Supramolecular interactions probed by 13C-13C solid-state NMR spectroscopy

We present a robust solid-state NMR approach for the accurate determination of molecular interfaces in insoluble and noncrystalline protein-protein complexes. The method relies on the measurement of intermolecular (13)C-(13)C distances in mixtures of [1-(13)C]glucose- and [2-(13)C]glucose-labeled proteins. We have applied this method to Parkinson's disease-associated α-synuclein fibrils and found that they are stacked in a parallel in-register arrangement. Additionally, intermolecular distance restraints for the structure determination of the fibrils at atomic resolution were measured.     

Protein structure determination by high-resolution solid-state NMR spectroscopy: application to microcrystalline ubiquitin

High-resolution solid-state NMR spectroscopy has become a promising method for the determination of three-dimensional protein structures for systems which are difficult to crystallize or exhibit low solubility. Here we describe the structure determination of microcrystalline ubiquitin using 2D (13)C-(13)C correlation spectroscopy under magic angle spinning conditions. High-resolution (13)C spectra have been acquired from hydrated microcrystals of site-directed (13)C-enriched ubiquitin. Inter-residue carbon-carbon distance constraints defining the global protein structure have been evaluated from 'dipolar-assisted rotational resonance' experiments recorded at various mixing times. Additional constraints on the backbone torsion angles have been derived from chemical shift analysis. Using both distance and dihedral angle constraints, the structure of microcrystalline ubiquitin has been refined to a root-mean-square deviation of about 1 A. The structure determination strategies for solid samples described herein are likely to be generally applicable to many proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy.     

Investigation of dipolar-mediated water-protein interactions in microcrystalline Crh by solid-state NMR spectroscopy

Water-protein interactions play a major role in protein folding, structure, and function, and solid-state NMR has recently been shown to be a powerful tool for the site-resolved observation of these interactions in solid proteins. In this article we report investigations on possible water-protein dipolar transfer mechanisms in the microcrystalline deuterated protein Crh by a set of solid-state NMR techniques. Double-quantum (DQ) filtered and edited heteronuclear correlation experiments are used to follow direct dipolar water-protein magnetization transfers. Experimental data reveal no evidence for "solid-like" water molecules, indicating that residence times of solvent molecules are shorter than required for DQ creation, typically a few hundred microseconds. An alternative magnetization pathway, intermolecular cross-relaxation via heteronuclear nuclear Overhauser effects (NOEs), is probed by saturation transfer experiments. The significant additional enhancements observed when irradiating at the water frequency can possibly be attributed to direct heteronuclear water-protein NOEs; however, a contribution from relayed magnetization transfer via chemical exchange or proton-proton dipolar mechanisms cannot be excluded.     

Characterization of folding intermediates of a domain-swapped protein by solid-state NMR spectroscopy

We have employed two-dimensional solid-state NMR to study structure and dynamics of insoluble folding states of the domain-swapped protein Crh. Starting from the protein precipitated at its pI, conformational changes due to a modest temperature increase were investigated at the level of individual residues and in real-time. As compared to the crystalline state, Crh pI-precipitates exhibited a higher degree of molecular mobility for several regions of the protein. A rigidly intact center was observed including a subset of residues of the hydrophobic core. Raising the temperature by 13 K to 282 K created a partially unfolded intermediate state that was converted into beta-sheet-rich aggregates that are mostly of spherical character according to electron microscopy. Residue-by-residue analysis indicated that two out of three alpha-helices in aggregated Crh underwent major structural rearrangements while the third helix was preserved. Residues in the hinge region exhibited major chemical-shift changes, indicating that the domain swap was not conserved in the aggregated form. Our study provides direct evidence that protein aggregates of a domain-swapped protein retain a significant fraction of native secondary structure and demonstrates that solid-state NMR can be used to directly monitor slow molecular folding events.     

Toward the characterization of peptidoglycan structure and protein-peptidoglycan interactions by solid-state NMR spectroscopy

Solid-state NMR spectroscopy is applied to intact peptidoglycan sacculi of the Gram-negative bacterium Escherichia coli. High-quality solid-state NMR spectra allow atom-resolved investigation of the peptidoglycan structure and dynamics as well as the study of protein-peptidoglycan interactions.     

Use of selective Trp side chain labeling to characterize protein-protein and protein-ligand interactions by NMR spectroscopy

Recent studies on amino acid occurrence in protein binding sites suggest that only a reduced number of residues are responsible for most interaction energy in protein-protein and protein-ligand interactions. Above all, tryptophan (Trp) seems to be the most frequent residue in protein's hot spots. Here we report a novel, efficient, and cost-effective method to selectively incorporate specific isotope labels into the side chains of Trp residues in recombinant proteins. We show that the method proposed allows selective NMR observation of Trp side chains that enables studies of ligand binding, protein-protein interactions, hydrogen binding, protein folding, and side chain dynamics. Examples with the protein BIR3 will be given.     

