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Recently, proton-detected magic-angle spinning (MAS) solid-state nuclear magnetic resonance (NMR) spectroscopy has become an attractive tool to study the structure and dynamics of insoluble proteins at atomic resolution. The sensitivity of the employed multidimensional experiments can be systematically improved when both transversal components of the magnetization are transferred simultaneously after an evolution period. The method of preservation of equivalent pathways has been explored in solution-state NMR; however, it does not find widespread application due to relaxation issues connected with increased molecular size. We present here for the first time heteronuclear transverse mixing sequences for correlation experiments at moderate and fast MAS frequencies. Optimal control allows to boost the signal-to-noise ratio (SNR) beyond the expected factor of 2 for each indirect dimension. In addition to the carbon-detected sensitivity-enhanced 2D NCA experiment, we present a novel proton-detected, doubly sensitivity-enhanced 3D hCANH pulse sequence for which we observe a 3-fold improvement in SNR compared to the conventional experimental implementation. The sensitivity gain turned out to be essential to unambiguously characterize a minor fibril polymorph of a human lambda-III immunoglobulin light chain protein that escaped detection so far.

Proton detection developed in the last 20 years as the method of choice to study biomolecules in the solid state. In perdeuterated proteins, proton dipolar interactions are strongly attenuated, which allows yielding of high-resolution proton spectra. Perdeuteration and backsubstitution of exchangeable protons is essential if samples are rotated with MAS rotation frequencies below 60 kHz. Protonated samples can be investigated directly without spin dilution using proton detection methods in case the MAS frequency exceeds 110 kHz. This review summarizes labeling strategies and the spectroscopic methods to perform experiments that yield assignments, quantitative information on structure, and dynamics using perdeuterated samples. Techniques for solvent suppression, H/D exchange, and deuterium spectroscopy are discussed. Finally, experimental and theoretical results that allow estimation of the sensitivity of proton detected experiments as a function of the MAS frequency and the external B0 field in a perdeuterated environment are compiled.

Magic-angle spinning (MAS) is an essential ingredient in a wide variety of solid-state NMR experiments. The standard procedures to adjust the rotor angle are not highly accurate, resulting in a slight misadjustment of the rotor from the magic angle ( θRL=tan-12 ) on the order of a few millidegrees. This small missetting has no significant impact on the overall spectral resolution, but is sufficient to reintroduce anisotropic interactions. Shown here is that site-specific 1 H-15 N dipolar couplings can be accurately measured in a heavily deuterated protein. This method can be applied at arbitrarily high MAS frequencies, since neither rotor synchronization nor particularly high radiofrequency field strengths are required. The off-MAS method allows the quantification of order parameters for very dynamic residues, which often escape an analysis using existing methods.

In this study, the effect of CurDAc, a water-soluble curcumin derivative, on the formation and stability of amyloid fibers is revealed. CurDAc interaction with amyloid is structurally selective, which is reflected in a strong interference with hIAPP aggregation while showing weaker interactions with human-calcitonin and amyloid-β(1–40) in comparison. Remarkably, CurDAc also exhibited potent fiber disaggregation for hIAPP generating a toxic oligomeric species.

Emulsion polymer isocyanate (EPI) adhesive is one of the most widely used structural adhesives in the woodworking industry. However, there has been a lack of knowledge on how the EPI-adhesive interacts with the chemical constituents of wood, in particular, with the wood extractives that are known to influence the bonding quality. In this study, the interactions of the EPI-adhesive with the water extracts and selected extractives from European wood species were systematically investigated using different analytical techniques. While the alterations in the curing properties of the pure EPI-adhesive and EPI-adhesive-extract mixtures were revealed by in situ rheology and Fourier-transform infrared spectroscopy, evolved gas analysis, and pyrolysis-gas chromatography/mass spectrometry, the solid-state 13C nuclear magnetic resonance were performed for analyzing the final chemical composition of the cured adhesive. Moreover, the influence of the extraction on the mechanical bonding performance was tested by tensile-shear tests. The study revealed significant interactions between the EPI-adhesive and tannin-rich, acidic wood extracts. The acidic chestnut and oak extracts catalyzed the curing reactions and led to a huge increase in the adhesive viscosity. These interactions might affect the bonding quality, for example, adhesive penetration depth and formation of bonding strength, therefore, careful attention is required when bonding acidic wood surfaces.

