Diederichs K. Welte W. Science. 1998; 282: 2215-2220Crossref PubMed Scopus (671) Google Scholar). The emphasis of this work was to obtain experimental data on the LPS·PMX complex, thereby allowing a detailed understanding of the interacting moieties. Because LPS in the outer membrane of Escherichia coli cells is integrated into a phospholipid bilayer, it was studied while integrated into phospholipid micelles to better mimic the natural environment. In our studies we used LPS from the deep rough mutant D31m4 of E. coli (Re-LPS). Biosynthetic production of the latter and its isolation and purification from the membrane fraction of the corresponding cells was described in literature. However, to facilitate purification by reversed-phase HPLC, the phosphate groups were methylated (17.Qureshi N. Takayama K. Mascagni P. Honovich J. Wong R. Cotter R.J. J. Biol. Chem. 1988; 263: 11971-11976Abstract Full Text PDF PubMed Google Scholar). The modified LPS was investigated in detail by NMR (18.Agrawal A.B. Qureshi N. Takayama K. Magn. Reson. Chem. 1998; 36: 1-7Crossref Scopus (6) Google Scholar). Because interactions with the charged phosphate groups were proposed to be important for binding antimicrobial peptides, we developed an HPLC-based method that allows purification of LPS in its natural (non-methylated) form (Fig. 1). 13C,15N-Labeled LPS from the deep rough mutant strain was isolated and purified to chemical homogeneity. Extensive use of heteronuclear solution NMR techniques allowed characterization of LPS embedded in DPC micelles and facilitated the study of its interactions with polymyxins from different organisms. The interaction studies relied on chemical shift mapping techniques and isotope-filtered NOEs and allowed direct probing for the interaction sites. Materials—15NH4Cl was purchased from Spectra Isotopes (Columbia, MD), perdeuterated DPC-d38 (99%-d), and D2O were ordered from Cambridge Isotope Laboratories (Andover, MA). Methyl-5-doxylstearic acid was bought from Aldrich (Buchs, Switzerland). The Re-LPS-producing strain D31m4 was purchased from the E. coli Genetic Resource Center, New Haven, CT. The PMX-M-producing strain Paenibacillus kobensis M was obtained from Prof. J. C. Vederas, and PMX-B and -E were purchased from Sigma-Aldrich. Production of 13C-Labeled LPS from the E. coli Strain D31m4—Cells from the D31m4 strain of E. coli were grown at 37 °C to an optical density of ∼1.0 at 600 nm on minimal medium M9 using 4 g of [13C]glucose and 1 g of 15N ammonium chloride supplemented with 100 mg of Trp, His, and Pro per liter. After harvest cells were resuspended in 50 ml of ice-cold water and pelleted down. To the pellet a minimum amount of cold water was added such that a thick paste was formed. LPS together with other components was precipitated through addition of 90 ml of ice-cold methanol and centrifuged at 8000 × g. The pellet was resuspended in 90 ml of ice-cold acetone, homogenized, and centrifuged again, followed by another acetone washing step. The lyophilized cells (∼0.7 g) were taken up in 50 ml of a phenol:chloroform: petroleum ether (4:10:16, v/v) solvent mixture and centrifuged at 9,200 × g, after which most of the LPS was contained in the supernatant. The remaining pellet was extracted once more to increase the yield in LPS. The supernatant was concentrated under a nitrogen stream, and 2 ml of water was added dropwise to the concentrate. A waxy precipitate was formed followed by three cycles of washing with methanol and subsequent centrifugation. Thereafter the pellet was dried and lyophilized, after which it could only be resuspended in water using repetitive additions of small amounts of water followed by sonication. Solubilization was improved upon adding aqueous 0.1 m EDTA in the first portions. The resulting solution was centrifuged at 200,000 × g overnight and then lyophilized. Chromatography used the following solvents: solvent A (methanol:chloroform:water, 57:12:31, v/v) and solvent B (methanol:chloroform, 29.8:70.2, v/v). The lyophilized pellet after resuspension in the mixture of solvent A and aqueous EDTA was directly loaded onto the HPLC column. For optimal purification of LPS a gradient system involving ternary solvent mixtures was used consisting of solvent