ABSTRACT
Engineering coordinated rotational motion in porous architectures enables the fabrication of molecular machines in solids. A flexible two-fold interpenetrated pillared Metal-Organic Framework precisely organizes fast mobile elements such as bicyclopentane (BCP) (107 â Hz regime at 85â K), two distinct pyridyl rotors and E-azo group involved in pedal-like motion. Reciprocal sliding of the two sub-networks, switched by chemical stimuli, modulated the sizes of the channels and finally the overall dynamical machinery. Actually, iodine-vapor adsorption drives a dramatic structural rearrangement, displacing the two distinct subnets in a concerted piston-like motion. Unconventionally, BCP mobility increases, exploring ultra-fast dynamics (107 â Hz) at temperatures as low as 44â K, while the pyridyl rotors diverge into a faster and slower dynamical regime by symmetry lowering. Indeed, one pillar ring gained greater rotary freedom as carried by the azo-group in a crank-like motion. A peculiar behavior was stimulated by pressurized CO2, which regulates BCP dynamics upon incremental site occupation. The rotary dynamics is intrinsically coupled to the framework flexibility as demonstrated by complementary experimental evidence (multinuclear solid-state NMR down to very low temperatures, synchrotron radiation XRD, gas sorption) and computational modelling, which helps elucidate the highly sophisticated rotor-structure interplay.
ABSTRACT
Structural analysis of macromolecular complexes within their natural cellular environment presents a significant challenge. Recent applications of solid-state NMR (ssNMR) techniques on living fungal cells and intact plant tissues have greatly enhanced our understanding of the structure of extracellular matrices. Here, we selectively highlight the most recent progress in this field. Specifically, we discuss how ssNMR can provide detailed insights into the chemical composition and conformational structure of pectin, and the consequential impact on polysaccharide interactions and cell wall organization. We elaborate on the use of ssNMR data to uncover the arrangement of the lignin-polysaccharide interface and the macrofibrillar structure in native plant stems or during degradation processes. We also comprehend the dynamic structure of fungal cell walls under various morphotypes and stress conditions. Finally, we assess how the combination of NMR with other techniques can enhance our capacity to address unresolved structural questions concerning these complex macromolecular assemblies.
Subject(s)
Plant Cells , Polysaccharides , Plant Cells/chemistry , Plant Cells/metabolism , Polysaccharides/chemistry , Magnetic Resonance Spectroscopy , Cell Wall/metabolism , Pectins/analysis , Pectins/chemistry , Pectins/metabolismABSTRACT
The advancement in the use of spectroscopic techniques to investigate coffee samples is of high interest especially considering the widespread problems with coffee adulteration and counterfeiting. In this work, the use of solid-state nuclear magnetic resonance (NMR) is investigated as a means to probe the various chemically-distinct phases existent in roasted coffee samples and to detect the occurrence of counterfeiting or adulterations in coffee blends. Routine solid-state 1H and 13C NMR spectra allowed the distinction between different coffee types (Arabica/Robusta) and the evaluation of the presence of these components in coffee blends. On the other hand, the use of more specialized solid-state NMR experiments revealed the existence of phases with different molecular mobilities (e.g., associated with lipids or carbohydrates). The results illustrate the usefulness of solid-state NMR spectroscopy to examine molecular mobilities and interactions and to aid in the quality control of coffee-related products.