Designed peptides derived from the islet amyloid polypeptide (IAPP) cross‐amyloid interaction surface with Aβ (termed interaction surface mimics or ISMs) have been shown to be highly potent inhibitors of Aβ amyloid self‐assembly. However, the molecular mechanism of their function is not well understood. Using solution‐state and solid‐state NMR spectroscopy in combination with ensemble‐averaged dynamics simulations and other biophysical methods including TEM, fluorescence spectroscopy and microscopy, and DLS, we characterize ISM structural preferences and interactions. We find that the ISM peptide R3‐GI is highly dynamic, can adopt a β‐like structure, and oligomerizes into colloid‐like assemblies in a process that is reminiscent of liquid–liquid phase separation (LLPS). Our results suggest that such assemblies yield multivalent surfaces for interactions with Aβ40. Sequestration of substrates into these colloid‐like structures provides a mechanistic basis for ISM function and the design of novel potent anti‐amyloid molecules., ISM inhibitors are highly dynamic assemblies and exchange between monomeric and high‐molecular‐weight states. In both states, the ISM peptide adopts a β‐loop‐like structure that provides a suitable surface for sequestration of Aβ40 in the colloidal state. ISM inhibitors thus exploit multivalency by self‐association to yield high substrate avidity.

Using NMR to probe transient binding of Aβ(1–40) monomers to fibers, we find partially bound conformations with the highest degree of interaction near F19-K28 and a lesser degree of interaction near the C-terminus (L34-G37). This represents a shift away from the KLVFFA recognition sequence (residues 16–21) currently used for inhibitor design.

In recent years, MAS solid-state NMR has emerged as a technique for the investigation of soluble protein complexes. It was found that high molecular weight complexes do not need to be crystallized in order to obtain an immobilized sample for solid-state NMR investigations. Sedimentation induced by sample rotation impairs rotational diffusion of proteins and enables efficient dipolar coupling based cross polarization transfers. In addition, viscosity contributes to the immobilization of the molecules in the sample. Natural Deep Eutectic Solvents (NADES) have very high viscosities, and can replace water in living organisms. We observe a considerable amount of cross polarization transfers for NADES solvents, even though their molecular weight is too low to yield significant sedimentation. We discuss how viscosity and sedimentation both affect the quality of the obtained experimental spectra. The FROSTY/sedNMR approach holds the potential to study large protein complexes, which are otherwise not amenable for a structural characterization using NMR. We show that using this method, backbone assignments of the symmetric proteasome activator complex (1.1MDa), and high quality correlation spectra of non-symmetric protein complexes such as the prokaryotic ribosome 50S large subunit binding to trigger factor (1.4MDa) are obtained.

In diabetes, hyperamylinemia contributes to cardiac dysfunction. The interplay between hIAPP, blood glucose and other plasma components is, however, not understood. We show that glucose and LDL interact with hIAPP, resulting in β-sheet rich oligomers with increased β-cell toxicity and hemolytic activity, providing mechanistic insights for a direct link between diabetes and cardiovascular diseases.

Polymorphism is a key feature of amyloid fibril structures but it remains challenging to explain these variations for a particular sample. Here, we report electron cryomicroscopy-based reconstructions from different fibril morphologies formed by a peptide fragment from an amyloidogenic immunoglobulin light chain. The observed fibril morphologies vary in the number and cross-sectional arrangement of a structurally conserved building block. A comparison with the theoretically possible constellations reveals the experimentally observed spectrum of fibril morphologies to be governed by opposing sets of forces that primarily arise from the β-sheet twist, as well as peptide–peptide interactions within the fibril cross-section. Our results provide a framework for rationalizing and predicting the structure and polymorphism of cross-β fibrils, and suggest that a small number of physical parameters control the observed fibril architectures., Amyloid fibril structures can display polymorphism. Here the authors reveal the cryo-EM structures of several different fibril morphologies of a peptide derived from an amyloidogenic immunoglobulin light chain and present a mathematical analysis of physical factors that influence fibril polymorphism.

