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Proteins fold into three-dimensional structures, which determine their diverse functions. The conformation of the backbone of each structure is locally at each C(α) effectively described by conformational angles resulting in Ramachandran plots. These, however, do not describe the conformations around hydrogen bonds, which can be non-local along the backbone and are of major importance for protein structure. Here, we introduce the spatial rotation between hydrogen bonded peptide planes as a new descriptor for protein structure locally around a hydrogen bond. Strikingly, this rotational descriptor sampled over high-quality structures from the protein data base (PDB) concentrates into 30 localized clusters, some of which correlate to the common secondary structures and others to more special motifs, yet generally providing a unifying systematic classification of local structure around protein hydrogen bonds. It further provides a uniform vocabulary for comparison of protein structure near hydrogen bonds even between bonds in different proteins without alignment.

Application of sets of (13)C-(13)C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important (13)C-(13)C distances in uniformly (13)C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl ((13)C') and aliphatic ((13)C(aliphatic)) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys. 130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly (13)C,(15)N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of (13)C'-(13)C(aliphatic) distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform (13)C,(15)N-labeling on the FGAIL fragment.

The process of resonance assignment represents a time-consuming and potentially error-prone bottleneck in structural studies of proteins by solid-state NMR (ssNMR). Software for the automation of this process is therefore of high interest. Procedures developed through the last decades for solution-state NMR are not directly applicable for ssNMR due to the inherently lower data quality caused by lower sensitivity and broader lines, leading to overlap between peaks. Recently, the first efforts towards procedures specifically aimed for ssNMR have been realized (Schmidt et al. in J Biomol NMR 56(3):243-254, 2013). Here we present a robust automatic method, which can accurately assign protein resonances using peak lists from a small set of simple 2D and 3D ssNMR experiments, applicable in cases with low sensitivity. The method is demonstrated on three uniformly (13)C, (15)N labeled biomolecules with different challenges on the assignments. In particular, for the immunoglobulin binding domain B1 of streptococcal protein G automatic assignment shows 100% accuracy for the backbone resonances and 91.8% when including all side chain carbons. It is demonstrated, by using a procedure for generating artificial spectra with increasing line widths, that our method, GAMES_ASSIGN can handle a significant amount of overlapping peaks in the assignment. The impact of including different ssNMR experiments is evaluated as well.

We present a novel sampling strategy, interleaving acquisition of multiple NMR spectra by exploiting initial polarization subsequently from (1)H and (2)H spins, taking advantage of their different T(1) relaxation times. Different (1)H- and (2)H-polarization based spectra are in this way simultaneously recorded improving either information content or sensitivity by adding spectra. The so-called Relaxation-optimized Acquisition of Proton Interleaved with Deuterium (RAPID) (1)H→(13)C/(2)H→(13)C CP/MAS multiple-acquisition method is demonstrated by 1D and 2D experiments using a uniformly (2)H, (15)N,(13)C-labeled α-spectrin SH3 domain sample with all or 30% back-exchanged labile (2)H to (1)H. It is demonstrated how 1D (13)C CP/MAS or 2D (13)C-(13)C correlation spectra initialized with polarization from either (1)H or (2)H may be recorded simultaneously with flexibility to be added or used individually for spectral editing. It is also shown how 2D (13)C-(13)C correlation spectra may be recorded interleaved with (2)H-(13)C correlation spectra to obtain (13)C-(13)C correlations along with information about dynamics from (2)H sideband patterns., (Copyright © 2011 Elsevier Inc. All rights reserved.)

