Molecular basis of secondary relaxation in stiff-chain glassy polymers.

Recent progress in establishing local order in polycarbonate-like glasses using rotational echo double resonance and centerband-only detection of exchange solid-state nuclear magnetic resonance (NMR) has stimulated a renewed attempt to connect molecular motion within glassy polymers and the mechanical properties of the glass. We have in fact established a correlation between molecular motion characterized by NMR and the mechanical secondary relaxation (tan δ) for nine polycarbonate-like glasses. All of the NMR and mechanical data are for T ≪ Tg. The resulting structural insights suggest that the chains of these polymers are simultaneously both Flory random coils and Vol'kenstein bundles. The cooperative motions of groups of bundles can be described qualitatively by a variety of constrained-kinetics models of the glass. All of the models share a common trait for large-amplitude motion: an exponential increase in the time required for an inter-bundle dilation event with a linear increase in bundle group size. This dependence and a locally ordered Vol'kenstein bundle lead to an understanding of the surprising 60° (K) shift of tan δ to higher temperature for ring-fluoro-polycarbonate relative to that of polycarbonate by the apparently minor substitution of a fluorine for a hydrogen on every fourth ring.

Lactate metabolism is associated with mammalian mitochondria.

It is well established that lactate secreted by fermenting cells can be oxidized or used as a gluconeogenic substrate by other cells and tissues. It is generally assumed, however, that within the fermenting cell itself, lactate is produced to replenish NAD+ and then is secreted. Here we explore the possibility that cytosolic lactate is metabolized by the mitochondria of fermenting mammalian cells. We found that fermenting HeLa and H460 cells utilize exogenous lactate carbon to synthesize a large percentage of their lipids. Using high-resolution mass spectrometry, we found that both 13C and 2-2H labels from enriched lactate enter the mitochondria. The lactate dehydrogenase (LDH) inhibitor oxamate decreased respiration of isolated mitochondria incubated in lactate, but not of isolated mitochondria incubated in pyruvate. Additionally, transmission electron microscopy (TEM) showed that LDHB localizes to the mitochondria. Taken together, our results demonstrate a link between lactate metabolism and the mitochondria of fermenting mammalian cells.

Desleucyl-Oritavancin with a Damaged d-Ala-d-Ala Binding Site Inhibits the Transpeptidation Step of Cell-Wall Biosynthesis in Whole Cells of Staphylococcus aureus.

We have used solid-state nuclear magnetic resonance to characterize the exact nature of the dual mode of action of oritavancin in preventing cell-wall assembly in Staphylococcus aureus. Measurements performed on whole cells labeled selectively in vivo have established that des-N-methylleucyl-N-4-(4-fluorophenyl)benzyl-chloroeremomycin, an Edman degradation product of [19F]oritavancin, which has a damaged d-Ala-d-Ala binding aglycon, is a potent inhibitor of the transpeptidase activity of cell-wall biosynthesis. The desleucyl drug binds to partially cross-linked peptidoglycan by a cleft formed between the drug aglycon and its biphenyl hydrophobic side chain. This type of binding site is present in other oritavancin-like glycopeptides, which suggests that for these drugs a similar transpeptidase inhibition occurs.

Characterization of the tertiary structure of the peptidoglycan of Enterococcus faecalis.

Solid-state NMR spectra of whole cells and isolated cell walls of Enterococcus faecalis grown in media containing combinations of 13C and 15N specific labels in d- and l-alanine and l-lysine (in the presence of an alanine racemase inhibitor alaphosphin) have been used to determine the composition and architecture of the cell-wall peptidoglycan. The compositional variables include the concentrations of (i) peptidoglycan stems without bridges, (ii) d-alanylated wall teichoic acid, (iii) cross-links, and (iv) uncross-linked tripeptide and tetra/pentapeptide stems. Connectivities of l-alanyl carbonyl­carbon bridge labels to d-[3-13C]alanyl and l-[ε-15N]lysyl stem labels prove that the peptidoglycan of E. faecalis has the same hybrid short-bridge architecture (with a mix of parallel and perpendicular stems) as the FemA mutant of Staphylococcus aureus, in which the cross-linked stems are perpendicular to one another and the cross-linking is close to the ideal 50% value. This is the first determination of the cell-wall chemical and geometrical architecture of whole cells of E. faecalis, a major source of nosocomial infections worldwide.

Peptidoglycan architecture of Gram-positive bacteria by solid-state NMR.