Subject(s)
Coffea , Coffee , Coffee/chemistry , Coffea/chemistry , Seeds/chemistry , Magnetic Resonance Spectroscopy/methods , Magnetic Resonance ImagingABSTRACT
Halogen bonding to phosphorus atoms remains uncommon, with relatively few examples reported in the literature. Here, the preparation and investigation of the cocrystal bis(dicyclohexylphenylphosphine)(1,6-diiodoperfluorohexane) by X-ray crystallography and solid-state multinuclear magnetic resonance spectroscopy is described. The crystal structure features two crystallographically unique C-I...P halogen bonds [dI...P = 3.090â (5)â Å, 3.264â (5)â Å] and crystallographic disorder of one of the 1,6-diiodoperfluorohexane molecules. The first of these is the shortest and most linear I...P halogen bond reported to date. 13C, 19F, and 31P magic angle spinning solid-state NMR spectra are reported. A 31P chemical shift change of -7.0 p.p.m. in the cocrystal relative to pure dicyclohexylphenylphosphine, consistent with halogen bond formation, is noted. This work establishes iodoperfluoroalkanes as viable halogen bond donors when paired with phosphorus acceptors, and also shows that dicyclohexylphenylphosphine can act as a practical halogen bond acceptor.
Subject(s)
Halogens , Iodine , Crystallography, X-Ray , Halogens/chemistry , Hydrogen Bonding , Iodides/chemistry , Magnetic Resonance Spectroscopy , Nuclear Magnetic Resonance, Biomolecular , Phosphorus , X-RaysABSTRACT
Atomic-scale description of surfaces and interfaces in core-shell aluminosilicate materials is not fully elucidated, partially due to their amorphous character and complex mechanisms that govern their properties. In this paper, new insights into nanostructured core-shell aluminosilicates have been demonstrated, by using different solid-state NMR methods, i.e 29Si, 29Si cross-polarization (CP), 27Al, 27Al triple-quantum (3Q), and 1H-27Al heteronuclear correlation (HETCOR) MAS NMR. For this purpose, nanostructured silica core-alumina shell microspheres, undoped and doped with gadolinium ions respectively, obtained by a chemical synthesis based on the Stöber method for the silica core and electrostatic attraction for developing the alumina shell were studied. As a result, a new alumino-silicate layer formation was proved at the interface between silica core, where aluminum diffuses, on small scale, in the silica network, and alumina shell, where silicon ions migrate, on a larger scale, in the alumina network, leading to a stable core-shell structure. Moreover, this process is accompanied by significant local structural changes in the transition zone, particularly at the aluminum neighborhood, which is quite well understood now, with the power of solid-state NMR spectroscopy.
Subject(s)
Aluminum Oxide , Silicon Dioxide , Aluminum/chemistry , Magnetic Resonance Spectroscopy , Microspheres , Silicon Dioxide/chemistryABSTRACT
Plant cell walls contain cellulose embedded in matrix polysaccharides. Understanding carbohydrate structures and interactions is critical to the production of biofuel and biomaterials using these natural resources. Here we present a solid-state NMR study of cellulose and pectin in 13C-labeled cell walls of Arabidopsis wild-type and mutant plants. Using 1D 13C and 2D 13C-13C correlation experiments, we detected a highly branched arabinan structure in qua2 and tsd2 samples, two allelic mutants for a pectin methyltransferase. Both mutants show close physical association between cellulose and the backbones of pectic homogalacturonan and rhamnogalacturonan-I. Relaxation and dipolar order parameters revealed enhanced microsecond dynamics due to polymer disorder in the mutants, but restricted motional amplitudes due to tighter pectin-cellulose associations. These molecular data shed light on polymer structure and packing in these two pectin mutants, helping to elucidate how pectin could influence cell wall architecture at the nanoscale, cell wall mechanics, and plant growth.