The design of inhibitors of protein-protein interactions mediating amyloid self-assembly is a major challenge mainly due to the dynamic nature of the involved structures and interfaces. Interactions of amyloidogenic polypeptides with other proteins are important modulators of self-assembly. Here we present a hot-segment-linking approach to design a series of mimics of the IAPP cross-amyloid interaction surface with Aβ (ISMs) as nanomolar inhibitors of amyloidogenesis and cytotoxicity of Aβ, IAPP, or both polypeptides. The nature of the linker determines ISM structure and inhibitory function including both potency and target selectivity. Importantly, ISMs effectively suppress both self- and cross-seeded IAPP self-assembly. Our results provide a novel class of highly potent peptide leads for targeting protein aggregation in Alzheimer's disease, type 2 diabetes, or both diseases and a chemical approach to inhibit amyloid self-assembly and pathogenic interactions of other proteins as well.

We introduce a labeling scheme for magic angle spinning (MAS) solid-state NMR that is based on deuteration in combination with dilution of the carbon spin system. The labeling strategy achieves spectral editing by simplification of the HαCα and aliphatic side chain spectral region. A reduction in both proton and carbon spin density in combination with fast spinning (≥50 kHz) is essential to retrieve artifact-free (13)C-R1 relaxation data for aliphatic carbons. We obtain good agreement between the NMR experimental data and order parameters extracted from a molecular dynamics (MD) trajectory, which indicates that carbon based relaxation parameters can yield complementary information on protein backbone as well as side chain dynamics.

Co-translational protein folding is not yet well understood despite the availability of high-resolution ribosome crystal structures. We present first solid-state NMR data on non-mobile regions of a prokaryotic ribosomal complex. Localized chemical shift perturbations and line broadening are observed for the backbone amide resonances corresponding to the regions in the trigger factor ribosome-binding domain that are involved in direct contact with the ribosome or undergo conformational changes upon ribosome binding. This large asymmetric protein complex (1.4 MDa) becomes accessible for NMR investigations by the combined use of proton detection and high MAS frequencies (60 kHz). The presented results open new perspectives for the understanding of the mechanism of large molecular machineries.

Bigger is better: Sequential backbone assignments are obtained by NMR spectroscopy for a 1 MDa proteasome complex. The method relies on immobilization of a soluble protein complex by magic-angle spinning. Deuteration and proton detection of exchangeable sites and paramagnetic relaxation enhancement enables exploration of structural and dynamic properties of supramolecular assemblies at atomic resolution.

For the first time, we have demonstrated the site-resolved measurement of reliable (i.e., free of interfering effects) (15)N R(1rho) relaxation rates from a solid protein to extract dynamic information on the microsecond time scale. (15)N R(1rho) NMR relaxation rates were measured as a function of the residue number in a (15)N,(2)H-enriched (with 10-20% back-exchanged protons at labile sites) microcrystalline SH3 domain of chicken alpha-spectrin. The experiments were performed at different temperatures and at different spin-lock frequencies, which were realized by on- and off-resonance spin-lock irradiation. The results obtained indicate that the interfering spin-spin contribution to the R(1rho) rate in a perdeuterated protein is negligible even at low spin-lock fields, in contrast to the case for normal protonated samples. Through correlation plots, the R(1rho) rates were compared with previous data for the same protein characterizing different kinds of internal mobility.

A reliable site-specific estimate of the individual N-H bond lengths in the protein backbone is the fundamental basis of any relaxation experiment in solution and in the solid-state NMR. The N-H bond length can in principle be influenced by hydrogen bonding, which would result in an increased N-H distance. At the same time, dynamics in the backbone induces a reduction of the experimental dipolar coupling due to motional averaging. We present a 3D dipolar recoupling experiment in which the (1)H,(15)N dipolar coupling is reintroduced in the indirect dimension using phase-inverted CP to eliminate effects from rf inhomogeneity. We find no variation of the N-H dipolar coupling as a function of hydrogen bonding. Instead, variations in the (1)H,(15)N dipolar coupling seem to be due to dynamics of the protein backbone. This is supported by the observed correlation between the H(N)-N dipolar coupling and the amide proton chemical shift. The experiment is demonstrated for a perdeuterated sample of the alpha-spectrin SH3 domain. Perdeuteration is a prerequisite to achieve high accuracy. The average error in the analysis of the H-N dipolar couplings is on the order of +/-370 Hz (+/-0.012 A) and can be as small as 150 Hz, corresponding to a variation of the bond length of +/-0.005 A.