Peptidoglycan is an essential component of cell wall in Gram-positive bacteria with unknown architecture. In this review, we summarize solid-state NMR approaches to address some of the unknowns in the Gram-positive bacteria peptidoglycan architecture: 1) peptidoglycan backbone conformation, 2) PG-lattice structure, 3) variations in the peptidoglycan architecture and composition, 4) the effects of peptidoglycan bridge-length on the peptidoglycan architecture in Fem mutants, 5) the orientation of glycan strands with respect to the membrane, and 6) the relationship between the peptidoglycan structure and the glycopeptide antibiotic mode of action. Solid-state NMR analyses of Staphylococcus aureus cell wall show that peptidoglycan chains are surprisingly ordered and densely packed. The peptidoglycan disaccharide backbone adopts 4-fold screw helical symmetry with the disaccharide unit periodicity of 40Å. Peptidoglycan lattice in the S. aureus cell wall is formed by cross-linked PG stems that have parallel orientations. The structural characterization of Fem-mutants of S. aureus with varying lengths of bridge structures suggests that the PG-bridge length is an important determining factor for the PG architecture.

REDOR constraints on the peptidoglycan lattice architecture of Staphylococcus aureus and its FemA mutant.

The peptidoglycan of Gram-positive bacteria consists of glycan chains with attached short peptide stems cross-linked to one another by glycyl bridges. The bridge of Staphylococcus aureus has five glycyl units and that of its FemA mutant has one. These long- and short-bridge cross-links create totally different cell-wall architectures. S. aureus and its FemA mutant grown in the presence of an alanine-racemase inhibitor were labeled with d-[1-¹³C]alanine, l-[3-¹³C]alanine, [2-¹³C]glycine, and l-[5-¹9F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between ¹³C-labeled alanines and ¹9F-labeled lysine incorporated in the peptidoglycan. The alanyl ¹³C labels in the parent strain were preselected for C{F} and C{P} REDOR measurement by their proximity to the glycine label using ¹³C¹³C spin diffusion. The observed ¹³C¹³C and ¹³C³¹P distances are consistent with a tightly packed architecture containing only parallel stems in a repeating structural motif within the peptidoglycan. Dante selection of d-alanine and l-alanine frequencies followed by ¹³C¹³C spin diffusion rules out scrambling of carbon labels. Cell walls of FemA were also labeled by a combination of d-[1-¹³C]alanine and l-[¹5N]alanine. Proximity of chains was measured by C{N} and N{C} REDOR distances and asymptotic plateaus, and both were consistent with a mixed-geometry model. Binding of an ¹9F-labeled eremomycin analog in the FemA cell wall matches that of binding to the parent-strain cell wall and reveals the proximity of parallel stems in the alternating parallel-perpendicular mixed-geometry model for the FemA peptidoglycan lattice.

The path to triacylglyceride obesity in the sta6 strain of Chlamydomonas reinhardtii.

When the sta6 (starch-null) strain of the green microalga Chlamydomonas reinhardtii is nitrogen starved in acetate and then "boosted" after 2 days with additional acetate, the cells become "obese" after 8 days, with triacylglyceride (TAG)-filled lipid bodies filling their cytoplasm and chloroplasts. To assess the transcriptional correlates of this response, the sta6 strain and the starch-forming cw15 strain were subjected to RNA-Seq analysis during the 2 days prior and 2 days after the boost, and the data were compared with published reports using other strains and growth conditions. During the 2 h after the boost, ∼425 genes are upregulated ≥2-fold and ∼875 genes are downregulated ≥2-fold in each strain. Expression of a small subset of "sensitive" genes, encoding enzymes involved in the glyoxylate and Calvin-Benson cycles, gluconeogenesis, and the pentose phosphate pathway, is responsive to culture conditions and genetic background as well as to boosting. Four genes-encoding a diacylglycerol acyltransferase (DGTT2), a glycerol-3-P dehydrogenase (GPD3), and two candidate lipases (Cre03.g155250 and Cre17.g735600)-are selectively upregulated in the sta6 strain. Although the bulk rate of acetate depletion from the medium is not boost enhanced, three candidate acetate permease-encoding genes in the GPR1/FUN34/YaaH superfamily are boost upregulated, and 13 of the "sensitive" genes are strongly responsive to the cell's acetate status. A cohort of 64 autophagy-related genes is downregulated by the boost. Our results indicate that the boost serves both to avert an autophagy program and to prolong the operation of key pathways that shuttle carbon from acetate into storage lipid, the combined outcome being enhanced TAG accumulation, notably in the sta6 strain.

Cross-link formation and peptidoglycan lattice assembly in the FemA mutant of Staphylococcus aureus.