A in 10 mm NH4Cl and solvent B in 50 mm NH4Cl. LPS (6 mg) was dispersed in a two phase system formed from 0.8 ml of solvent A and 0.2 ml of 0.1 m aqueous EDTA (pH 7) and loaded directly onto the RP-C8 column. Chromatographic separation was achieved using the following gradient of solvents A and B: 2 column volumes of 2% B, 3 column volumes (2-17% B), 3.5 column volumes (17-27% B). UV detection was impossible, and hence fractions were lyophilized and their content checked by MALDI-TOF using 6-aza-2-thiothymine as the matrix. Elution of the desired LPS occurred around 20-23% of solvent B. Production of 13C,15N-Labeled PMX-M from P. kobensis M—The producer strain, P. kobensis M, was grown aerobically at 30 °C on tryptic soy agar. A 1-liter batch of M9 medium was inoculated with a 10-ml P. kobensis M preculture (1% inoculum). After a total growth time of 16-24 h at 30 °C with shaking (200 rpm), the cells were removed by centrifugation (1 h, 10,000 rpm), and the supernatant was then passed through a Amberlite XAD-16 column. After washing with 30% ethanol, active peptide was then eluted with 70% isopropanol, which was adjusted to pH 2 (pH meter reading) with 12 n HCl. All fractions were assessed for antimicrobial activity using a well plate assay. The contents of the active fraction were applied to a Superdex peptide 10/300 column (Amersham Biosciences). Fractions were collected for 3 column volumes with pure Milli-Q Water and each assayed for activity. All active fractions were pooled, concentrated, and applied as 20% isopropanol solutions to C18 reversed-phase HPLC. Complete purification required two separate steps of C18 HPLC. The first separation used a gradient of water/isopropanol (0.1% trifluoroacetic acid), from 20% to 50% isopropanol, and the second step a water/methanol gradient (0.1% trifluoroacetic acid), from 45% to 85% methanol. PMX-M eluted at around 55%. Finally, 8-10 mg of pure PMX-M was obtained as slightly yellowish powder from a 1-liter culture, and its chemical nature was verified by electrospray ionization-mass spectrometry (experimental mass: 1224.73 Da; theoretical mass: 1223.57 Da). During all steps of expression and purification, antimicrobial activity was monitored by inhibition of growth of an indicator strain. Agar plates were prepared by inoculating molten tryptic soy agar (40 g/liter) with a culture of the indicator organism E. coli (1.0% inoculum). Small wells (∼4.6-mm diameter) were made in the seeded agar plates, and 50 μl of filtered culture supernatant was added to the wells. Plates were incubated at 30 °C, and the growth of the indicator organism was visible after ∼3 h (19.Martin N.I. Hu H.J. Moake M.M. Churey J.J. Whittal R. Worobo R.W. Vederas J.C. J. Biol. Chem. 2003; 278: 13124-13132Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). NMR Spectroscopy—LPS samples used for assignment purposes contained ∼1 mm LPS, 300 mm d38-DPC in 40 mm d13-MES D2O buffer, pD = 5.8. Resonance positions required very small changes to adapt to 13C,1H HSQC spectra in 40 mm acetate buffer at pH 4.4. All interaction studies were performed in 40 mm acetate buffer, 300 mm d38-DPC in D2O, or H2O/D2O 9/1 (pH 4.4). Measurements of interactions between LPS and PMX by chemical shift mapping observing LPS resonances utilized a 350 μm solution of 15N,13C Re-LPS and equimolar unlabeled peptides. Chemical shift changes in PMX-M were monitored on a 200 μm solution of 15N,13C-labeled PMX-M and equimolar unlabeled Re-LPS. No further salt was present in the measurements except for initial attempts to optimize conditions for 15N,1H HSQC spectra in the PMX-M·Re-LPS sample (see below). To satisfy requirements for better sensitivity higher concentrations of doubly labeled LPS (500 μm) were used in the isotope-filtered NOESY experiments (200-ms mixing time). Due to the moderate dissociation constant the experiment in fact monitored transferred NOEs; therefore, 3-fold excess of unlabeled PMX-E or PMX-B (2 mm solutions) were used with conditions of pH, detergent, and temperature otherwise identical to those of the shift mapping studies. Spectra were recorded on Bruker AV-600 or AV-700 NMR spectrometer at T = 310 K. Proton and carbon chemical shifts were calibrated to 2,2-dimethyl-2-silapentane-5-sulfonic