In this paper, we seek to compare the internal dynamics of a small globular protein, SH3 domain from alpha-spectrin, in solution and in a crystalline state. The comparison involves side-chain methyl 13C R1 relaxation rates that are highly sensitive to local dynamics in the vicinity of the methyl site. To conduct the relaxation measurements, protein samples have been prepared using specially labeled alpha-ketoisovalerate precursors, resulting in selective incorporation of the 1H-13C spin pair in one or both methyl groups of the valine and leucine side chains. The sparse labeling pattern in an otherwise deuterated sample makes it possible to record high-resolution 13C, 1H solid-state spectra using magic angle spinning experiment with a MAS frequency of 22 kHz. Furthermore, this labeling scheme avoids proton-driven 13C-13C spin-diffusion effects, thus allowing for accurate measurements of 13C R1 relaxation in the individual methyl groups. While the relaxation response from a polycrystalline sample is generally expected to be multiexponential, we demonstrate both theoretically and experimentally that in this particular case the relaxation profiles are, in excellent approximation, monoexponential. In fact, solid-state relaxation data can be interpreted in a model-free fashion, similar to solution data. Direct comparison between the experimentally measured solid and solution rates reveals a strong correlation, r = 0.94. Furthermore, when solution rates are corrected for the effect of the overall molecular tumbling (quantified on the basis of the solution 15N relaxation data), the results are in one-to-one agreement with the solid-state rates. This finding indicates that methyl dynamics in the solution and solid samples are quantitatively similar. More broadly, it suggests that the entire dynamic network, including motions of side chains in the protein hydrophobic core and backbone motions, is similar. This result opens interesting possibilities for combined interpretation of solid- and solution-state relaxation data, potentially leading to a detailed characterization of internal protein dynamics on a wide range of time scales.

Many MAS (magic angle spinning) solid-state NMR investigations of biologically relevant protein samples are hampered by poor resolution, particularly in the 15N chemical shift dimension. We show that dynamics in the nanosecond-microsecond time scale in solid-state samples can induce significant line broadening of 15N resonances in solid-state NMR experiments. Averaging of 15NH(alpha/beta) multiplet components due to 1H decoupling induces effective relaxation of the 15N coherence in case the N-H spin pair undergoes significant motion. High resolution solid-state NMR spectra can then only be recorded by application of TROSY (Transverse Relaxation Optimized Spectroscopy) type techniques which select the narrow component of the multiplet pattern. We speculate that this effect has been the major obstacle to the NMR spectroscopic characterization of many membrane proteins and fibrillar aggregates so far. Only in very favorable cases, where dynamics are either absent or very fast (picosecond), high-resolution spectra were obtained. We expect that this approach which requires intense deuteration will have a significant impact on the quality and the rate at which solid-state NMR spectroscopic investigations will emerge in the future.

Side-chain 2H and backbone 15N relaxation data have been collected at multiple temperatures in the samples of the SH3 domain from alpha-spectrin. Combined analyses of the data allowed for determination of the temperature-dependent correlation times tauf characterizing fast methyl motion. Molecular dynamics simulations confirmed that tauf are dominated by methyl rotation; the corresponding activation energies approximate methyl rotation barriers. For 33 methyl groups in the alpha-spectrin SH3 domain the average barrier height was thus determined to be 2.8 +/- 0.9 kcal/mol. This value is deemed representative of the "fluid" hydrophobic protein core where some barriers are increased and others are lowered because of the contacts with surrounding atoms, but there is no local order that could produce systematically higher (lower) barriers. For comparison, the MD simulation predicts the average barrier of 3.1 kcal/mol (calculated via the potential of mean force) or 3.4-3.5 kcal/mol (rigid barriers after appropriate averaging over multiple MD snapshots). The latter result prompted us to investigate rigid methyl rotation barriers in a series of NMR structures from the Protein Databank. In most cases the barriers proved to be higher than expected, 4-6 kcal/mol. To a certain degree, this is caused by tight packing of the side chains in the NMR structures and stems from the structure calculation procedure where the coordinates are first annealed toward the temperature of 0 K and then subjected to energy minimization. In several cases the barriers10 kcal/mol are indicative of van der Waals violations. The notable exceptions are (i) the structures solved using the GROMOS force field where tight methyl packing is avoided (3.0-3.6 kcal/mol) and (ii) the structure solved by means of the dynamic ensemble refinement method (Lindorff-Larsen, K.; Best, R. B.; DePristo, M. A.; Dobson, C. M.; Vendruscolo, M. Nature 2005, 433, 128) (3.5 kcal/mol). These results demonstrate that methyl rotation barriers, derived from the experiments that are traditionally associated with studies of protein dynamics, can be also used in the context of structural work. This is particularly interesting in view of the recent efforts to incorporate dynamics data in the process of protein structure elucidation.

