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The transforming growth factor beta induced protein (TGFBIp) is a major protein component of the human cornea. Mutations occurring in TGFBIp may cause corneal dystrophies, which ultimately lead to loss of vision. The majority of the disease-causing mutations are located in the C-terminal domain of TGFBIp, referred as the fourth fascilin-1 (FAS1-4) domain. In the present study the FAS1-4 Ala546Thr, a mutation that causes lattice corneal dystrophy, was investigated in dimethylsulfoxide using liquid-state NMR spectroscopy, to enable H/D exchange strategies for identification of the core formed in mature fibrils. Isotope-labeled fibrillated FAS1-4 A546T was dissolved in a ternary mixture 95/4/1 v/v/v% dimethylsulfoxide/water/trifluoroacetic acid, to obtain and assign a reference 2D (1)H-(15)N HSQC spectrum for the H/D exchange analysis. Here, we report the near-complete assignments of backbone and aliphatic side chain (1)H, (13)C and (15)N resonances for unfolded FAS1-4 A546T at 25 °C.

Co-solute paramagnetic relaxation enhancement (PRE) is an attractive way to speed up data acquisition in NMR spectroscopy by shortening the T 1 relaxation time of the nucleus of interest and thus the necessary recycle delay. Here, we present the rationale to utilize high-spin iron(III) as the optimal transition metal for this purpose and characterize the properties of its neutral chelate form Fe(DO3A) as a suitable PRE agent. Fe(DO3A) effectively reduces the T 1 values across the entire sequence of the intrinsically disordered protein α-synuclein with negligible impact on line width. The agent is better suited than currently used alternatives, shows no specific interaction with the polypeptide chain and, due to its high relaxivity, is effective at low concentrations and in 'proton-less' NMR experiments. By using Fe(DO3A) we were able to complete the backbone resonance assignment of a highly fibrillogenic peptide from α1-antitrypsin by acquiring the necessary suite of multidimensional NMR datasets in 3 h.

NMR spectroscopy is a widely used technique for characterizing the structure and dynamics of macromolecules. Often large amounts of NMR data are required to characterize the structure of proteins. To save valuable time and resources on data acquisition, simulated data is useful in the developmental phase, for data analysis, and for comparison with experimental data. However, existing tools for this purpose can be difficult to use, are sometimes specialized for certain types of molecules or spectra, or produce too idealized data. Here we present a fast, flexible and robust tool, VirtualSpectrum, for generating peak lists for most multi-dimensional NMR experiments for both liquid and solid state NMR. It is possible to tune the quality of the generated peak lists to include sources of artifacts from peak overlap, noise and missing signals. VirtualSpectrum uses an analytic expression to represent the spectrum and derive the peak positions, seamlessly handling overlap between signals. We demonstrate our tool by comparing simulated and experimental spectra for different multi-dimensional NMR spectra and analyzing systematically three cases where overlap between peaks is particularly relevant; solid state NMR data, liquid state NMR homonuclear (1)H and (15)N-edited spectra, and 2D/3D heteronuclear correlation spectra of unstructured proteins. We analyze the impact of protein size and secondary structure on peak overlap and on the accuracy of structure determination based on data of different qualities simulated by VirtualSpectrum.

We present here a simple refocused modification, r TPPM, of the Two-Pulse Phase-Modulation (TPPM) heteronuclear decoupling method, which improves decoupling and makes the sequence much more robust with respect to essential experimental parameters. The modified sequence is compared with the established TPPM sequence and a variety of other decoupling sequences at low to moderate magic-angle spinning frequencies. Simulations are shown to compare TPPM and r TPPM with respect to various experimental parameters. The observations from simulations are corroborated with experimental findings at two spinning frequencies on U-(13)C-glycine and U-(13)C-L-histidine.HCl.H2O., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

(1)H-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 (2)H excitation instead of (1)H 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 (2)H-excitation and (2)H-(13)C cross-polarization (CP) step, combined with (1)H detection. We show that by using (2)H-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., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

We present a scheme that allows the simultaneous detection of PAR and PAIN correlation spectra in a single two-dimensional experiment. For both spectra, we obtain almost the same signal-to-noise ratio as if a PAR or PAIN spectrum is recorded separately, which in turn implies that one of the spectra may be considered additional information for free. The experiment is based on the observation that in a PAIN experiment, the PAR condition is always also fulfilled. The performance is demonstrated experimentally using uniformly (13)C,(15)N-labeled samples of N-f-MLF-OH and ubiquitin.

