Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several amyloid fibril systems, thus providing valuable information regarding the forces and interactions that ...confer the extraordinary stability of the amyloid architecture. Despite these advances, however, obtaining atomic resolution information describing the higher levels of structural organization within the fibrils remains a significant challenge. Here, we detail MAS NMR experiments and sample labeling schemes designed specifically to probe such higher order amyloid structure, and we have applied them to the fibrils formed by an eleven-residue segment of the amyloidogenic protein transthyretin (TTR(105–115)). These experiments have allowed us to define unambiguously not only the arrangement of the peptide β-strands into β-sheets but also the β-sheet interfaces within each protofilament, and in addition to identify the nature of the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts have resulted in 111 quantitative distance and torsion angle restraints (10 per residue) that describe the various levels of structure organization. The experiments benefited extensively from the use of dynamic nuclear polarization (DNP), which in some cases allowed us to shorten the data acquisition time from days to hours and to improve significantly the signal-to-noise ratios of the spectra. The β-sheet interface and protofilament interactions identified here revealed local variations in the structure that result in multiple peaks for the exposed N- and C-termini of the peptide and in inhomogeneous line-broadening for the residues buried within the interior of the fibrils.
Comprehensive multiphase NMR is a novel NMR technique that permits all components (solutions, gels, and solids) to be studied in unaltered natural samples. In this study a wide range of CMP-NMR ...interaction and editing-based experiments are combined to follow contaminants (pentafluorophenol (PFP) and perfluorooctanoic acid (PFOA)) from the solution state (after a spill) through the gel-state and finally into the true solid-state (sequestered) in an intact water-swollen soil. Kinetics experiments monitoring each phase illustrate PFOA rapidly transfers from solution to the solid phase while for PFP the process is slower with longer residence times in the solution and gel phase. Interaction-based experiments reveal that PFOA enters the soil via its hydrophobic tails and selectively binds to soil microbial protein. PFP sorption shows less specificity exhibiting interactions with a range of gel and solid soil components with a preference toward aromatics (mainly lignin). The results indicate that in addition to more traditional measurements such as K oc, other factors including the influence of the contaminant on the soil–water interface, specific biological interactions, soil composition (content of lignin, protein, etc.) and physical accessibility/swellability of soil organic components will likely be central to better explaining and predicting the true behavior of contaminants in soil.
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► A comprehensive multiphase (CMP)-NMR probe has been developed. ► It is susceptibility matched, has high power circuits, MAS, lock and gradients. ► It can handle the full range of ...solution-, gel- and solid-state experiments. ► CMP-NMR permits all bonds in all phases to be studied in unaltered samples. ► It possesses a great potential for the in situ study of heterogeneous systems.
Heterogeneous samples, such as soils, sediments, plants, tissues, foods and organisms, often contain liquid-, gel- and solid-like phases and it is the synergism between these phases that determine their environmental and biological properties. Studying each phase separately can perturb the sample, removing important structural information such as chemical interactions at the gel-solid interface, kinetics across boundaries and conformation in the natural state. In order to overcome these limitations a Comprehensive Multiphase-Nuclear Magnetic Resonance (CMP-NMR) probe has been developed, and is introduced here, that permits all bonds in all phases to be studied and differentiated in whole unaltered natural samples. The CMP-NMR probe is built with high power circuitry, Magic Angle Spinning (MAS), is fitted with a lock channel, pulse field gradients, and is fully susceptibility matched. Consequently, this novel NMR probe has to cover all HR-MAS aspects without compromising power handling to permit the full range of solution-, gel- and solid-state experiments available today. Using this technology, both structures and interactions can be studied independently in each phase as well as transfer/interactions between phases within a heterogeneous sample. This paper outlines some basic experimental approaches using a model heterogeneous multiphase sample containing liquid-, gel- and solid-like components in water, yielding separate 1H and 13C spectra for the different phases. In addition, 19F performance is also addressed. To illustrate the capability of 19F NMR soil samples, containing two different contaminants, are used, demonstrating a preliminary, but real-world application of this technology. This novel NMR approach possesses a great potential for the in situ study of natural samples in their native state.