Biological magic angle spinning (MAS) solid-state nuclear magnetic resonance spectroscopy has developed rapidly over the past two decades. For the structure determination of a protein by solid-state NMR, routinely (13)C,(13)C distance restraints as well as dihedral restraints are employed. In protonated samples, this is achieved by growing the bacterium on a medium which contains [1,3]-(13)C glycerol or [2]-(13)C glycerol to dilute the (13)C spin system. Labeling schemes, which rely on heteronuclei, are insensitive both for detection and in terms of quantification of distances, since they are relying on low-γ nuclei. Proton detection can in principle provide a gain in sensitivity by a factor of 8 and 31, compared to the (13)C or (15)N detected version of the experiment. We report here a new labeling scheme, which enables (1)H-detection of aliphatic resonances with high resolution in MAS solid-state NMR spectroscopy. We prepared microcrystals of the SH3 domain of chicken α-spectrin with 5% protonation at nonexchangeable sites and obtained line widths on the order of 25 Hz for aliphatic (1)H resonances. We show further that (13)C resolved 3D-(1)H,(1)H correlation experiments yield access to long-range proton-proton distances in the protein.

Structural investigations are a prerequisite to understand protein function. Intermediate time scale motional processes (ns-micros) are deleterious for NMR of biological solids and obscure the detection of amide moieties in traditional CP based solid-state NMR approaches as well as in regular scalar coupling based experiments. We show that this obstacle can be overcome by using TROSY type techniques in triple resonance experiments, which enable the assignment of resonances in loop regions of a microcrystalline protein. The presented approach provides an exemplified solution for the analysis of secondary structure elements undergoing slow dynamics that might be particularly crucial for understanding protein function.

A new method is introduced to measure the backbone torsion angles α and ζ in 13C-labeled oligonucleotides. The experiments relies on the quantification of the cross-correlated relaxation of C,P double and zero quantum coherence caused by the C,H dipolar coupling and the P chemical shift anisotropy. Two-dimensional surfaces that reveal the angular dependence of the cross-correlated relaxation rates depend on the backbone angles α and β as well as e and ζ and are interpreted using torsion angle information for the angles β and e from experiments measuring 3J(H,P) and 3J(C,P) coupling constants. The experiments have been carried out on the 10mer RNA 5‘-CGCUUUUGCG-3‘ that forms a hairpin and in which the four uridine residues are 13C-labeled in the ribofuranoside moiety.

The structure of a catalytic intermediate with important implications for the interpretation of the stereochemical outcome of the palladium complex catalyzed allylic substitution with phosphino–oxazoline (PHOX) ligands is determined by liquid state NMR. The complex displays a novel structure that is highly distorted compared with other palladium η2-olefin complexes known so far. The structure has been determined from nuclear overhauser data (NOE), scalar coupling constants, and long range projection angle restraints derived from dipole dipole cross-correlated relaxation of multiple quantum coherence. The latter restraints have been implemented into a distance geometry protocol. The projection angle restraints yield a higher precision in the determination of the relative orientation of the two molecular moieties and are essential to provide an exact structural definition of the olefinic part of the catalytic intermediate with respect to the ligand.

In vitro protein-folding studies using chemical denaturants such as urea are indispensible in elucidating the forces and mechanisms determining the stability, structure, and dynamics of water-soluble proteins. By contrast, α-helical membrane-associated proteins largely evade such approaches because they are resilient to extensive unfolding. We have used optical and NMR spectroscopy to provide an atomistic-level dissection of the effects of urea on the structure and dynamics of the α-helical membrane-associated protein Mistic as well as its interactions with detergent and solvent molecules. In the presence of the zwitterionic detergent lauryl dimethylamine oxide, increasing concentrations of urea result in a complex sequence of conformational changes that go beyond simple two-state unfolding. Exploiting this finding, we report the first high-resolution structural models of the urea denaturation process of an α-helical membrane-associated protein and its completely unfolded state, which contains almost no regular secondary structure but nevertheless retains a topology close to that of the folded state.