The exquisite sensitivity of chemical shifts as reporters of structural information, and the ability to measure them routinely and accurately, gives great import to formulations that elucidate the structure-chemical-shift relationship. Here we present a new and highly accurate, precise, and robust formulation for the prediction of NMR chemical shifts from protein structures. Our approach, shAIC (shift prediction guided by Akaikes Information Criterion), capitalizes on mathematical ideas and an information-theoretic principle, to represent the functional form of the relationship between structure and chemical shift as a parsimonious sum of smooth analytical potentials which optimally takes into account short-, medium-, and long-range parameters in a nuclei-specific manner to capture potential chemical shift perturbations caused by distant nuclei. shAIC outperforms the state-of-the-art methods that use analytical formulations. Moreover, for structures derived by NMR or structures with novel folds, shAIC delivers better overall results; even when it is compared to sophisticated machine learning approaches. shAIC provides for a computationally lightweight implementation that is unimpeded by molecular size, making it an ideal for use as a force field., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We present design of novel low-power homonuclear dipolar recoupling experiments for magic-angle-spinning solid-state NMR studies of proteins. The pulse sequences are developed by combining principles of symmetry-based dipolar recoupling and optimal control-based pulse sequence design. The scaffold of the pulse sequences is formed by known CN-type recoupling sequences, while the intrinsic sequence elements are designed using optimal control. This procedure allows for the development of high-performance pulse sequences demanding significantly weaker rf fields than previous symmetry-based pulse sequences while compensating for rf inhomogeneity and providing excitation over relevant ranges of chemical shifts for biological applications. The new recoupling experiments, referred to as optimal control CN ((OC)CN), are demonstrated numerically and experimentally by two-dimensional (2D) (13)C-(13)C and three-dimensional (3D) (15)N-(13)C-(13)C chemical shift correlation experiments on uniformly (13)C, (15)N-labeled ubiquitin. Exploiting the double-quantum, band-selective dipolar recoupling properties of the (OC)CN experiments, we demonstrate significant sensitivity enhancement for 2D and 3D correlation spectra showing exclusively one- or two-bond correlations.

Dipolar truncation prevents accurate measurement of long-range internuclear distances between nuclei of the same spin species, e.g., within (13)C-(13)C spin pairs in uniformly (13)C-isotope-labeled proteins, using magic-angle spinning solid-state NMR spectroscopy. Accordingly, one of the richest sources of accurate structure information is at present not exploited fully, leaving the bulk part of the experimentally derived structural constraints to less accurate long-range (13)C-(13)C dipolar couplings estimated from methods based on spin diffusion through proton spins in the close environment. In this paper, we extend our previous triple-oscillating field technique [N. Khaneja and N. C. Nielsen, J. Chem. Phys. 128, 015103 (2008)] for dipolar recoupling without dipolar truncation in homonuclear spin systems to a more advanced rf modulation with four independent oscillations and rotations involving nonorthogonal axes. This provides important new degrees of freedom, which are used to improve the scaling factor of the recoupled dipole-dipole couplings by a factor of 2.5 relative to the triple-oscillating field approach. This significant improvement, obtained by refocusing of otherwise defocused parts of the residual dipolar coupling Hamiltonian, may be exploited to measure much weaker (13)C-(13)C dipolar couplings (and thereby longer distances) with much higher accuracy. We present a detailed theoretical description of multiple-field oscillating recoupling experiments, along with numerical simulations and experimental results on U-(13)C, (15)N-L-threonine and U-(13)C,(15)N-ubiquitin.

Application of sets of 13C-13C internuclear distance restraints constitutes a typical key element in determining the structure of peptides and proteins by magic-angle-spinning solid-state NMR spectroscopy. Accurate measurements of the structurally highly important 13C-13C distances in uniformly 13C-labeled peptides and proteins, however, pose a big challenge due to the problem of dipolar truncation. Here, we present novel two-dimensional (2D) solid-state NMR experiments capable of extracting distances between carbonyl (13C′) and aliphatic (13Caliphatic) spins with high accuracy. The method is based on an improved version of the four-oscillating field (FOLD) technique [L. A. Straasø, M. Bjerring, N. Khaneja, and N. C. Nielsen, J. Chem. Phys.130, 225103 (2009)] which circumvents the problem of dipolar truncation, thereby offering a base for accurate extraction of internuclear distances in many-spin systems. The ability to extract reliable accurate distances is demonstrated using one- and two-dimensional variants of the FOLD experiment on uniformly 13C,15N-labeled-L-isoleucine. In a more challenging biological application, FOLD 2D experiments are used to determine a large number of 13C′-13Caliphatic distances in amyloid fibrils formed by the SNNFGAILSS fibrillating core of the human islet amyloid polypeptide with uniform 13C,15N-labeling on the FGAIL fragment. [ABSTRACT FROM AUTHOR]