Nuclear magnetic resonance (NMR) spectroscopy is arguably one the most powerful tools to study the interactions and molecular structure within plants. Traditionally, however, NMR has developed as two ...separate fields, one dealing with liquids and the other dealing with solids. Plants in their native state contain components that are soluble, swollen, and true solids. Here, a new form of NMR spectroscopy, developed in 2012, termed comprehensive multiphase (CMP)-NMR is applied for plant analysis. The technology composes all aspects of solution, gel, and solid-state NMR into a single NMR probe such that all components in all phases in native unaltered samples can be studied and differentiated in situ. The technology is evaluated using wild-type Arabidopsis thaliana and the cellulose-deficient mutant ectopic lignification1 (eli1) as examples. Using CMP-NMR to study intact samples eliminated the bias introduced by extraction methods and enabled the acquisition of a more complete structural and metabolic profile; thus, CMP-NMR revealed molecular differences between wild type (WT) and eli1 that could be overlooked by conventional methods. Methanol, fatty acids and/or lipids, glutamine, phenylalanine, starch, and nucleic acids were more abundant in eli1 than in WT. Pentaglycine was present in A. thaliana seedlings and more abundant in eli1 than in WT.
The chemical nature of xenobiotic binding sites in soils is of vital importance to environmental biogeochemistry. Interactions between xenobiotics and the naturally occurring organic constituents of ...soils are strongly correlated to environmental persistence, bioaccessibility, and ecotoxicity. Nevertheless, because of the complex structural and chemical heterogeneity of soils, studies of these interactions are most commonly performed indirectly, using correlative methods, fractionation, or chemical modification. Here we identify the organic components of an unmodified peat soil where some organofluorine xenobiotic compounds interact using direct molecular-level methods. Using 19F→1H cross-polarization magic angle spinning (CP-MAS) nuclear magnetic resonance (NMR) spectroscopy, the 19F nuclei of organofluorine compounds are used to induce observable transverse magnetization in the 1H nuclei of organic components of the soil with which they interact after sorption. The observed 19F→1H CP-MAS spectra and dynamics are compared to those produced using model soil organic compounds, lignin and albumin. It is found that lignin-like components can account for the interactions observed in this soil for heptafluoronaphthol (HFNap) while protein structures can account for the interactions observed for perfluorooctanoic acid (PFOA). This study employs novel comprehensive multi-phase (CMP) NMR technology that permits the application of solution-, gel-, and solid-state NMR experiments on intact soil samples in their swollen state.
Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core ...formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20–64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4–18.8 T) and temperature (109–180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.
Comprehensive multiphase (CMP) NMR is a novel technology that integrates all the hardware from solution-, gel- and solid-state into a single NMR probe, permitting all phases to be studied in intact ...samples. Here comprehensive multiphase (CMP) NMR is used to study all components in a living organism for the first time. This work describes 4 new scientific accomplishments summarized as: (1) CMP-NMR is applied to a living animal, (2) an effective method to deliver oxygen to the organisms is described which permits longer studies essential for in-depth NMR analysis in general, (3) a range of spectral editing approaches are applied to fully differentiate the various phases solutions (metabolites) through to solids (shell) (4)
13
C isotopic labelling and multidimensional NMR are combined to provide detailed assignment of metabolites and structural components
in vivo
. While not explicitly studied here the multiphase capabilities of the technique offer future possibilities to study kinetic transfer between phases (
e.g.
nutrient assimilation, contaminant sequestration), molecular binding at interfaces (
e.g.
drug or contaminant binding) and bonding across and between phases (
e.g.
muscle to bone)
in vivo
. Future work will need to focus on decreasing the spinning speed to reduce organism stress during analysis.
Comprehensive Multiphase NMR provides an overview as to all components (liquids, gels, solids) in a living organism.
Comprehensive Multiphase NMR provides an overview as to all components (liquids, gels, solids) in a living organism.
Comprehensive multiphase (CMP) NMR is a novel technology that integrates all the ...hardware from solution-, gel- and solid-state into a single NMR probe, permitting all phases to be studied in intact samples. Here comprehensive multiphase (CMP) NMR is used to study all components in a living organism for the first time. This work describes 4 new scientific accomplishments summarized as: (1) CMP-NMR is applied to a living animal, (2) an effective method to deliver oxygen to the organisms is described which permits longer studies essential for in-depth NMR analysis in general, (3) a range of spectral editing approaches are applied to fully differentiate the various phases solutions (metabolites) through to solids (shell) (4)
13
C isotopic labelling and multidimensional NMR are combined to provide detailed assignment of metabolites and structural components
in vivo
. While not explicitly studied here the multiphase capabilities of the technique offer future possibilities to study kinetic transfer between phases (
e.g.
nutrient assimilation, contaminant sequestration), molecular binding at interfaces (
e.g.
drug or contaminant binding) and bonding across and between phases (
e.g.
muscle to bone)
in vivo
. Future work will need to focus on decreasing the spinning speed to reduce organism stress during analysis.
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