acid, and nitrogen shifts were referenced indirectly to liquid NH3 (20.Live D.H. Davis D.G. Agosta W.C. Cowburn D. J. Am. Chem. Soc. 1984; 106: 6104-6105Crossref Scopus (129) Google Scholar). The spectra were processed using the Bruker Topspin 2.0 software and transferred into CARA (21.Keller R. The Computer Aided Resonance Assignment. Cantina Verlag, Goldau2004Google Scholar) or SPARKY (22.Goddard T.D. Kneller D.G. Sparky 3, Version 3.113. University of California, San Francisco, CA2006Google Scholar) programs for further analysis. For chemical shift assignments of 13C,15N-labeled LPS two-dimensional versions of three-dimensional double- and triple-resonance experiments were recorded. In general, experiments used coherence selection schemes via pulsed-field gradients (23.Keeler J. Clowes R.T. Davis A.L. Laue E.D. Methods Enzymol. 1994; 239: 145-207Crossref PubMed Scopus (203) Google Scholar) and sensitivity-enhancement building blocks (24.Palmer A.G. Cavanagh J. Wright P.E. Rance M. J. Magn. Reson. 1991; 93: 151-170Google Scholar, 25.Kay L.E. Keifer P. Saarien T. J. Am. Chem. Soc. 1992; 114: 10663-10665Crossref Scopus (2453) Google Scholar) whenever possible. For assignments of the carbon spin systems in the lipid chains and the sugar units (H)CCH experiments recorded with 4- and 12-ms DIPSI-2 C-C mixing cycles were used. Linkage of the lipid chains onto the glucosamine parts of lipid A was achieved via correlations with the amide nitrogens using HNCA and HN(CO)CA experiments. To distinguish the two Kdo units, key NOEs derived from a 13C-resolved NOESY were exploited. Assignment of all resonances of polymyxin was done using HN(CO)CACB (26.Yamazaki T. Lee W. Arrowsmith C.H. Muhandiram D.R. Kay L.E. J. Am. Chem. Soc. 1994; 116: 11655-11666Crossref Scopus (502) Google Scholar), HNCACB (27.Wittekind M. Mueller L. J. Magn. Reson. Ser. B. 1993; 101: 201-205Crossref Scopus (860) Google Scholar), and (H)CCH experiments (28.Bax A. Clore G.M. Driscoll P.C. Gronenborn A.M. Ikura M. Kay L.E. J. Magn. Reson. 1990; 87: 620-627Google Scholar, 29.Olejniczak E.T. Xu R.X. Fesik S.W. J. Biomol. NMR. 1992; 2: 655-659Crossref PubMed Scopus (77) Google Scholar) analogous to the procedure used for proteins. Because of the small size of the peptide two-dimensional versions were recorded with a total of <12-h measuring time for acquiring all spectra. Assignments of polymyxin-B and -E were based on assignments from PMX-M adjusted by using additional two-dimensional heteronuclear spectra. In the spin-label experiments, a 0.5 mm solution of LPS was separated into two aliquots, and to one of these 5-doxylstearate methyl ester was added so that the final concentration corresponded to approximately one spin-label per micelle. Signal intensities from the two corresponding constant-time 13C,1H HSQC were extracted, and the ratio of signal intensities from the samples with and without spin label was calculated. Molecular Dynamics Calculations—All calculations were performed within the program GROMACS (30.Hess B. Kutzner C. van d.S. David Lindahl E. J. Chem. Theory Comput. 2008; 4: 435-447Crossref PubMed Scopus (12813) Google Scholar). Briefly, coordinates of LPS were adapted from the pdb entry 1QFF, and coordinates of polymyxin B were built using the program Ghemical (31.Hassinen T. Perakyla M. J. Comput. Chem. 2001; 22: 1229-1242Crossref Scopus (121) Google Scholar). Parameters and topologies of PMX-B and LPS as well as partial charges of PMX-B for GROMACS were established based on data from the PRODRG server (32.Schüttelkopf A.W. van Aalten D.M.F. Acta Crystallogr. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4469) Google Scholar) and the GROMOS 53a6 force field (33.Oostenbrink C. Villa A. Mark A.E. van Gunsteren W.F. J. Comp. Chem. 2004; 25: 1656-1676Crossref PubMed Scopus (3071) Google Scholar). Partial charges of LPS were assigned for a protonation state corresponding to experimental conditions of pH by the MPEOP method (34.Gasteiger J. Marsili M. Tetrahedron. 