Staphylococcus aureus FemA mutant grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-(13)C]alanine, l-[3-(13)C]alanine, [2-(13)C]glycine, and l-[5-(19)F]lysine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance (REDOR) NMR of isolated cell walls was used to measure internuclear distances between (13)C-labeled alanines and (19)F-labeled lysine incorporated in the peptidoglycan. The alanyl (13)C labels were preselected for REDOR measurement by their proximity to the glycine label using (13)C-(13)C spin diffusion. The observed (13)C-(13)C and (13)C-(19)F distances are consistent with a tightly packed, hybrid architecture containing both parallel and perpendicular stems in a repeating structural motif within the peptidoglycan.

Staphylococcus aureus peptidoglycan stem packing by rotational-echo double resonance NMR spectroscopy.

Staphylococcus aureus grown in the presence of an alanine-racemase inhibitor was labeled with d-[1-(13)C]alanine and l-[(15)N]alanine to characterize some details of the peptidoglycan tertiary structure. Rotational-echo double-resonance NMR of intact whole cells was used to measure internuclear distances between (13)C and (15)N of labeled amino acids incorporated in the peptidoglycan, and from those labels to (19)F of a glycopeptide drug specifically bound to the peptidoglycan. The observed (13)C-(15)N average distance of 4.1-4.4 Å between d- and l-alanines in nearest-neighbor peptide stems is consistent with a local, tightly packed, parallel-stem architecture for a repeating structural motif within the peptidoglycan of S. aureus.

The isotridecanyl side chain of plusbacin-A3 is essential for the transglycosylase inhibition of peptidoglycan biosynthesis.

Plusbacin-A3 (pb-A3) is a cyclic lipodepsipeptide that exhibits antibacterial activity against multidrug-resistant Gram-positive pathogens. Plusbacin-A3 is thought not to enter the cell cytoplasm, and its lipophilic isotridecanyl side chain is presumed to insert into the membrane bilayer, thereby facilitating either lipid II binding or some form of membrane disruption. Analogues of pb-A3, [(2)H]pb-A3 and deslipo-pb-A3, were synthesized to test membrane insertion as a key to the mode of action. [(2)H]pb-A3 has an isotopically (2)H-labeled isopropyl subunit of the lipid side chain, and deslipo-pb-A3 is missing the isotridecanyl side chain. Both analogues have the pb-A3 core structure. The loss of antimicrobial activity in deslipo-pb-A3 showed that the isotridecanyl side chain is crucial for the mode of action of the drug. However, rotational-echo double-resonance nuclear magnetic resonance characterization of [(2)H]pb-A3 bound to [1-(13)C]glycine-labeled whole cells of Staphylococcus aureus showed that the isotridecanyl side chain does not insert into the lipid membrane but instead is found in the staphylococcal cell wall, positioned near the pentaglycyl cross-bridge of the cell-wall peptidoglycan. Addition of [(2)H]pb-A3 during the growth of S. aureus resulted in the accumulation of Park's nucleotide, consistent with the inhibition of the transglycosylation step of peptidoglycan biosynthesis.

The effects of daptomycin on cell wall biosynthesis in Enterococcal faecalis.

Daptomycin is a cyclic lipodepsipeptide antibiotic reserved for the treatment of serious infections by multidrug-resistant Gram-positive pathogens. Its mode of action is considered to be multifaceted, encompassing the targeting and depolarization of bacterial cell membranes, alongside the inhibition of cell wall biosynthesis. To characterize the daptomycin mode of action, 15N cross-polarization at magic-angle spinning NMR measurements were performed on intact whole cells of Staphylococcus aureus grown in the presence of a sub-inhibitory concentration of daptomycin in a chemically defined media containing L-[ϵ-15N]Lys. Daptomycin-treated cells showed a reduction in the lysyl-ε-amide intensity that was consistent with cell wall thinning. However, the reduced lysyl-ε-amine intensity at 10 ppm indicated that the daptomycin-treated cells did not accumulate in Park's nucleotide, the cytoplasmic peptidoglycan (PG) precursor. Consequently, daptomycin did not inhibit the transglycosylation step of PG biosynthesis. To further elucidate the daptomycin mode of action, the PG composition of daptomycin-susceptible Enterococcus faecalis grown in the presence of daptomycin was analyzed using liquid chromatography-mass spectrometry. Sixty-nine muropeptide ions correspond to PG with varying degrees of modifications including crosslinking, acetylation, alanylation, and 1,6-anhydrous ring formation at MurNAc were quantified. Analysis showed that the cell walls of daptomycin-treated E. faecalis had a significant reduction in PG crosslinking which was accompanied by an increase in lytic transglycosylase activities and a decrease in PG-stem modifications by the carboxypeptidases. The changes in PG composition suggest that daptomycin inhibits cell wall biosynthesis by impeding the incorporation of nascent PG into the cell walls by transpeptidases and maturation by carboxypeptidases. As a result, the newly formed cell walls become highly susceptible to degradation by the autolysins, resulting in thinning of the cell wall.