Structural studies of sialon ceramics by high-resolution solid-state NMR

High-resolution solide-state ²⁷Al NMR with magic-angle spinning (MASNMR) readily monitors the quantity and coordination (four- and six-fold) of aluminium in two ceramic materials of the SiAlON system. Sialon X-phase, of approximate composition Si₃Al₆O₁₂N₂, contains aluminium-centred octahedra and tetrahedra in the ratio ca. 1.9:1.0, while another sample containing a mixture of sialon polytypoids shows AlO₆ octahedra and a large quantity of what is most probably nitrogen-coordinated tetrahedral aluminium. In addition, ²⁹Si MASNMR detects two different kinds of silicon in the latter sample in a 2:1 ratio. These observations are interpreted satisfactorily in terms of the crystal structures of the compounds and provide further examples of the potential of MASNMR in the investigation of complex ceramic systems.     

Longer-range distances by spinning-angle-encoding solid-state NMR spectroscopy

A new spinning-angle-encoding spin-echo solid-state NMR approach is used to accurately determine the dipolar coupling corresponding to a C-C distance over 4 ? in a fully labelled dipeptide. The dipolar coupling dependent spin-echo modulation was recorded off magic angle, switching back to the magic angle for the acquisition of the free-induction decay, so as to obtain optimum sensitivity. The retention of both ideal resolution and long-range distance sensitivity was achieved by redesigning a 600 MHz HX MAS NMR probe to provide fast angle switching during the NMR experiment: for 1.8 mm rotors, angle changes of up to ～5° in ～10 ms were achieved at 12 kHz MAS. A new experimental design that combines a reference and a dipolar-modulated experiment and a master-curve approach to data interpretation is presented.     

Protein 3D structure determination by high-resolution solid-state NMR

Developments in NMR technology, sample preparation, pulse sequence methodology and structure calculation protocols have recently allowed one to progress towards structure determination at high-resolution of proteins by solid-state NMR spectroscopy. We here report solid-state NMR protocols based on magic-angle-spinning experiments, combined with modified structure calculation protocols, for structure determination of uniformly ¹³C, ¹⁵N isotopically labeled proteins. We demonstrate the use of these protocols to obtain high-resolution structures for the example of the microcrystalline Crh protein. The CHHC, DARR and PAR solid-state NMR experiments, as well as the calculation protocols using the program ARIA, are presented.     

The crystalline structures of ethylene-dimethylaminoethyl methacrylate copolymers as studied by solid-state high-resolution 13C NMR spectroscopy

The crystalline structures of ethylene-dimethylaminoethyl methacrylate (EDAM) copolymers, which were either melt-quenched (mq) or isothermally crystallized (iso), were studied by solid-state high-resolution ¹³C NMR spectroscopy. It revealed that the crystalline structures of EDAM copolymers are greatly dependent on the comonomer content, crystallization condition and the storage time after treatment. The ratio of monoclinic to orthorhombic crystal (M/O) increases with the increase in the dimethylaminoethyl methacrylate content. Higher crystallinity and lower monoclinic content were observed for iso samples compared to the mq ones. The monoclinic crystal was found to melt at lower temperatures compared to the orthorhombic one during the heating process. The degree of crystallinity as well as the contents of monoclinic and orthorhombic crystals and the M/O value are found to increase after storage at room temperature for a month.     

Powder crystallography by proton solid-state NMR spectroscopy

The investigation of 1H-1H spin-diffusion build-up curves using a rate matrix analysis approach shows that high-resolution magic angle spinning NMR of protons, applied to powdered organic compounds, provides a method to probe crystalline arrangements. The comparison between experimental 1H data and simulation is shown to depend strongly on the parameters of the crystal structure, for example on the unit cell parameters or the orientation of the molecule in the unit cell, and those parameters are experimentally determined for a model organic compound.     

Water-protein interactions in microcrystalline crh measured by 1H-13C solid-state NMR spectroscopy

Using solid-state NMR carbon-proton dipolar correlation spectroscopy, we observed hydrogen exchange on the millisecond time scale between water molecules and protein protons in a solid sample. These interactions are shown to be related to important structural features of the protein such as hydrogen-bonding or salt-bridge networks.     

Characterization and quantitation of clarithromycin polymorphs by powder X-ray diffractometry and solid-state NMR spectroscopy

Characterization of clarithromycin polymorph was performed by solid-state cross polarization and magic angle spinning (CP/MAS) 13C-NMR spectroscopy. Two polymorphs, form II and form I, of clarithromycins indicated characteristic resonances of C1 carbonyl carbon at 176.2 and 175.2 ppm, respectively. Since each peak of C1 carbon was well separated in the spectrum of the two polymorphs, we performed quantitative analysis of the polymorphic fraction from the peak area of these peaks. The peak area of form I was found to linearly increase with an increase of its content, with a correlation coefficient of above 0.99. Solid-state NMR was found to be a useful technique to determine the characteristics of the polymorphic forms.     

