Metabolic Fate of 13C-Labeled Polydextrose and Impact on the Gut Microbiome: A Triple-Phase Study in a Colon Simulator.

The present study introduces a novel triple-phase (liquids, solids, and gases) approach, which employed uniformly labeled [U-13C] polydextrose (PDX) for the selective profiling of metabolites generated from dietary fiber fermentation in an in vitro colon simulator using human fecal inocula. Employing 13C NMR spectroscopy, [U-13C] PDX metabolism was observed from colonic digest samples. The major 13C-labeled metabolites generated were acetate, butyrate, propionate, and valerate. In addition to these short-chain fatty acids (SCFAs), 13C-labeled lactate, formate, succinate, and ethanol were detected in the colon simulator samples. Metabolite formation and PDX substrate degradation were examined comprehensively over time (24 and 48 h). Correlation analysis between 13C NMR spectra and gas production confirmed the anaerobic fermentation of PDX to SCFAs. In addition, 16S rRNA gene analysis showed that the level of Erysipelotrichaceae was influenced by PDX supplementation and Erysipelotrichaceae level was statistically correlated with SCFA formation. Overall, our study demonstrates a novel approach to link substrate fermentation and microbial function directly in a simulated colonic environment.

The cell-wall active mannuronan C5-epimerases in the model brown alga Ectocarpus: From gene context to recombinant protein.

Mannuronan C5-epimerases (ManC5-Es) catalyze in brown algae the remodeling of alginate, a major cell-wall component which is involved in many biological functions in these organisms. ManC5-Es are present as large multigenic families in brown algae, likely indicating functional specificities and specializations. ManC5-Es control the distribution pattern of (1-4) linked ß-d-mannuronic acid (M) and α-l-guluronic acid (G) residues in alginates, giving rise to widely different polysaccharide compositions and sequences, depending on tissue, season, age, or algal species. As such they are also a source of powerful new tools for the biotechnological and enzymatic processing of alginates, to match the growing interest for food hydrocolloids and in biomedical and nanotechnological applications. We report here the first heterologous production of a ManC5-E of brown algal origin that is successfully refolded in an active form. The activity was measured by 1H NMR and by an indirect enzymatic assay using a known bacterial alginate lyase. The transcript expression as a function of the developmental program of the brown alga Ectocarpus, together with the bioinformatic analyses of the corresponding gene context of this multigenic family, is also presented.

High Throughput Identification and Quantification of Phospholipids in Complex Mixtures.

We present a fully automatic method, autoP, for identification and quantification of lipids in complex lipid mixtures from 1D (31)P and 2D (1)H-(31)P NMR spectra. The (31)P chemical shifts in lipids are highly sensitive to experimental conditions such as pH and temperature, so the present method uses the much more unambiguous (1)H chemical shifts for assignment and (31)P intensities for quantification. By using 2D (1)H-(31)P total correlation spectroscopy (TOCSY) correlation experiments, we demonstrate that approximately 20 different lipids can be automatically and unambiguously assigned and quantified by this automatic method.

Solid-state NMR methods for oriented membrane proteins.

Oriented-sample solid-state NMR represents one of few experimental methods capable of characterising the membrane-bound conformation of proteins in the cell membrane. Since the technique was developed 25 years ago, the technique has been applied to study the structure of helix bundle membrane proteins and antimicrobial peptides, characterise protein-lipid interactions, and derive information on dynamics of the membrane anchoring of membrane proteins. We will review the major developments in various aspects of oriented-sample solid-state NMR, including sample-preparation methods, pulse sequences, theory required to interpret the experiments, perspectives for and guidelines to new experiments, and a number of representative applications.

Facile synthesis of phosphatidyl saccharides for preparation of anionic nanoliposomes with enhanced stability.