Subject(s)
Arabidopsis/chemistry , Cell Wall/chemistry , Cellulose/chemistry , Methyltransferases/chemistry , Pectins/chemistry , Arabidopsis/enzymology , Cell Wall/enzymology , Cellulose/metabolism , Magnetic Resonance Spectroscopy/methods , Methyltransferases/metabolism , Pectins/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolismABSTRACT
Nucleoside triphosphates (NTPs) are used as chemical energy source in a variety of cell systems. Structural snapshots along the NTP hydrolysis reaction coordinate are typically obtained by adding stable, nonhydrolyzable adenosine triphosphate (ATP) -analogues to the proteins, with the goal to arrest a state that mimics as closely as possible a physiologically relevant state, e.g., the pre-hydrolytic, transition and post-hydrolytic states. We here present the lessons learned on two distinct ATPases on the best use and unexpected pitfalls observed for different analogues. The proteins investigated are the bacterial DnaB helicase from Helicobacter pylori and the multidrug ATP binding cassette (ABC) transporter BmrA from Bacillus subtilis, both belonging to the same division of P-loop fold NTPases. We review the magnetic-resonance strategies which can be of use to probe the binding of the ATP-mimics, and present carbon-13, phosphorus-31, and vanadium-51 solid-state nuclear magnetic resonance (NMR) spectra of the proteins or the bound molecules to unravel conformational and dynamic changes upon binding of the ATP-mimics. Electron paramagnetic resonance (EPR), and in particular W-band electron-electron double resonance (ELDOR)-detected NMR, is of complementary use to assess binding of vanadate. We discuss which analogues best mimic the different hydrolysis states for the DnaB helicase and the ABC transporter BmrA. These might be relevant also to structural and functional studies of other NTPases.
Subject(s)
ATP-Binding Cassette Transporters/metabolism , Adenosine Triphosphate/chemistry , Bacillus subtilis/enzymology , DnaB Helicases/metabolism , Helicobacter pylori/enzymology , Adenosine Triphosphatases/metabolism , Adenosine Triphosphate/analogs & derivatives , Adenylyl Imidodiphosphate/chemistry , Aluminum Compounds/chemistry , Bacterial Proteins/metabolism , Electron Spin Resonance Spectroscopy , Electrons , Fluorides/chemistry , Hydrolysis , Magnetic Resonance Spectroscopy , Protein ConformationABSTRACT
We present an approach towards the in situ solid state NMR monitoring of mechanochemical reactions in a ball mill. A miniaturized vibration ball mill is integrated into the measuring coil of a home-built solid state NMR probe, allowing for static solid state NMR measurements during the mechanochemical reaction within the vessel. The setup allows to quantitatively follow the product evolution of a prototypical mechanochemical reaction, the formation of zinc phenylphosphonate from zinc acetate and phenylphosphonic acid. MAS NMR investigations on the final reaction mixture confirmed a reaction yield of 89% in a typical example. Thus, NMR spectroscopy may in the future provide complementary information about reaction mechanisms of mechanochemical reactions and team up with other analytical methods which have been employed to follow reactions in situ, such as Raman spectroscopy or X-ray diffraction.
ABSTRACT
In plants, many natural defense mechanisms include cellular membrane fusion as a way to resist infection by external pathogens. Several plant proteins mediate membrane fusion, but the detailed mechanism by which they promote fusion is less clear. Understanding this process could provide valuable insights into these proteins' physiological functions and guide bioengineering applications (i.e. the design of antimicrobial proteins). The plant-specific insert (PSI) from Solanum tuberosum can help reduce certain pathogen attack via membrane fusion. To gain new insights into the process of PSI-induced membrane fusion, a combined approach of NMR, FRET, and in silico studies was used. Our results indicate that (i) under acidic conditions, the PSI experiences a monomer-dimer equilibrium, and the dimeric PSI induces membrane fusion below a certain critical pH; (ii) after fusion, the PSI resides in a highly dehydrated environment with limited solvent accessibility, suggesting its capability in reducing repulsive dehydration forces between liposomes to facilitate fusion; and (iii) as shown by molecular dynamics simulations, the PSI dimer can bind stably to membrane surfaces and can bridge liposomes in close proximity, a critical step for the membrane fusion. In summary, this study provides new and unique insights into the mechanisms by which the PSI and similar proteins induce membrane fusion.