1980; 36: 3219-3228Crossref Scopus (3685) Google Scholar, 35.No K.T. Grant J.A. Scheraga H.A. J. Phys. Chem. 1990; 94: 4732-4739Crossref Scopus (115) Google Scholar). Parameters for DPC were derived from values for dipalmitoylphosphatidylcholine from the GROMOS force field library. A detailed description of the methodology pursued can be found in the supplemental materials. Briefly, the system was prepared as follows: (i) An initial complex between LPS and polymyxin B was prepared and equilibrated in the presence of a DPC micelle. (ii) A set of simulated annealing calculations was performed yielding 450 structures. The DPC molecules were explicitly included in the system, water molecules were substituted by implicit solvent, and the dominant NOE-derived upper distance limits were included (the force constant was set to 1000 kJ mol-1nm-2). (iii) Those structures were selected, which displayed best agreement with the chemical shift mapping data. (iv) These were then equilibrated with explicit solvent and subjected to further refinement and analysis. The latter included an assessment of the stability of the MD trajectory and a comparison of the average intermolecular distances with chemical shift mapping data. Production and Purification of 13C-Labeled LPS—LPS from the deep rough E. coli mutant D31m4 was isolated from the membrane fraction of cells grown on minimal medium containing [13C]glucose and [15N]H4Cl as the sole carbon and nitrogen sources, respectively. After pentachlorphenol extraction and further purification using published protocols (3.Raetz C.R. Garrett T.A. Reynolds C.M. Shaw W.A. Moore J.D. Smith D.C. Ribeiro A.A. Murphy R.C. Ulevitch R.J. Fearns C. Reichart D. Glass C.K. Benner C. Subramaniam S. Harkewicz R. Bowers-Gentry R.C. Buczynski M.W. Cooper J.A. Deems R.A. Dennis E.A. J. Lipid Res. 2006; 47: 1097-1111Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar, 36.Galanos C. Luderitz O. Westphal O. Eur. J. Biochem. 1969; 9: 245-249Crossref PubMed Scopus (1372) Google Scholar) the yield was ∼129 mg/liter of culture. Remaining impurities were removed by reversed-phase HPLC using a ternary solvent mixture. In this procedure the solvent system was carefully adapted to form a single phase over the whole gradient range of solvent A (methanol:chloroform:water) and solvent B (methanol:chloroform) at room temperature. Importantly, any mixture of these two solvent systems is relatively close to a two-phase system, and this condition proved to have favorable properties for dissolving LPS. MALDI-TOF spectra of LPS before and after this HPLC purification step are depicted in Fig. 2. Sufficient quantities (40 mg) of chemically pure LPS for the NMR studies could be produced from 1 liter of culture using this protocol. As demonstrated in supplemental Fig. S4 this method is capable of separating pyrophosphate from the monophosphate derivatives. Assignment of LPS and Polymyxin Resonances—Chemical shift mapping (37.Shuker S.B. Hajduk P.J. Meadows R.P. Fesik S.W. Science. 1996; 274: 1531-1534Crossref PubMed Scopus (1846) Google Scholar) or NOE-based methods (38.Clore G.M. Gronenborn A.M. J. Magn. Reson. 1982; 48: 402-417Google Scholar, 39.Clore G.M. Gronenborn A.M. J. Magn. Reson. 1983; 53: 423-442Google Scholar, 40.Otting G. Wüthrich K. Q. Rev. Biophys. 1990; 23: 39-96Crossref PubMed Scopus (349) Google Scholar) can be used to study biomolecular interactions (see also Refs. 41.Marchioro C. Davalli S. Provera S. Heller M. Ross A. Senn H. Zerbe O. BioNMR in Drug Research. Wiley-VCH, Weinheim, Germany2003: 321-340Google Scholar, 42.Blommers M.J.J. Rüdisser S. Zerbe O. BioNMR in Drug Research. Wiley-VCH, Weinheim, Germany2003: 355-372Google Scholar, 43.Gemmecker G. Zerbe O. BioNMR in Drug Research. Wiley-VCH, Weinheim, Germany2003: 373-390Google Scholar). Both methods potentially deliver information on interacting moieties but require assignments of chemical shifts. The best chemical shift dispersion is usually available in heteronuclear shift correlation spectra (e.g. 15N,1H HSQC or 13C,1H HSQC spectra). Importantly, these experiments still work well in the presence of the increased line widths that are usually present in systems that are stably anchored into phospholipids micelles. In addition, as was unfortunately the case in some of our applications, additional exchange broadening occurred upon complex formation. To probe integration of LPS into DPC micelles and to study its interaction with peptides, we decided to label it with 13C and 15N isotopes and use the corresponding HSQC spectra for chemical shift mapping. To assign all signals in the constant-time 13C,1H HSQC spectrum (44.Vuister G.W. Bax A. J. Magn. Reson. 