A pentasaccharide for monitoring pharmacodynamic response to gene therapy in GM1 gangliosidosis.

BACKGROUND: GM1 gangliosidosis is a rare, fatal, neurodegenerative disease caused by mutations in the GLB1 gene and deficiency in ß-galactosidase. Delay of symptom onset and increase in lifespan in a GM1 gangliosidosis cat model after adeno-associated viral (AAV) gene therapy treatment provide the basis for AAV gene therapy trials. The availability of validated biomarkers would greatly improve assessment of therapeutic efficacy. METHODS: The liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to screen oligosaccharides as potential biomarkers for GM1 gangliosidosis. The structures of pentasaccharide biomarkers were determined with mass spectrometry, as well as chemical and enzymatic degradations. Comparison of LC-MS/MS data of endogenous and synthetic compounds confirmed the identification. The study samples were analyzed with fully validated LC-MS/MS methods. FINDINGS: We identified two pentasaccharide biomarkers, H3N2a and H3N2b, that were elevated more than 18-fold in patient plasma, cerebrospinal fluid (CSF), and urine. Only H3N2b was detectable in the cat model, and it was negatively correlated with ß-galactosidase activity. Following intravenous (IV) AAV9 gene therapy treatment, reduction of H3N2b was observed in central nervous system, urine, plasma, and CSF samples from the cat model and in urine, plasma, and CSF samples from a patient. Reduction of H3N2b accurately reflected normalization of neuropathology in the cat model and improvement of clinical outcomes in the patient. INTERPRETATIONS: These results demonstrate that H3N2b is a useful pharmacodynamic biomarker to evaluate the efficacy of gene therapy for GM1 gangliosidosis. H3N2b will facilitate the translation of gene therapy from animal models to patients. FUNDING: This work was supported by grants U01NS114156, R01HD060576, ZIAHG200409, and P30 DK020579 from the National Institutes of Health (NIH) and a grant from National Tay-Sachs and Allied Diseases Association Inc.

Carbon partitioning in soybean (Glycine max) leaves by combined (11) C and (13) C labeling.

We labeled soybean (Glycine max) leaves with 200 and 600 ppm (13) CO(2) spiked with (11) CO(2) and examined the effects of light intensity and water stress on metabolism by using a combination of direct positron imaging and solid-state (13) C nuclear magnetic resonance (NMR) of the same leaf. We first made 60-min movies of the transport of photosynthetically assimilated (11) C labels. The positron imaging identified zones or patches within which variations in metabolism could be probed later by NMR. At the end of each movie, the labeled leaf was frozen in liquid nitrogen to stop metabolism, the leaf was lyophilized, and solid-state NMR was used either on the whole leaf or on various leaf fragments. The NMR analysis determined total (13) C incorporation into sugars, starch, proteins, and protein precursors. The combination of (11) C and (13) C analytical techniques has led to three major conclusions regarding photosynthetically heterogeneous soybean leaves: transient starch deposition is not the temporary storage of sucrose excluded from a saturated sugar-transport system; peptide synthesis is reduced under high-light, high CO(2) conditions; and all glycine from the photorespiratory pathway is routed to proteins within photosynthetically active zones when the leaf is water stressed and under high-light and low CO(2) conditions.

Chain packing in glassy polymers by natural-abundance 13C-13C spin diffusion using 2D centerband-only detection of exchange.

The proximities of specific subgroups of nearest-neighbor chains in glassy polymers are revealed by distance-dependent (13)C-(13)C dipolar couplings and spin diffusion. The measurement of such proximities is practical even with natural-abundance levels of (13)C using a 2D version of centerband-only detection of exchange (CODEX). Two-dimensional CODEX is a relaxation-compensated experiment that avoids the problems associated with variations in T(1)(C)'s due to dynamic site heterogeneity in the glass. Isotropic chemical shifts are encoded in the t(1) preparation times before and after mixing, and variations in T(2)'s are compensated by an S(0) reference (no mixing). Data acquisition involves acquisition of an S(0) reference signal on alternate scans, and the active control of power amplifiers, to achieve stability and accuracy over long accumulation times. The model system to calibrate spin diffusion is the polymer itself. For a mixing time of 200 ms, only (13)C-(13)C pairs separated by one or two bonds (2.5 Å) show cross peaks, which therefore identify reference intrachain proximities. For a mixing time of 1200 ms, 5 Å interchain proximities appear. The resulting cross peaks are used in a simple and direct way to compare nonrandom chain packing for two commercial polycarbonates with decidedly different mechanical properties.