Subject(s)
Membrane Fusion , Plant Proteins/metabolism , Solanum tuberosum/metabolism , Hydrogen-Ion Concentration , Liposomes/metabolism , Molecular Dynamics Simulation , Plant Proteins/chemistry , Protein Aggregates , Protein Multimerization , Solanum tuberosum/chemistryABSTRACT
Materials often contain minor heterogeneous phases that are difficult to characterize yet nonetheless significantly influence important properties. Here we describe a solid-state NMR strategy for quantifying minor heterogenous sample regions containing dilute, essentially uncoupled nuclei in materials where the remaining nuclei experience heteronuclear dipolar couplings. NMR signals from the coupled nuclei are dephased while NMR signals from the uncoupled nuclei can be amplified by one or two orders of magnitude using Carr-Meiboom-Purcell-Gill (CPMG) acquisition. The signal amplification by CPMG can be estimated allowing the concentration of the uncoupled spin regions to be determined even when direct observation of the uncoupled spin NMR signal in a single pulse experiment would require an impractically long duration of signal averaging. We use this method to quantify residual graphitic carbon using 13C CPMG NMR in poly(carbon monofluoride) samples synthesized by direct fluorination of carbon from various sources. Our detection limit for graphitic carbon in these materials is better than 0.05 mol%. The accuracy of the method is discussed and comparisons to other methods are drawn.
Subject(s)
Carbon/chemistry , Magnetic Resonance Spectroscopy/methods , Signal Processing, Computer-Assisted , Algorithms , Fluorine/chemistry , Fluorocarbon Polymers/chemistry , Graphite/chemistry , Limit of Detection , Materials Testing , Petroleum , Programming Languages , Reproducibility of ResultsABSTRACT
The rat has been considered as an appropriate animal model for the study of the mineralization process in humans. In this work, we found that the phosphorus species in human dentin characterized by solid-state NMR spectroscopy consist mainly of orthophosphate and hydrogen phosphate. Some orthophosphates are found in a disordered phase, where the phosphate ions are hydrogen-bonded to structural water, some present a stoichiometric apatite structure, and some a hydroxyl-depleted apatite structure. The results of this study are largely the same as those previously obtained for rat dentin. However, the relative amounts of the various phosphorus species in human and rat dentin are dramatically different. In particular, stoichiometric apatite is more abundant in human dentin than in rat dentin, whereas the converse is true for disordered-phase orthophosphates. Furthermore, spatial proximity among all phosphorus species in human dentin is identical within experimental error, in contrast to what observed for rat dentin. Although it is not clear how these spectroscopic data could relate to the hierarchical structure or the mechanical properties of teeth, our data reveal that the molecular structures of human and rat dentin at different growth stages are not exactly the same.
Subject(s)
Apatites/chemistry , Dentin/chemistry , Magnetic Resonance Spectroscopy , Phosphates/analysis , Phosphorus/analysis , HumansABSTRACT
NMR relaxation theory and NMR lineshape calculations were used to characterize the rates of C2 symmetry jumps of deuterium nuclei in partly deuterated gypsum powder. The experimental data consisted of variable temperature deuterium NMR powder line shapes and deuterium T1 relaxation times. All of the Mathematica© notebooks used to simulate the spectra and match the experimental T1 values are included as supplementary material, and are suitable templates for similar calculations on other systems. Our simulations show that the deuterium nuclei of D2O in Gypsum undergo a two-site C2 180° jump about the D-O-D bisector angle of 54.8°. The jump rate stays in the fast motion regime down to about 218â¯K. Below 193â¯K the powder lineshapes change, the spectral intensities drop significantly, and the motion slows into the intermediate motion regime. The best fit quadrupole coupling constants (QCC's) vary between 216â¯kHz at the highest temperatures to 235â¯kHz at the lowest temperatures. The asymmetry parameters (ɳ) vary between 0.11 at the highest temperatures to 0.15 at the lowest temperatures. Knowledge of the C2 jump rates allowed us to calculate activation parameters for the jumps, namely ΔH = 22â¯kJ/mol, and ΔS = -10â¯J/mol·K which indicate a non-spontaneous activation process, an activation energy of Eaâ¯=â¯23â¯kJ/mol, and a pre-exponential factor of Aâ¯=â¯3.6â¯×â¯1012. As expected, there was no evidence of quantum tunneling.