1H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using 2H excitation instead of 1H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, 'quadruple-resonance NMR spectroscopy', is presented which relies on an efficient 2H-excitation and 2H-13C cross-polarization (CP) step, combined with 1H detection. We show that by using 2H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties. [ABSTRACT FROM AUTHOR]

Abstract: The structure of the chlorosome baseplate protein CsmA from Chlorobium tepidum in a 1:1 chloroform:methanol solution was determined using liquid-state NMR spectroscopy. The data reveal that the 59-residue protein is predominantly α-helical with a long helical domain extending from residues V6 to L36, containing a putative bacteriochlorophyll a binding domain, and a short helix in the C-terminal part extending from residues M41 to G49. These elements are compatible with a model of CsmA having the long N-terminal α-helical stretch immersed into the lipid monolayer confining the chlorosome and the short C-terminal helix protruding outwards, thus available for interaction with the Fenna–Matthews–Olson antenna protein. [Copyright &y& Elsevier]

Based on exact numerical simulations, taking into account isotropic and conformation-dependent anisotropic nuclear spin interactions, we systematically analyse the prospects for high-resolution solid-state NMR on large isotope-labeled membrane proteins macroscopically oriented in phospholipid bilayers. Using the known X-ray structures of rhodopsin and porin as models for large membrane proteins with typical α-helical and β-barrel structural motifs, the analysis considers all possible one- to six-dimensional spectra comprised of frequency dimensions with evolution under any combination of amide 1H, amide 15N, and carbonyl 13C chemical shifts as well as 1H-15N dipole-dipole couplings. Under consideration of typical nuclear spin interaction and experimental line-shape parameters, the analysis provides new insight into the resolution capability and orientation-dependent transfer efficiency of existing experiments as well as guidelines as to improved experimental approaches for the study of large uniformly 15N- and [13C,15N]-labeled membrane proteins. On basis of these results and numerical optimizations of coherence-transfer efficiencies, we propose several new high-resolution experiments for sequential protein backbone assignment and structure determination. [ABSTRACT FROM AUTHOR]

Low-field NMR sensor technology is proposed for accurate and operationally simple on-farm or laboratory determination of protein and phosphorus constituents in livestock feed. The total phosphorus content is determined directly on the native sample, while quantification of digestible protein involves enzymatic digestion here adapted to provide the total protein content. Comparison with traditional laboratory reference analysis for the total protein and phosphorus contents of 31 feed samples (including various grains, feed mixtures, and commercial feed products for cattle, pigs, horses, poultry, and sheep) demonstrate the feasibility and good accuracy of the NMR measurements. Table of contents entry – for TOC only. [Display omitted] • Mobile, multinuclear, low-field NMR for on-farm measurements. • Total/digestible protein and total phosphorus contents. • Direct analysis of bulk sample (raw material for phosphorus, digested for protein). • Feasibility and accuracy demonstrated on feed for a wide range of animals. [ABSTRACT FROM AUTHOR]

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D9k (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca2+, the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca2+-loaded calbindin D9k does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca2+ is bound to the protein. A reduction in pH in the absence of Ca2+ increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca2+ affinity causes a transition to the micelle-associated state also in the presence of Ca2+ when the pH is lowered. Site-specific analysis of the data indicates that calbindin D9k has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca2+-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D9k in the cell is discussed, along with the broader implications for the function of the S100 protein family. [ABSTRACT FROM AUTHOR]