1992; 98: 428-435Google Scholar), three-dimensional (H)CCH-TOCSY spectra (28.Bax A. Clore G.M. Driscoll P.C. Gronenborn A.M. Ikura M. Kay L.E. J. Magn. Reson. 1990; 87: 620-627Google Scholar, 29.Olejniczak E.T. Xu R.X. Fesik S.W. J. Biomol. NMR. 1992; 2: 655-659Crossref PubMed Scopus (77) Google Scholar) served for assignment of spin systems (Fig. 3). The C,H-plane of the HNCA (45.Grzesiek S. Bax A. J. Magn. Reson. 1992; 96: 432-440Google Scholar, 46.Kay L.E. Ikura M. Tschudin R. Bax A. J. Magn. Reson. 1990; 89: 496Google Scholar) and HN(CO)CA (45.Grzesiek S. Bax A. J. Magn. Reson. 1992; 96: 432-440Google Scholar) experiments was used to establish scalar connectivities between terminal carbons of the fatty acid chains and C-2 of the glucosamine moieties. Unsubstituted hydroxymyristoyl (HM) can be distinguished from lauroxymyristoyl (LM) and thereby helps to differentiate between GlcN-A from GlcN-B. Due to chemical shift degeneracies, it was impossible to assign chains of the fatty acids (myristoyl of myristoxy-myristoyl and lauroyl of LM). The two Kdo units were linked and thereby distinguished from each other using several key NOEs in the three-dimensional 13C-NOESY spectra. The unique chemical shift of the C3 moiety of Kdo is located in a region separated from all other sugar resonances and was used as a starting point for sequential assignment. Only the methylene group C3 of Kdo-C is expected to receive an NOE from H(C6) of GlcN-B. This assignment was additionally supported by the fact that H(C6) and H(C7) of Kdo-C displayed an NOE to H(C3) of both Kdo units, which is unlikely to be the case for H(C6) of Kdo-D. The 1H,13C and 15N chemical shifts of LPS in DPC micelles are reported in Table 1.TABLE 1Carbon and proton chemical shifts of LPS from the deep-rough mutant of E. coli, 0.35 mm LPS in 300 mm DPC (pH 4.4), 40 mm acetate, T = 310 KSugar moietiesGlcN-AGlcN-BKdo-CKdo-DH15.3464.681H23.9283.861H35.1435.0721.9002.104H31.9371.731H43.6893.8794.0864.022H54.0403.6544.0793.989H64.0183.4093.6473.550H63.8263.734H73.8693.920H83.8723.926H83.5993.697C195.56104.41C254.0555.61C375.4376.0335.3336.50C468.6374.7570.5868.00C573.3276.1966.6368.35C670.5064.7373.6674.63C771.8971.77C865.5065.32Lipid chainsLMMMHM-4HM-3LHM2MHM1HA2.306aResonances that cannot be distinguished between L and M because of overlap.2.562-aResonances that cannot be distinguished between L and M because of overlap.2.6432.3122.383HA2.585-aResonances that cannot be distinguished between L and M because of overlap.2.6702.3662.433HB1.563aResonances that cannot be distinguished between L and M because of overlap.5.283-aResonances that cannot be distinguished between L and M because of overlap.5.1283.8903.910HC1.210aResonances that cannot be distinguished between L and M because of overlap.1.519-aResonances that cannot be distinguished between L and M because of overlap.1.5481.3031.383HC1.559-aResonances that cannot be distinguished between L and M because of overlap.1.6011.3801.471HD1.2811.392CA36.51aResonances that cannot be distinguished between L and M because of overlap.43.15-aResonances that cannot be distinguished between L and M because of overlap.41.1045.3844.18CB27.34aResonances that cannot be distinguished between L and M because of overlap.72.37-aResonances that cannot be distinguished between L and M because of overlap.72.5069.7769.61CG31.50aResonances that cannot be distinguished between L and M because of overlap.37.18-aResonances that cannot be distinguished between L and M because of overlap.36.2538.3040.30CD27.9828.08a Resonances that cannot be distinguished between L and M because of overlap. Open table in a new tab Topology of LPS and Polymyxin Insertion into the DPC Micelle—To probe whether LPS properly inserts into the DPC micelles, and whether the sugar moieties really protrude into the aqueous phase, the micelle-integrating spin label methyl 5-doxylstearate was used. The paramagnetic moiety of the spin label resides in the headgroup region (47.Papavoine C.H. Konings R.N. Hilbers C.W. van de Veen F.J. Biochemistry. 1994; 33: 12990-12997