When applied to biomolecules, solid-state NMR suffers from low sensitivity and resolution. The major obstacle to applying proton detection in the solid state is the proton dipolar network, and deuteration can help avoid this problem. In the past, researchers had primarily focused on the investigation of exchangeable protons in these systems. In this Account, we review NMR spectroscopic strategies that allow researchers to observe aliphatic non-exchangeable proton resonances in proteins with high sensitivity and resolution. Our labeling scheme is based on u-[(2)H,(13)C]-glucose and 5-25% H2O (95-75% D2O) in the M9 bacterial growth medium, known as RAP (reduced adjoining protonation). We highlight spectroscopic approaches for obtaining resonance assignments, a prerequisite for any study of structure and dynamics of a protein by NMR spectroscopy. Because of the dilution of the proton spin system in the solid state, solution-state NMR (1)HCC(1)H type strategies cannot easily be transferred to these experiments. Instead, we needed to pursue ((1)H)CC(1)H, CC(1)H, (1)HCC or ((2)H)CC(1)H type experiments. In protonated samples, we obtained distance restraints for structure calculations from samples grown in bacteria in media containing [1,3]-(13)C-glycerol, [2]-(13)C-glycerol, or selectively enriched glucose to dilute the (13)C spin system. In RAP-labeled samples, we obtained a similar dilution effect by randomly introducing protons into an otherwise deuterated matrix. This isotopic labeling scheme allows us to measure the long-range contacts among aliphatic protons, which can then serve as restraints for the three-dimensional structure calculation of a protein. Due to the high gyromagnetic ratio of protons, longer range contacts are more easily accessible for these nuclei than for carbon nuclei in homologous experiments. Finally, the RAP labeling scheme allows access to dynamic parameters, such as longitudinal relaxation times T1, and order parameters S(2) for backbone and side chain carbon resonances. We expect that these measurements will open up new opportunities to obtain a more detailed description of protein backbone and side chain dynamics.

Magic-angle spinning (MAS) solid-state NMR becomes an increasingly important tool for the determination of structures of membrane proteins and amyloid fibrils. Extensive deuteration of the protein allows multidimensional experiments with exceptionally high sensitivity and resolution to be obtained. Here we present an experimental strategy to measure highly unambiguous spatial correlations for distances up to 13 Å. Two complementary three-dimensional experiments, or alternatively a four-dimensional experiment, yield highly unambiguous cross-peak assignments, which rely on four encoded chemical shift dimensions. Correlations to residual aliphatic protons are accessible via synchronous evolution of the (15)N and (13)C chemical shifts, which encode valuable amide-methyl distance restraints. On average, we obtain six restraints per residue. Importantly, 50% of all restraints correspond to long-range distances between residues i and j with |i - j|5, which are of particular importance in structure calculations. Using ARIA, we calculate a high-resolution structure for the microcrystalline 7.2 kDa α-spectrin SH3 domain with a backbone precision of ∼1.1 Å.

Structural characterization of insoluble proteins often relies on solid-state NMR spectroscopy. Perdeuteration and partial back-substitution of exchangeable protons, as proposed for crystalline model proteins, is now shown to lead to beneficial proton spectra for heterogeneous systems, such as fibrils formed by the Alzheimer's disease β-amyloid peptide Aβ40, the lipid reconstituted β-barrel membrane protein OmpG, and the α-helical membrane protein bacteriorhodopsin.

Heteronuclear correlation experiments employing perdeuterated proteins enable the observation of all hydroxyl protons in a microcrystalline protein by MAS solid-state NMR. Dipolar-based sequences allow magnetization transfers that are >50 times faster compared to scalar-coupling-based sequences, which significantly facilitates their assignment. Hydroxyl exchange rates were measured using EXSY-type experiments. We find a biexponential decay behavior for those hydroxyl groups that are involved in side chain-side chain C-O-H...O horizontal lineC hydrogen bonds. The quantification of the distances between the hydroxyl proton and the carbon atoms in the hydrogen-bonding donor as well as acceptor group is achieved via a REDOR experiment. In combination with X-ray data and isotropic proton chemical shifts, availability of (1)H,(13)C distance information can aid in the quantitative description of the geometry of these hydrogen bonds. Similarly, correlations between backbone amide proton and carbonyl atoms are observed, which will be useful in the analysis of the registry of beta-strand arrangement in amyloid fibrils.