Physical stability during storage and against processing such as dehyration/rehydration are the cornerstone in designing delivery vehicles. In this work, mono-, di- and tri-saccharides were enzymatically conjugated to phosphatidyl group through a facile approach namely phospholipase D (PLD) mediated transphosphatidylation in a biphasic reaction system. The purified products were structurally identified and the connectivities of carbohydrate to phosphatidyl moiety precisely mapped by (1)H, (31)P, (13)C NMR pulse sequences and LC-ESI-FTMS. The synthetic phosphatidyl saccharides were employed as the sole biomimetic component for preparation of nanoliposomes. It was found that the critical micelle concentration (CMC) of phosphatidyl saccharides increases as more bulky sugar moiety (mono- to tri-) is introduced. Phosphatidyl di-saccharide had the largest membrane curvature. In comparison to the zwitterionic phosphatidylcholine liposome, all phosphatidyl saccharides liposomes are anionic and demonstrated significantly enhanced stability during storage. According to the confocal laser scan microscopy (CLSM) and atom force microscopy (AFM) analyses, the nanoliposomes formed by the synthetic phosphatidyl saccharides also show excellent stability against dehydration/rehydration process in which most of the liposomal structures remained intact. The abundance hydroxyl groups in the saccharide moieties might provide sufficient H-bondings for stabilization. This work demonstrated the synthesized phosphatidyl saccharides are capable of functioning as enzymatically liable materials which can form stable nanoliposomes without addition of stabilizing excipients.

Mechanisms of peptide-induced pore formation in lipid bilayers investigated by oriented 31P solid-state NMR spectroscopy.

There is a considerable interest in understanding the function of antimicrobial peptides (AMPs), but the details of their mode of action is not fully understood. This motivates extensive efforts in determining structural and mechanistic parameters for AMP's interaction with lipid membranes. In this study we show that oriented-sample (31)P solid-state NMR spectroscopy can be used to probe the membrane perturbations and disruption by AMPs. For two AMPs, alamethicin and novicidin, we observe that the majority of the lipids remain in a planar bilayer conformation but that a number of lipids are involved in the peptide anchoring. These lipids display reduced dynamics. Our study supports previous studies showing that alamethicin adopts a transmembrane arrangement without significant disturbance of the surrounding lipids, while novicidin forms toroidal pores at high concentrations leading to more extensive membrane disturbance.

Phospholipase D (PLD) catalyzed synthesis of phosphatidyl-glucose in biphasic reaction system.

Presence of saccharides in glycophospholipids may increase its potential to form supramolecular structures, which are not only stable for an extended period of time as compared to other PLs like phosphatidylcholine, but may also confer an antioxidative property. Most syntheses routes for glycophospholipid involved the usage of toxic chemicals or solvents, complicated steps and low yield. The present work attempted to develop an enzymatic method for the production of glycophospholipids. Phosphatidyl-glucose (PL-Glu) was synthesized as a model glycophospholipid. The effects of organic solvents, water content, substrate ratio, pH and temperature on glycophospholipid yield (mol%) were examined in this study. Under optimum reaction conditions, more than 95 mol% of PL-Glu was obtained in 1.5 h. The conversion rate is significantly higher than previously reported findings. The established model reaction system in the present work was used to synthesize other types of glycophospholipid.

Long-term-stable ether-lipid vs conventional ester-lipid bicelles in oriented solid-state NMR: altered structural information in studies of antimicrobial peptides.

Recently, ether lipids have been introduced as long-term stable alternatives to the more natural, albeit easier degradable, ester lipids in the preparation of oriented lipid bilayers and bicelles for oriented-sample solid-state NMR spectroscopy. Here we report that ether lipids such as the frequently used 14-O-PC (1,2-di-O-tetradecyl-sn-glycero-3-phosphocholine) may induce significant changes in the structure and dynamics, including altered interaction between peptides and lipids relative to what is observed with the more conventionally used DMPC (1,2-dimyristoyl-sn-glycero-3-phosphocholine) bilayers. Such effects are demonstrated for the antimicrobial peptide novicidin, for which 2D separate-local-field NMR and circular dichroism experiments reveal significant structural/conformational differences for the peptide in the two different lipid systems. Likewise, we observe altered secondary structure and different temperature-dependent membrane anchoring for the antimicrobial peptide alamethicin depending on whether the peptide is reconstituted into ester or ether lipids. Such observations are not particularly surprising considering the significant difference of the lipids in the phosphorus headgroup and they may provide important new insight into the delicate peptide-membrane interactions in the systems studied. In contrast, these observations reinforce the need to carefully consider potential structural changes in addition to long-term stability prior to the selection of membrane environment of membrane proteins in the analysis of their structure and dynamics. In more general terms, the results underscore the necessity in structural biology to address both the protein and its environments in studies relating structure to function.