ABSTRACT
Ganglioside lipids have been associated with several physiological processes, including cell signaling. They have also been associated with amyloid aggregation in Parkinson's and Alzheimer's disease. In biological systems, gangliosides are present in a mix with other lipid species, and the structure and properties of these mixtures strongly depend on the proportions of the different components. Here, we study self-assembly in model mixtures composed of ganglioside GM1 and a zwitterionic phospholipid, 1,2-Dioleoyl-sn-glycero-3-phosphocholine (DOPC). We characterize the structure and molecular dynamics using a range of complementary techniques, including cryo-TEM, polarization transfer solid state NMR, diffusion NMR, small-angle X-ray scattering (SAXS), dynamic light scattering (DLS), and calorimetry. The main findings are: (1) The lipid acyl chains are more rigid in mixtures containing both lipid species compared to systems that only contain one of the lipids. (2) The system containing DOPC with 10 mol % GM1 contains both vesicles and micelles. (3) At higher GM1 concentrations, the sample is more heterogenous and also contains small disc-like or rod-like structures. Such a co-existence of structures can have a strong impact on the overall properties of the lipid system, including transport, solubilization, and partitioning, which can be crucial to the understanding of the role of gangliosides in biological systems.
Subject(s)
G(M1) Ganglioside/chemistry , Phosphatidylcholines/chemistry , Micelles , Molecular Dynamics Simulation , Scattering, Small Angle , Water/chemistry , X-Ray DiffractionABSTRACT
Understanding the nature of the drug-polymer interactions in micellar drug delivery systems and what happens with the drug and the polymer once the complex has formed is essential for the rational design of the polymeric matrices suitable for a particular drug. In this work, glycopolymeric vesicles-a block copolymer, poly(1-O-methacryloyl-ß-d-fructopyranose)-b-poly(methyl methacrylate), (PFru36-PMMA160),-designed to target tumor cells loaded with two drugs, ellipticine and curcumin, were characterized. Advanced solid-state NMR spectroscopy and single-crystal/powder X-ray diffraction (XRD) combined with CASTEP calculations shed light on the nature of the drug, the polymer, and their interactions. While the low drug loading (ca. 5%) ensured that the structure, size, and shape of the polymeric vesicles did not change significantly, the solid-state forms of the drugs changed markedly. Upon loading into the vesicles, ellipticine favored a highly ordered form distinctly different from the bulk drug as indicated by 13C solid-state NMR spectroscopy. A detailed analysis of the CASTEP-calculated 13C spectra derived from crystallographic data based on the lowest mean absolute error showed the best match with form I. Moreover, ellipticine before loading was found as a new polymorph and was described by single-crystal XRD as a new orthorhombic Form III. Likewise, curcumin, originally present in monoclinic Form I was found to recrystallize as metastable orthorhombic Form II inside the vesicles. Intermolecular interactions between the polymeric vesicles and the drugs, ellipticine as well as curcumin, were detected using 2D 1H magic angle spinning experiments, indicating that the drugs are localized in the hydrophobic layer of the vesicles.