We show that large protein complexes can be investigated in solution using magic-angle-spinning (MAS) NMR spectroscopy without the need for sample crystallization or precipitation. In order to efficiently average anisotropic interactions with MAS, the rotational diffusion of the molecule has to be suppressed. This can be readily achieved by lowering the sample temperature and by adding glycerol to the protein solution. The approach is demonstrated using the human small heat shock protein (sHSP) alphaB-Crystallin, which forms oligomeric assemblies of approximately 600 kDa. We suggest this scheme as an approach for overcoming size limitations imposed by overall tumbling in solution-state NMR investigations of large protein complexes.

We present a site-resolved study of slow (ms to s) motions in a protein in the solid (microcrystalline) state performed with the use of a modified version of the centerband-only detection of exchange (CODEX) NMR experiment. CODEX was originally based on measuring changes in molecular orientation by means of the chemical shift anisotropy (CSA) tensor, and in our modification, angular reorientations of internuclear vectors are observed. The experiment was applied to the study of slow (15)N-(1)H motions of the SH3 domain of chicken alpha-spectrin. The protein was perdeuterated with partial back-exchange of protons at labile sites. This allowed indirect (proton) detection of (15)N nuclei and thus a significant enhancement of sensitivity. The diluted proton system also made negligible proton-driven spin diffusion between (15)N nuclei, which interferes with the molecular exchange (motion) and hampers the acquisition of dynamic parameters. The experiment has shown that approximately half of the peaks in the 2D (15)N-(1)H correlation spectrum exhibit exchange in a different extent. The correlation time of the slow motion for most peaks is 1 to 3 s. This is the first NMR study of the internal dynamics of proteins in the solid state on the millisecond to second time scale with site-specific spectral resolution that provides both time-scale and geometry information about molecular motions.

We show in this manuscript that (2)H,(13)C correlation spectra in uniformly (2)H,(13)C isotopically enriched peptides and proteins can be recorded in MAS solid-state NMR with site specific resolution. A resolved deuterium dimension is obtained by evolving (2)H double-quantum coherences. Experimental (2)H line widths are obtained that are as small as 16 Hz (0.17 ppm at 600 MHz) in the double-quantum dimension. The unprecedented resolution in the deuterium dimension obtained for proteins opens new perspectives for correlation experiments and, in particular, for the characterization of dynamics of proteins in the solid-state.

Dynamic parameters become more and more accessible in the study of uniformly isotopically enriched proteins by MAS solid-state NMR. We demonstrate that T(2)-related relaxation properties can quantitatively be determined in a sample of a perdeuterated microcrystalline protein by the measurement of (15)N,(1)H dipole, (15)N CSA cross-correlated relaxation rates. We find that the measured cross-correlated relaxation rates are independent of the MAS rotation frequency, and therefore reflect local dynamic fluctuations of the protein structure.

It is well-known that structures of globular proteins in liquid and in crystalline solid are essentially identical. Many lines of evidence suggest that internal dynamics are also similar (assuming ...

The conformation of the cyclic pentapeptide cyclo(-D-Pro-Ala(4)-) in solution and in the solid state was reinvestigated using modern NMR techniques. To allow unequivocal characterization of hydrogen bonds, relaxation behavior, and intramolecular distances, differently labeled isotopomers were synthesized. The NMR results, supported by extensive MD simulations, demonstrate unambiguously that the preferred conformation previously described by us, but recently questioned, is indeed correct. The validation of the conformational preferences of this cyclic peptide is important given that this system is a template for several bioactive compounds and for controlled "spatial screening" for the search of bioactive conformations.