Characterization of nonderivatized plant cell walls using high-resolution solution-state NMR spectroscopy

A recently described plant cell wall dissolution system has been modified to use perdeuterated solvents to allow direct in-NMR-tube dissolution and high-resolution solution-state NMR of the whole cell wall without derivatization. Finely ground cell wall material dissolves in a solvent system containing dimethylsulfoxide-d(6) and 1-methylimidazole-d(6) in a ratio of 4:1 (v/v), keeping wood component structures mainly intact in their near-native state. Two-dimensional NMR experiments, using gradient-HSQC (heteronuclear single quantum coherence) 1-bond (13)C--(1)H correlation spectroscopy, on nonderivatized cell wall material from a representative gymnosperm pinus taeda (loblolly pine), an angiosperm Populus tremuloides (quaking aspen), and a herbaceous plant Hibiscus cannabinus (kenaf) demonstrate the efficacy of the system. We describe a method to synthesize 1-methylimidazole-d(6) with a high degree of perdeuteration, thus allowing cell wall dissolution and NMR characterization of nonderivatized plant cell wall structures.     

Structural and dynamical studies of poly(vinyl alcohol) gels by high-resolution solid-state C NMR spectroscopy

The ¹³C CP/MAS NMR spectra of isotactic, syndiotactic and atactic poly(vinyl alcohol) (PVA) gels were measured in order to clarify the structure of the immobile component of PVA gel. In the ¹³C CP/MAS NMR spectra, the three CH carbon peaks I, II and III (at about 77, 71 and 65 ppm) were clearly observed, which originate from the formation of strong intermolecular or intramolecular hydrogen bonds between hydroxyl groups like solid PVA. It has been assigned that these peaks originate from the crosslinked region in the gel state. On the basis of the experimental results, intermolecular hydrogen bonds play an important role in the formation of the crosslinked-region in the gel state. Further, the effect of PVA's tacticity on the amount of the crosslinked regions by intermolecular interactions was discussed. In addition, molecular motion in the immobile and mobile region of PVA gel was discussed through the observation of ¹³C spin-lattice relaxation time T₁.     

Characterization of PDMS model junctions and networks by solution and solid-state silicon-29 NMR spectroscopy

The application of ²⁹Si solution and solid-state cross-polarization/magic-angle spinning (CP/MAS) nuclear magnetic resonance (NMR) techniques to the study of structural features in polydimethylsiloxane (PDMS) model endlinked elastomeric networks is explored. The relationship between the topological (network) functionality of a structural moiety, which determines network mechanical properties, and its chemical (spectral) functionality, which is reported by the NMR, is discussed. The second-order spectral shifts corresponding to topographical functionality variation within a chemical functionality class are usually sufficiently well resolved in these networks to allow positive identification of a variety of structural features. The basic PDMS repeat unit, ? OSi(CH₃)₂? , is found to possess an axially symmetric chemical shift tensor with σ_∥ = ?56.8 ppm downfield from TMS, and σ_⊥ = ?4.4 ppm. This axial symmetry does not result from rapid reorientation about the chain axis. The NMR spectrum reveals defects in model endlinked networks. In the case of vinyl-endlinked systems, the defects are ascribed to the formation of elastically ineffective loops. Hydroxyl-endlinked systems contain either loops or else trifunctional junctions (hydrolyzed before chain coupling could take place) and dangling chain ends. The CP/MAS technique provides an order-of-magnitude reduction over standard solution techniques in the time to acquire a spectrum from a network not containing paramagnetic doping. ¹³C spectra of PDMS systems are not as informative as ²⁹Si spectra.     

Low-power high-resolution solid-state NMR of peptides and proteins

We demonstrate that two-dimensional solid-state NMR chemical-shift correlation spectra can be recorded under low-power conditions. Except for the cross-polarization period, no rf-field amplitudes above 40 kHz are used. Such experiments require the use of fast (>50 kHz) magic-angle spinning (MAS). A comparison with the high-power version of the experiment shows no general line broadening but some changes in the polarization-transfer dynamics.     

On-line detection of emulsion polymerization by solid-state NMR spectroscopy

 The on-line detection of emulsion polymerization processes by means of solid-state NMR spectroscopy is demonstrated for the first time using poly(butyl acrylate) as a model system. Relatively short time intervals are accessible via ¹H detection while the use of ¹³C NMR spectroscopy results in an increased spectral resolution. Details of sample preparation and experimental techniques are given, while remaining artifacts of the preliminary results will be addressed in further investigations. Received: 7 November 1997 Accepted: 5 January 1998