Subject(s)
Antineoplastic Agents/chemistry , Curcumin/chemistry , Drug Carriers/chemistry , Glycoconjugates/chemistry , Nuclear Magnetic Resonance, Biomolecular , Hydrophobic and Hydrophilic InteractionsABSTRACT
Polyphosphate (poly-P) is a major constituent in activated sludge from wastewater treatment plants with enhanced biological phosphorus removal due to poly-P synthesis by poly-P accumulating organisms where it plays an important role for recovery of phosphorus from waste water. Our aim was to develop a reliable protocol for poly-P quantification by 31P NMR spectroscopy. This has so far been complicated by the risks of inefficient extraction and poly-P hydrolysis in the extracts. A protocol for complete extraction, identification and quantification of poly-P in activated sludge from a waste water treatment plant was identified based on test and evaluation of existing extraction protocols in combination with poly-P determination and quantification by solution and solid state 31P NMR spectroscopy. The total poly-P middle group content was quantified by solid state NMR for comparison with the poly-P middle groups quantified by solution NMR, which is novel. Three different extraction protocols previously used in literature were compared: 1) a single 0.25â¯M NaOH-0.05â¯M EDTA extraction, 2) a 0.05â¯M EDTA pre-extraction followed by a 0.25â¯M NaOH main extraction and 3) a 0.05â¯M EDTA pre-extraction followed by a 0.25â¯M NaOH-0.05â¯M EDTA main extraction. The results showed that the extraction protocol 2 was optimal for fresh activated sludge, extracting 10.8⯱â¯0.4 to 11.4⯱â¯1.2 mgP/gDW poly-P. Extraction protocols 1 and 3 extracted less than 9.4⯱â¯0.5 mgP/gDW poly-P. A comparison of the quantification of poly-P by 31P solution NMR and by 31P solid state NMR spectroscopy of lyophilised activated sludge showed 86⯱â¯9% extraction efficiency of poly-P, which confirms that the extraction protocol recovered most of the poly-P from the samples without pronounced poly-P degradation.
Subject(s)
Sewage , Water Purification , Magnetic Resonance Spectroscopy , Phosphorus , Polyphosphates , Wastewater , WaterABSTRACT
Phosphate, which contains the essential element phosphorous (P), is a necessary fertilizer for agriculture, but the current phosphate deposits are running out and alternative sources are needed. Sludge obtained from wastewater treatment plants contains high concentrations of phosphorus and represents an alternative, sustainable source. In this study, sludge obtained from a wastewater treatment plant with biological and chemical phosphorus removal was acidified (pHâ¯=â¯3, 4, 5 and 6) to release orthophosphate followed by sequestration of the orthophosphate by a zinc aluminum layered double hydroxide (Zn2Al-LDH). Sulfuric acid (H2SO4), nitric acid (HNO3), and hydrochloric acid (HCl) was tested, which showed that only sulfate anions compete with phosphate and results in reduced phosphate recovery (25-35%). The orthophosphate concentration in the liquid phase increased from 20% (raw sludge) to 75% of the total phosphorus concentration at a pH of 3, which enhanced the phosphate uptake by the ZnAl-LDH from 1.7⯱â¯0.2% to 60.3⯱â¯0.6%. During acidification, the competing anion carbonate is degassed as CO2, which further improved the phosphate uptake. PXRD showed the intercalation of carbonate in the LDH in the raw sludge at pHâ¯=â¯8, whereas orthophosphate was intercalated at lower pH values. 27Al MAS NMR spectroscopy and powder X-ray diffraction (PXRD) proved preservation of the LDH at all pH values. Furthermore, about a fourth of the Al is present as an amorphous aluminum phosphate (AlPO4) upon exposure to phosphate at low pH (pHâ¯=â¯3 and 5) based on 27Al MAS NMR spectroscopy. At a pH of 6 about a third of the P is present as brushite (CaHPO4·2H2O).
Subject(s)
Phosphorus , Sewage , Aluminum , Aluminum Hydroxide , HydroxidesABSTRACT
In-cell NMR offers great insight into the characterization of the effect of toxins and antimicrobial peptides on intact cells. However, the complexity of intact live cells remains a significant challenge for the analysis of the effect these agents have on different cellular components. Here we show that 31P solid-state NMR can be used to quantitatively characterize the dynamic behaviour of DNA within intact live bacteria. Lipids were also identified and monitored, although 31P dynamic filtering methods indicated a range of dynamic states for phospholipid headgroups. We demonstrate the usefulness of this methodology for monitoring the activity of the antibiotic ampicillin and the antimicrobial peptide (AMP) maculatin 1.1 (Mac1.1) against Gram-negative bacteria. Perturbations in the dynamic behaviour of DNA were observed in treated cells, which indicated additional mechanisms of action for the AMP Mac1.1 not previously reported. This work highlights the value of 31P in-cell solid-state NMR as a tool for assessing the antimicrobial activity of antibiotics and AMPs in bacterial cells.