MAS solid-state NMR experiments applied to biological solids are still hampered by low sensitivity and resolution. In this work, we employ a deuteration scheme in which individual methyl groups are selectively protonated. This labeling scheme allows the acquisition of proton carbon correlation spectra with a resolution comparable to that in solution-state NMR experiments. We observe an increase in resolution by a factor of 10−15 compared to standard heteronuclear correlation experiments using PMLG for 1H,1H dipolar decoupling in the indirect dimension. At the same time, the full sensitivity of the proton-based experiment is retained. In comparison to the heteronuclear detected version of the experiment, a gain in sensitivity of a factor of approximately 4.7 is achieved.

We show in this communication that dynamic information for uniformly 2H,13C,15N isotopically enriched, crystalline proteins can be obtained by MAS solid-state NMR spectroscopy. The experiments make use of the deuterium quadrupolar tensor, which is the dominant interaction mechanism. Dynamic properties are accessed by measurement of the size of the quadrupolar coupling constant, Cq, and the value of the asymmetry parameter, eta, via evolution of the deuterium chemical shift, as well as by measurement of deuterium T1 relaxation times. Three-dimensional experiments are performed in order to obtain site-specific resolution. Due to proton dilution, no proton decoupling is required in the carbon evolution periods at MAS rotation frequencies of 10 kHz.

We demonstrate constraint of peptide backbone and side-chain conformation with 3D (1)H-(15)N-(13)C-(1)H dipolar chemical shift, magic-angle spinning NMR experiments. In these experiments, polarization is transferred from (15)N[i] by ramped SPECIFIC cross polarization to the (13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1] resonances and evolves coherently under the correlated (1)H-(15)N and (1)H-(13)C dipolar couplings. The resulting set of frequency-labeled (15)N(1)H-(13)C(1)H dipolar spectra depend strongly upon the molecular torsion angles phi[i], chi1[i], and psi[i - 1]. To interpret the data with high precision, we considered the effects of weakly coupled protons and differential relaxation of proton coherences via an average Liouvillian theory formalism for multispin clusters and employed average Hamiltonian theory to describe the transfer of (15)N polarization to three coupled (13)C spins ((13)C(alpha)[i], (13)C(beta)[i], and (13)C(alpha)[i - 1]). Degeneracies in the conformational solution space were minimized by combining data from multiple (15)N(1)H-(13)C(1)H line shapes and analogous data from other 3D (1)H-(13)C(alpha)-(13)C(beta)-(1)H (chi1), (15)N-(13)C(alpha)-(13)C'-(15)N (psi), and (1)H-(15)N[i]-(15)N[i + 1]-(1)H (phi, psi) experiments. The method is demonstrated here with studies of the uniformly (13)C,(15)N-labeled solid tripeptide N-formyl-Met-Leu-Phe-OH, where the combined data constrains a total of eight torsion angles (three phi, three chi1, and two psi): phi(Met) = -146 degrees, psi(Met) = 159 degrees, chi1(Met) = -85 degrees, phi(Leu) = -90 degrees, psi(Leu) = -40 degrees, chi1(Leu) = -59 degrees, phi(Phe) = -166 degrees, and chi1(Phe) = 56 degrees. The high sensitivity and dynamic range of the 3D experiments and the data analysis methods provided here will permit immediate application to larger peptides and proteins when sufficient resolution is available in the (15)N-(13)C chemical shift correlation spectra.

In this communication, we demonstrate the feasibility of 1H detection in MAS solid-state NMR for a microcrystalline, uniformly 2H,15N-labeled sample of a SH3 domain of chicken alpha-spectrin, using pulsed field gradients for suppression of water magnetization. Today, B0 gradients are employed routinely in solution-state NMR for coherence order selection and solvent suppression. We suggest to use gradients to purge water magnetization which cannot be suppressed using conventional water suppression schemes. The achievable gain in sensitivity for 1H detection is in the order of 5 compared to the 15N detected version of the experiment (at a MAS rotation frequency of 13.5 kHz). We expect that this labeling concept which achieves high sensitivity due to 1H detection, in combination with the possibility to measure long range 1H-1H distances as we have shown previously, to be a useful tool for the determination of protein structures in the solid state.

Membrane proteins in their native cellular membranes are accessible by dynamic nuclear polarization magic angle spinning solid-state NMR spectroscopy without the need of purification and reconstitution (see picture). Dynamic nuclear polarization is essential to achieve the required gain in sensitivity to observe the membrane protein of interest.