Observation of 1000-fold enhancement of nitrogen-15 NMR via proton-detected multiquantum coherences: studies of large peptides

Live, David; Davis, Donald G.; Agosta, William C.; Cowburn, David

doi:10.1021/ja00332a071

Cited by 111 publications

(52 citation statements)

References 1 publication

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“…Spectra were recorded on Bruker AV-600 or AV-700 NMR spectrometer at T ϭ 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 NH 3 (20). The spectra were processed using the Bruker Topspin 2.0 software and transferred into CARA (21) or SPARKY (22) programs for further analysis.…”

Section: Production Ofmentioning

confidence: 99%

Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

Mareš

Kumaran

Gobbo

et al. 2009

Journal of Biological Chemistry

111

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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 isotopelabeled 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.

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Section: Production Ofmentioning

confidence: 99%

Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

Mareš

Kumaran

Gobbo

et al. 2009

Journal of Biological Chemistry

111

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“…The water peak was assigned a value of 4.71 ppm and thus used as an internal reference. The "N chemical shift reference was CH3N02, which was assigned a value of 380.23 ppm (Live et al, 1984). Two-dimensional sensitivity-enhanced pulse sequences (Cavanagh et al, 1991;Palmer et al, 1991;Skelton et al, 1993) were employed for the inversion-recovery (Vold et al, 1968), CPMG spin-echo (Meiboom & Gill, 1958), and steady-state NOE (Noggle & Shirmer, 1971) experiments to measure the spin-lattice relaxation rate constants @'), spin-spin relaxation rate constants (R2).…”

Section: Nmr Spectroscopymentioning

confidence: 99%

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family

et al. 1998

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Serine proteinase protein inhibitors follow the standard mechanism of inhibition (Laskowski M Jr, Kato I, 1980, Annu Rev Biochem 49593‐626), whereby an enzyme‐catalyzed equilibrium between intact (I) and reactive‐site hydrolyzed inhibitor (I*) is reached. The hydrolysis constant, Khyd is defined as [I*]/[I]. Here, we explore the role of internal dynamics in the resynthesis of the scissile bond by comparing the internal mobility data of intact and cleaved inhibitors belonging to two different families. The inhibitors studied are recombinant Cucurbita maxima trypsin inhibitor III (rCMTI‐III; Mr 3 kDa) of the squash family and rCMTI‐V (Mr ∼ 7 kDa) of the potato I family. These two inhibitors have different binding loop‐scaffold interactions and different Khyd values—2.4 (CMTI‐III) and 9 (CMTI‐V)—at 25°C. The reactive‐site peptide bond (P1‐P'1) is that between Arg5 and Ile6 in CMTI‐III, and that between Lys44 and Asp45 in CMTI‐V. The order parameters (S2) of backbone NHs of uniformly 15N‐labeled rCMTI‐III and rCMTI‐III* were determined from measurements of 15N spin‐lattice and spin‐spin relaxation rates, and {1H}‐15N steady‐state heteronuclear Overhauser effects, using the model‐free formalism, and compared with the data reported previously for rCMTI‐V and rCMTI‐V*. The backbones of rCMTI‐III (〈S2〉 = 0.71) and rCMTI‐III* (〈S2) = 0.63) are more flexible than those of rCMTI‐V (〈S2〉 = 0.83) and rCMTI‐V* (〈S2) = 0.85). The binding loop residues, P4‐P1, in the two proteins show the following average order parameters: 0.57 (rCMTI‐III) and 0.44 (rCMTI‐III*); 0.70 (rCMTI‐V) and 0.40 (rCMTI‐V*). The P1′‐P4′ residues, on the other hand, are associated with (S2) values of 0.56 (rCMTI‐III) and 0.47 (rCMTI‐III*); and 0.73 (rCMTI‐V) and 0.83 (rCMTI‐V*). The newly formed C‐terminal (Pn residues) gains a smaller magnitude of flexibility in rCMTI‐III* due to the Cys3‐Cys20 crosslink. In contrast, the newly formed N‐terminal (Pn′ residues) becomes more flexible only in rCMTI‐III*, most likely due to lack of an interaction between the P1′ residue and the scaffold in rCMTI‐III. Thus, diminished flexibility gain of the Pn residues and, surprisingly, increased flexibility of the Pn′ residues seem to facilitate the resynthesis of the P1‐P1′ bond, leading to a lower Khyd value.

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“…Chemical shifts were referenced to 3-(2,2,3,3-2H4) trimethylsilyl propionate (TSP) (IH), liquid ammonia (15N) (Live et al, 1984), and TSP (13C) (Bax & Subramanian, 1986). Quadrature detection using TPPI-States ) was employed for all experiments except for 3D NOESY-HMQC and 3D TOCSY-HMQC, in which case TPPI ) was used.…”

Section: N M R Sdectroscodv Methodsmentioning

confidence: 99%

Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

Lee

1996

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Observation of 1000-fold enhancement of nitrogen-15 NMR via proton-detected multiquantum coherences: studies of large peptides

Cited by 111 publications

References 1 publication

Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

Interactions of Lipopolysaccharide and Polymyxin Studied by NMR Spectroscopy

NMR studies of internal dynamics of serine proteinase protein inhibitors: Binding region mobilities of intact and reactive‐site hydrolyzed Cucurbita maxima trypsin inhibitor (CMTI)‐III of the squash family and comparison with those of counterparts of CMTI‐V of the potato I family

Structural and dynamic characterization of eukaryotic gene regulatory protein domains in solution

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