Subject(s)
Anti-Infective Agents/pharmacology , Escherichia coli/drug effects , Magnetic Resonance Spectroscopy , Phosphorus/chemistry , Stress, Physiological/drug effects , Ampicillin/pharmacology , DNA, Bacterial/metabolism , Escherichia coli/ultrastructure , Microbial Viability/drug effects , Nucleic Acids/metabolism , TemperatureABSTRACT
Whole-cell protein profiling, spatial localization, and quantification of activities such as gene transcription and protein translation are possible with modern biochemical and biophysical techniques. Yet, addressing questions of overall compositional changes within a cell - capturing the relative amounts of protein and ribosomal RNA levels and lipid content simultaneously - would require extractions and purifications with caveats due to isolation yields and detection methods. A holistic view of cellular composition would aid in the study of cellular composition and function. Here, solid state NMR is used to identify 13C NMR signatures for cellular organelles in HeLa cells without the use of any isotopic labeling. Comparisons are made with carbon spectra of subcellular assemblies including DNA, lipids, ribosomes, nuclei and mitochondria. Whole-cell comparisons are made with different mammalian cells lines, with red blood cells that lack nuclei and organelles, and with Gram-negative and Gram-positive bacteria. Furthermore, treatment of mammalian cells with cycloheximide, a commonly used protein synthesis inhibitor, revealed unanticipated changes consistent with a significant increase in protein glycosylation, obvious at the whole cell level. Thus, we demonstrate that solid-state NMR serves as a unique analytical tool to catalog and compare the ratios of distinct carbon types in cells and serves as a discovery tool to reveal the workings of inhibitors such as cycloheximide on whole-cell biochemistry.
Subject(s)
Cell Nucleus/metabolism , Endoplasmic Reticulum/metabolism , Golgi Apparatus/metabolism , Magnetic Resonance Spectroscopy/methods , Mitochondria/metabolism , Cycloheximide/pharmacology , Erythrocytes/cytology , Erythrocytes/drug effects , Erythrocytes/metabolism , Escherichia coli/chemistry , Escherichia coli/isolation & purification , Glycoproteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Macromolecular Substances/isolation & purification , Macromolecular Substances/metabolism , Protein Synthesis Inhibitors/pharmacology , Staphylococcus aureus/chemistry , Staphylococcus aureus/isolation & purificationABSTRACT
Potato native and wound healing periderms contain an external multilayered phellem tissue (potato skin) consisting of dead cells whose cell walls are impregnated with suberin polymers. The phellem provides physical and chemical barriers to tuber dehydration, heat transfer, and pathogenic infection. Previous RNAi-mediated gene silencing studies in native periderm have demonstrated a role for a feruloyl transferase (FHT) in suberin biosynthesis and revealed how its down-regulation affects both chemical composition and physiology. To complement these prior analyses and to investigate the impact of FHT deficiency in wound periderms, a bottom-up methodology has been used to analyze soluble tissue extracts and solid polymers concurrently. Multivariate statistical analysis of LC-MS and GC-MS data, augmented by solid-state NMR and thioacidolysis, yields two types of new insights: the chemical compounds responsible for contrasting metabolic profiles of native and wound periderms, and the impact of FHT deficiency in each of these plant tissues. In the current report, we confirm a role for FHT in developing wound periderm and highlight its distinctive features as compared to the corresponding native potato periderm.