Pardaxin permeabilizes vesicles more efficiently by pore formation than by disruption.

Pardaxin is a 33-amino-acid neurotoxin from the Red Sea Moses sole Pardachirus marmoratus, whose mode of action shows remarkable sensitivity to lipid chain length and charge, although the effect of pH is unclear. Here we combine optical spectroscopy and dye release experiments with laser scanning confocal microscopy and natural abundance (13)C solid-state nuclear magnetic resonance to provide a more complete picture of how pardaxin interacts with lipids. The kinetics and efficiency of release of entrapped calcein is highly sensitive to pH. In vesicles containing zwitterionic lipids (PC), release occurs most rapidly at low pH, whereas in vesicles containing 20% anionic lipid (PG), release occurs most rapidly at high pH. Pardaxin forms stable or transient pores in PC vesicles that allow release of contents without loss of vesicle integrity, whereas the inclusion of PG promotes total vesicle collapse. In agreement with this, solid-state nuclear magnetic resonance reveals that pardaxin takes up a trans-membrane orientation in 14-O-PC/6-O-PC bicelles, whereas the inclusion of 14-0-PG restricts it to contacts with lipid headgroups, promoting membrane lysis. Pore formation in zwitterionic vesicles is more efficient than lysis of anionic vesicles, suggesting that electrostatic interactions may trap pardaxin in several suboptimal interconverting conformations on the membrane surface.

Divorcing folding from function: how acylation affects the membrane-perturbing properties of an antimicrobial peptide.

Many small cationic peptides, which are unstructured in aqueous solution, have antimicrobial properties. These properties are assumed to be linked to their ability to permeabilize bacterial membranes, accompanied by the transition to an alpha-helical folding state. Here we show that there is no direct link between folding of the antimicrobial peptide Novicidin (Nc) and its membrane permeabilization. N-terminal acylation with C8-C16 alkyl chains and the inclusion of anionic lipids both increase Nc's ability to form alpha-helical structure in the presence of vesicles. Nevertheless, both acylation and anionic lipids reduce the extent of permeabilization of these vesicles and lead to slower permeabilization kinetics. Furthermore, acylation significantly decreases antimicrobial activity. Although acyl chains of increasing length also increase the tendency of the peptides to aggregate in solution, this cannot rationalize our results since permeabilization and antimicrobial activities are observed well below concentrations where aggregation occurs. This suggests that significant induction of alpha-helical structure is not a prerequisite for membrane perturbation in this class of antimicrobial peptides. Our data suggests that for Nc, induction of alpha-helical structure may inhibit rather than facilitate membrane disruption, and that a more peripheral interaction may be the most efficient permeabilization mechanism. Furthermore, acylation leads to a deeper embedding in the membrane, which could lead to an anti-permeabilizing "plugging" effect.

Residue-specific information about the dynamics of antimicrobial peptides from (1)H-(15)N and (2)H solid-state NMR spectroscopy.

We present a new method to obtain information about the conformational dynamics of membrane-proteins using solid-state NMR experiments of oriented samples. By measuring the orientation-dependent (1)H-(15)N dipole-dipole coupling, (15)N anisotropic chemical shift, and (2)H quadrupole coupling parameters for a single residue, it is possible to obtain information about the local dynamics of each residue in the protein. This may be interpreted on an individual basis or through models extended to study conformational motion of membrane-protein segments. The method is demonstrated for the antimicrobial peptaibol alamethicin for which combined analysis of anisotropic interactions for the Aib(8) residue provides detailed information about helix-tilt angle, wobbling, and oscillatory rotation around the helix axis in the membrane bound state. This information is in very good agreement with coarse-grained MD simulations of the peptide in lipid bilayers.