Highlights: [•] Heteronuclear polarization transfer by adiabatic RESPIRATIONCP. [•] Increased transfer efficiency and robustness towards rf inhomogeneity. [•] Demonstrated improved performance for 13C→ 15N polarization transfer in proteins. [ABSTRACT FROM AUTHOR]

Mutations in the transforming growth factor β-induced protein (TGFBIp) are linked to the development of corneal dystrophies in which abnormal protein deposition in the cornea leads to a loss of corneal transparency and ultimately blindness. Different mutations give rise to phenotypically distinct corneal dystrophies. Most mutations are located in the fourth fasciclin-1 domain (FAS1-4). The amino acid substitution A546T in the FAS1-4 domain is linked to the development of lattice corneal dystrophy with amyloid deposits in the superficial and deep stroma, classifying it as an amyloid disease. Here we provide a detailed description of the fibrillation of the isolated FAS1-4 domain carrying the A546T substitution. The A546T substitution leads to a significant destabilization of FAS1-4 and induces a partially folded structure with increased surface exposure of hydrophobic patches. The mutation also leads to two distinct fibril morphologies. Long straight fibrils composed of pure β-sheet structure are formed at lower concentrations, whereas short and curly fibrils containing a mixture of α-helical and β-sheet structures are formed at higher concentrations. The formation of short and curly fibrils is preceded by the formation of a small number of oligomeric species with high membrane permeabilization potential and rapid fibril formation. The long straight fibrils are formed more slowly and through progressively bigger oligomers that lose their membrane permeabilization potential as fibrillation proceeds beyond the lag phase. These different fibril classes and associated biochemical differences may lead to different clinical symptoms associated with the mutation. [ABSTRACT FROM AUTHOR]

Abstract: We present a novel sampling strategy, interleaving acquisition of multiple NMR spectra by exploiting initial polarization subsequently from 1H and 2H spins, taking advantage of their different T 1 relaxation times. Different 1H- and 2H-polarization based spectra are in this way simultaneously recorded improving either information content or sensitivity by adding spectra. The so-called Relaxation-optimized Acquisition of Proton Interleaved with Deuterium (RAPID) 1H→ 13C/2H→ 13C CP/MAS multiple-acquisition method is demonstrated by 1D and 2D experiments using a uniformly 2H, 15N,13C-labeled α-spectrin SH3 domain sample with all or 30% back-exchanged labile 2H to 1H. It is demonstrated how 1D 13C CP/MAS or 2D 13C–13C correlation spectra initialized with polarization from either 1H or 2H may be recorded simultaneously with flexibility to be added or used individually for spectral editing. It is also shown how 2D 13C–13C correlation spectra may be recorded interleaved with 2H–13C correlation spectra to obtain 13C–13C correlations along with information about dynamics from 2H sideband patterns. [Copyright &y& Elsevier]

Abstract: Heteronuclear dipolar recoupling experiments without 1H decoupling based on simple cross polarization are introduced for applications in biological solid-state NMR. It is shown that standard or adiabatic variants of the cross-polarization experiment with irradiation on the low-γ (e.g., 13C,15N) spins even at modest spinning frequencies enable efficient band-selective or broadband dipolar recoupling without the need for intense 1H decoupling. This facilitates experiments on expensive isotope-labelled protein samples for which sample heating by intense 1H decoupling may lead to sample detoriation. The principle is demonstrated numerically and experimentally on uniformly 13C,15N-labelled samples of GB1 and fibrils of hIAPP from the human islet amyloid labelled on the FGAIL part. [Copyright &y& Elsevier]

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

Abstract: We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of 13C,15N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically 15N to 13C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly 13C,15N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical 15N–13C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D 15N→ 13C coherence transfer experiments, as well as 2D and 3D 15N,13C and 15N,13C,13C chemical shift correlation experiments on uniformly 13C,15N-labeled ubiquitin. [Copyright &y& Elsevier]