Oxygen-17 appears only in protein in water-stressed soybean leaves labeled by (17)O2.

We have used a rotational-echo adiabatic-passage double-resonance (13)C{(17)O} solid-state NMR experiment to prove that the glycine produced in the oxygenase reaction of ribulose bisphosphate carboxylase-oxygenase is incorporated exclusively into protein (or protein precursors) of intact, water-stressed soybean leaves exposed to (13)CO(2) and (17)O(2). The water stress increased stomatal resistance and decreased gas exchange so that the Calvin cycle in the leaf chloroplasts was no more than 35% (13)C isotopically enriched. Labeled O(2) levels were sufficient, however, to increase the (17)O isotopic concentration of oxygenase products 20-fold over the natural-abundance level of 0.04%. The observed direct incorporation of glycine into protein shows that water stress suppresses photorespiration in soybean leaves.

Variability in C(3)-plant cell-wall biosynthesis in a high-CO(2) atmosphere by solid-state NMR spectroscopy.

We have used a frequency-selective rotational-echo double-resonance (REDOR) solid-state NMR experiment to measure the concentrations of glycine-glycine pairs in proteins (and protein precursors) of intact leaves of plants exposed to both high- and low-CO(2) atomospheres. The results are interpreted in terms of differences in cell-wall biosynthesis between plant species. We illustrate this variability by comparing the assimilation of label in cheatgrass and soybean leaves labeled using (15)N-fertilizer and (13)CO(2) atmospheres. Cheatgrass and soybean are both C(3) plants but differ in their response to a high-CO(2) environment. Based on REDOR results, we determined that cheatgrass (a plant that seems likely to flourish in future low-water, high-CO(2) environments) routes 2% of the assimilated carbon label that remains in the leaf after 1 h in a 600-ppm (13)CO(2) atmosphere to glycine-rich protein (or its precursors), a structural component of cell walls cross-linked to lignins. In contrast, soybean under the same conditions routes none of its assimilated carbon to glycine-rich protein.

Plant cell-wall cross-links by REDOR NMR spectroscopy.

We present a new method that integrates selective biosynthetic labeling and solid-state NMR detection to identify in situ important protein cross-links in plant cell walls. We have labeled soybean cells by growth in media containing l-[ring-d(4)]tyrosine and l-[ring-4-(13)C]tyrosine, compared whole-cell and cell-wall (13)C CPMAS spectra, and examined intact cell walls using (13)C{(2)H} rotational echo double-resonance (REDOR) solid-state NMR. The proximity of (13)C and (2)H labels shows that 25% of the tyrosines in soybean cell walls are part of isodityrosine cross-links between protein chains. We also used (15)N{(13)C} REDOR of intact cell walls labeled by l-[ε-(15)N,6-(13)C]lysine and depleted in natural-abundance (15)N to establish that the side chains of lysine are not significantly involved in covalent cross-links to proteins or sugars.

Staphylococcus aureus peptidoglycan tertiary structure from carbon-13 spin diffusion.

The cell-wall peptidoglycan of Staphylococcus aureus is a heterogeneous, highly cross-linked polymer of unknown tertiary structure. We have partially characterized this structure by measuring spin diffusion from (13)C labels in pentaglycyl cross-linking segments to natural-abundance (13)C in the surrounding intact cell walls. The measurements were performed using a version of centerband-only detection of exchange (CODEX). The cell walls were isolated from S. aureus grown in media containing [1-(13)C]glycine. The CODEX spin diffusion rates established that the pentaglycyl bridge of one peptidoglycan repeat unit of S. aureus is within 5 A of the glycan chain of another repeat unit. This surprising proximity is interpreted in terms of a model for the peptidoglycan lattice in which all peptide stems in a plane perpendicular to the glycan mainchain are parallel to one another.

Humidity-swing mechanism for CO2 capture from ambient air.

A humidity-swing polymeric sorbent captures CO2 from ambient air at room temperature simply by changing the humidity level. To date there has been no direct experimental evidence to characterize the chemical mechanism for this process. In this report we describe the use of solid-state NMR to study the humidity-swing CO2 absorption/desorption cycle directly. We find that at low humidity levels CO2 is absorbed as HCO3-. At high humidity levels, HCO3- is replaced by hydrated OH- and the absorbed CO2 is released.