Incorporation of antimicrobial peptides into membranes: a combined liquid-state NMR and molecular dynamics study of alamethicin in DMPC/DHPC bicelles.

Detailed insight into the interplay between antimicrobial peptides and biological membranes is fundamental to our understanding of the mechanism of bacterial ion channels and the action of these in biological host-defense systems. To explore this interplay, we have studied the incorporation, membrane-bound structure, and conformation of the antimicrobial peptide alamethicin in lipid bilayers using a combination of 1H liquid-state NMR spectroscopy and molecular dynamics (MD) simulations. On the basis of experimental NMR data, we evaluate simple in-plane and transmembrane incorporation models as well as pore formation for alamethicin in DMPC/DHPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine/1,2-dihexanoyl-sn-glycero-3-phosphatidylcholine) bicelles. Peptide-lipid nuclear Overhauser effect (NOE) and paramagnetic relaxation enhancement (PRE) data support a transmembrane configuration of the peptide in the bilayers, but they also reveal that the system cannot be described by a single simple conformational model because there is a very high degree of dynamics and heterogeneity in the three-component system. To explore the origin of this heterogeneity and dynamics, we have compared the NOE and PRE data with MD simulations of an ensemble of alamethicin peptides in a DMPC bilayer. From all-atom MD simulations, the contacts between peptide, lipid, and water protons are quantified over a time interval up to 95 ns. The MD simulations provide a statistical base that reflects our NMR data and even can explain some initially surprising NMR results concerning specific interactions between alamethicin and the lipids.

Resolution enhancement in solid-state NMR of oriented membrane proteins by anisotropic differential linebroadening.

We demonstrate that a significant improvement in the spectral resolution may be achieved in solid-state NMR experiments of proteins in inhomogeneously disordered oriented lipid bilayers. Using 1H homonuclear decoupling instead of standard 1H heteronuclear decoupling, the 15N line widths may be reduced by up to seven times for such samples. For large oriented membrane proteins, such resolution enhancements may be crucial for assignment and structural interpretation.

Membrane-bound conformation of peptaibols with methyl-deuterated alpha-amino isobutyric acids by 2H magic angle spinning solid-state NMR spectroscopy.

We present the use of 2H magic-angle spinning (MAS) NMR on methyl-deuterated alpha-amino isobutyric acid (Aib) as a new method to obtain fast and accurate structural constraints on peptaibols in membrane-bound environments. Using nonoriented vesicle-reconstituted samples we avoid the delicate preparation of oriented samples, and the use of MAS ensures high sensitivity and thereby very fast acquisition of experimental spectra. Furthermore, given the high content ( approximately 40%) of Aib in peptaibols and the fact that the amino acid Aib may be synthesized from cheap starting materials, even in the case of 2H, 13C, or 15N labeling, this method is ideally suited for studies of the membrane-bound conformation of peptaibols.

2D separated-local-field spectra from projections of 1D experiments.

A novel procedure for reconstruction of 2D separated-local-field (SLF) NMR spectra from projections of 1D NMR data is presented. The technique, dubbed SLF projection reconstruction from one-dimensional spectra (SLF-PRODI), is particularly useful for uniaxially oriented membrane protein samples and represents a fast and robust alternative to the popular PISEMA experiment which correlates (1)H-(15)N dipole-dipole couplings with (15)N chemical shifts. The different 1D projections in the SLF-PRODI experiment are obtained from 1D spectra recorded under influence of homonuclear decoupling sequences with different scaling factors for the heteronuclear dipolar couplings. We demonstrate experimentally and numerically that as few as 2-4 1D projections will normally be sufficient to reconstruct a 2D SLF-PRODI spectrum with a quality resembling typical PISEMA spectra, leading to significant reduction of the acquisition time.