Biomedical subjects

Lynette Cegelski

Publications and source records attributed to Lynette Cegelski.

10 recordsLinked to original sources

PubMed2007

Rotational-echo double-resonance NMR distance measurements for the tubulin-bound Paclitaxel conformation.

The important anticancer drug Taxol (paclitaxel, PTX) owes its unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. The conformation of the microtubule-bound drug has been the focus of numerous research efforts, since the inability of polymerized tubulin to form crystals precludes structure proof by X-ray crystallography. Likewise, although the alpha,beta-tubulin dimer structure has been solved by electron crystallography, the 3.7 A resolution is too low to permit direct determination of either ligand conformation or binding pose. In this article, we present experimental results from 2H{19F} REDOR NMR that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.

Animals↗

PubMed2006

Structures of Staphylococcus aureus cell-wall complexes with vancomycin, eremomycin, and chloroeremomycin derivatives by 13C{19F} and 15N{19F} rotational-echo double resonance.

Solid-state NMR has been used to examine isolated cell walls and intact whole cells of Staphylococcus aureus complexed to five different vancomycin, eremomycin, and chloroeremomycin derivatives. The cell walls and whole cells were specifically labeled with d-[1-(13)C]alanine, or a combination of [1-(13)C]glycine and [epsilon-(15)N]lysine. Each of the bound glycopeptides had a (19)F-labeled substituent at either its C-terminus or its disaccharide position. The (13)C{(19)F} rotational-echo double-resonance (REDOR) dephasing for the cell-wall (13)C-labeled bridging pentaglycyl segment connecting a glycopeptide-complexed peptidoglycan stem with its neighboring stem indicates that the fluorine labels for all bound glycopeptides are positioned at one or the other end of the bridge. An exception is N'-(p-trifluoromethoxybenzyl)chloroeremomycin, whose hydrophobic substituent differs in length by one phenyl group compared to that of oritavancin, N'-4-[(4-chlorophenyl)benzyl)]chloroeremomycin. For this drug, the fluorine label is near the middle of the pentaglycyl segment. (15)N{(19)F} REDOR dephasing shows the proximity of the fluorine to the bridge-link site of the pentaglycyl bridge for C-terminus-substituted moieties and the cross-link site for disaccharide-substituted moieties. Full-echo REDOR spectra of cell-wall complexes from cells labeled by d-[1-(13)C]alanine (in the presence of an alanine racemase inhibitor) reveal three different carbonyl carbon chemical-shift environments, arising from the d-Ala-d-Ala binding site and the d-Ala-Gly-1 cross-link site. The REDOR results indicate a single fluorine dephasing center in each peptidoglycan complex. Molecular models of the mature cell-wall complexes that are consistent with internuclear distances obtained from (13)C{(19)F} and (15)N{(19)F} REDOR dephasing allow a correlation of structure and antimicrobial activity of the glycopeptides.

Alanine↗

PubMed2006

Peptide antibiotics in action: investigation of polypeptide chains in insoluble environments by rotational-echo double resonance.

Rotational-echo double resonance (REDOR) is a solid-state NMR technique that has the capability of providing intra- and intermolecular distance and orientational restraints in non-crystallizable, poorly soluble heterogeneous molecular systems such as cell membranes and cell walls. In this review, we will present two applications of REDOR: the investigation of a magainin-related antimicrobial peptide in lipid bilayers and the study of a vancomycin-like glycopeptide in the cell walls of Staphylococcus aureus.

Amino Acid Sequence↗

PubMed2006

LRP6 holds the key to the entry of anthrax toxin.

In this issue of Cell, it is demonstrated that the low-density lipoprotein receptor-related protein 6 (LRP6) promotes endocytosis of the anthrax toxin into cells. LRP6 acts as a coreceptor with either TEM8 or CMG2, the two previously identified receptors for anthrax toxin.

Animals↗

PubMed2006

Conformational and quantitative characterization of oritavancin-peptidoglycan complexes in whole cells of Staphylococcus aureus by in vivo 13C and 15N labeling.

Solid-state NMR has been used to examine the cell walls of intact whole cells of Staphyloccus aureus grown on media containing D-[1-(13)C]alanine, [(15)N]glycine, and the alanine racemase inhibitor, alaphosphin. The results of in situ site-selective, four-frequency NMR experiments show directly for the first time that (i) 54% of the cell-wall peptidoglycan stems have D-alanine termini and 46%, D-alanine-D-alanine termini; (ii) the molar ratio of stems ending in D-alanine to esterified alditol repeats of cell-wall teichoic and lipoteichoic acids is 3:2; and (iii) 50% of the mature cell-wall binding sites for a fluorinated oritavancin analogue consist of two nearest-neighbor peptide stems of different glycan strands. The drug is bound to the D-Ala-D-Ala terminus of one stem and is proximate to the bridging pentaglycyl segment that cross-links the two stems. Structural details of the binding site are revealed in a model of the glycopeptide-peptidoglycan interaction produced by molecular dynamics simulations with internuclear distance restraints determined by NMR.

Anti-Bacterial Agents↗

PubMed2005

NMR determination of photorespiration in intact leaves using in vivo 13CO2 labeling.

Solid-state 13C NMR measurements of intact soybean leaves labeled by 13CO2 lead to the conclusion that photorespiration is 17% of photosynthesis for a well-watered and fertilized plant. This is the first direct assessment of the level of photorespiration in a functioning plant. A 13C{31P} rotational-echo double-resonance (REDOR) measurement tracked the incorporation of 13C label into intermediates in the Calvin cycle as a function of time. For labeling times of 5 min or less, the isotopic enrichment of the Calvin cycle depends on the flux of labeled carbon from 13CO2, relative to the flux of unlabeled carbon from glycerate returned from the photorespiratory cycle. Comparisons of these two rates for a fixed value of the 13CO2 concentration indicate that the ratio of the rate of photosynthesis to the rate of photorespiration of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in soybean leaves is 5.7. This translates into a photorespiratory CO2 loss that is 21% of net CO2 assimilation, about 80% of the value estimated from Rubisco kinetics parameters. The ratio of rates is reduced at low external CO2 concentrations, as measured by net carbon assimilation rates. The carbon assimilation was determined from 13C-label spin counts converted into total carbon by the REDOR-determined isotopic enrichments of the Calvin cycle. The net carbon assimilation rates indicate that the rate of decarboxylation of glycine is not directly proportional to the oxygenase activity of Rubisco as is commonly assumed.

Carbon Dioxide↗

PubMed2005

Glycine metabolism in intact leaves by in vivo 13C and 15N labeling.

Solid-state (13)C NMR measurements of intact soybean leaves labeled by (13)CO(2) (at subambient concentrations) show that excess glycine from the photorespiratory C(2) cycle (i.e. glycine not part of the production of glycerate in support of photosynthesis) is either fully decarboxylated or inserted as (13)C-labeled glycyl residues in proteins. This (13)C incorporation in leaf protein, which is uniformly (15)N labeled by (15)NH(4)(15)NO(3), occurs as soon as 2 min after the start of (13)CO(2) labeling. In those leaves with lower levels of available nitrogen (as measured by leaf nitrate and glutamine-glutamate concentrations), the excess glycine is used primarily as glycyl residues in protein.

Carbon Dioxide↗

PubMed2003

REDOR with a relative full-echo reference.

REDOR and REDOR-like 13C[19F] and 2H[19F] NMR experiments have been performed on lyophilized whole cells of Staphylococcus aureus. The bacteria were grown to maturity on media containing L-[13C(3)]alanine or L-[methyl-d(3)]alanine, and then complexed with the 4-fluorobiphenyl derivative of chloroeremomycin, an analogue of the widely used antibiotic, vancomycin. The position of the 19F of the drug bound in the bacterial cell wall was determined relative to L-alanine 13C and 2H labels in the peptidoglycan peptide stem that was closest to the fluorinated biphenyl moiety of the drug. These determinations were made by dipolar recoupling methods that do not require an absolute measurement of the REDOR full echo (the signal observed without rotor-synchronized dephasing pulses) of the labels in the peptide stem.

Alanine↗

PubMed2002

Rotational-echo double resonance characterization of the effects of vancomycin on cell wall synthesis in Staphylococcus aureus.

Cross-polarization magic-angle spinning and rotational-echo double resonance 13C and 15N NMR experiments have been performed on intact cells of Staphylococcus aureus labeled with D-[1-13C]alanine and [15N]glycine or with [1-13C]glycine and L-[epsilon-15N]lysine. The cells were harvested during stationary or exponential growth conditions, the latter in media with and without the addition of vancomycin. The results of these experiments allowed the in situ determination of the relative concentrations of peptidoglycan cross-links (the number of peptide-stem D-alanines covalently linked to a pentaglycyl bridge) and bridge-links (the number of peptide-stem lysines covalently linked to a pentaglycyl bridge). The concentration of cross-links remained constant in the presence of vancomycin, whereas the number of bridge-links decreased. These changes suggest that vancomycin (at therapeutic levels) interrupts peptidoglycan synthesis in S. aureus by interference with transglycosylation.

Alanine↗

PubMed2002

Rotational-echo double resonance characterization of vancomycin binding sites in Staphylococcus aureus.

Solid-state NMR experiments with stable isotope-labeled Staphylococcus aureus have provided insight into the structure of the peptidoglycan binding site of a potent fluorobiphenyl derivative of chloroeremomycin (Eli Lilly LY329332). Rotational-echo double resonance (REDOR) NMR provided internuclear distances from the 19F of this glycopeptide antibiotic to natural-abundance 31P and to specific 13C and 15N labels biosynthetically incorporated into the bacteria from labeled alanine, glycine, or lysine in the growth medium. Results from experiments with intact late log phase bacteria and cell walls indicated homogeneous drug-peptidoglycan binding. Drug dimers were not detected in situ, and the hydrophobic fluorobiphenyl group of LY329332 did not insert into the bilayer membrane. A model of the binding site consistent with the REDOR results positions the vancomycin cleft around an un-cross-linked D-Ala-D-Ala peptide stem with the fluorobiphenyl moiety of the antibiotic near the base of a second, proximate stem in a locally ordered peptidoglycan matrix.

Anti-Bacterial Agents↗