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.)

A new solid-state NMR method, the double-oscillating field technique (DUO), that under magic-angle-spinning conditions produces an effective Hamiltonian proportional to the native high-field homonuclear dipole-dipole coupling operator is presented. The method exploits one part of the radio frequency (rf) field to recouple the dipolar coupling interaction with a relatively high scaling factor and to eliminate offset effects over a reasonable bandwidth while in the recoupling frame, the other part gives rise to a sufficiently large longitudinal component of the residual rf field that averages nonsecular terms and in addition ensures stability toward rf inhomogeneity and rf miscalibration. The capability of the DUO experiment to mediate transfer of polarization is described theoretically and compared numerically and experimentally with finite pulse rf driven recoupling and experimentally with dipolar-assisted rotational resonance. Two-dimensional recoupling experiments were performed on antiparallel amyloid fibrils of the decapeptide SNNFGAILSS with the FGAIL fragment uniformly labeled with (13)C and (15)N.

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.

The fibril structure formed by the amyloidogenic fragment SNNFGAILSS of the human islet amyloid polypeptide (hIAPP) is determined with 0.52 A resolution. Symmetry information contained in the easily obtainable resonance assignments from solid-state NMR spectra (see picture), along with long-range constraints, can be applied to uniquely identify the supramolecular organization of fibrils.

A new experiment, clean HMBC, is introduced for suppression of strong-coupling induced artifacts in HMBC spectra. The culprits of these artifacts are an inherent shortcoming of low-pass J filters in the presence of strong coupling and the (1)H pi pulse in the middle of the evolution period aimed at suppressing evolution under heteronuclear J couplings and (1)H chemical shifts. A pi pulse causes coherence transfer in strongly coupled spin systems and, as is well known in e.g., homonuclear J spectra, this leads to peaks that would not be there in the absence of strong coupling. Similar artifacts occur in HMBC spectra, but they have apparently been overlooked, presumably because they have been assigned to inefficiency of low-pass J filters or not noticed because of a coarse digital resolution in the spectra. Clean HMBC is the HMBC technique of choice for molecules notorious for strong coupling among protons, such as carbohydrates, and the new technique is demonstrated on D-mannose. Finally, a fundamental difference between HMBC and H2BC explains why strong-coupling artifacts are much less of a problem in the latter type of spectra.

We demonstrate that a significant improvement in the spectral resolution may be achieved in solid-state NMR experiments of proteins in inhomogeneously disordered oriented lipid bilayers. Using 1H homonuclear decoupling instead of standard 1H heteronuclear decoupling, the 15N line widths may be reduced by up to seven times for such samples. For large oriented membrane proteins, such resolution enhancements may be crucial for assignment and structural interpretation.

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-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 (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D(9k) (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca(2+), the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca(2+)-loaded calbindin D(9k) 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 Ca(2+) is bound to the protein. A reduction in pH in the absence of Ca(2+) increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca(2+) affinity causes a transition to the micelle-associated state also in the presence of Ca(2+) when the pH is lowered. Site-specific analysis of the data indicates that calbindin D(9k) 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 Ca(2+)-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D(9k) in the cell is discussed, along with the broader implications for the function of the S100 protein family.

We present the first solid-state NMR experiments developed using optimal control theory. Taking heteronuclear dipolar recoupling in magic-angle-spinning NMR as an example, it proves possible to significantly improve the efficiency of the experiments while introducing robustness toward instrumental imperfections such as radio frequency inhomogeneity. The improvements are demonstrated by numerical simulations as well as practical experiments on a 13Calpha,15N-labeled powder of glycine. The experiments demonstrate a gain of 53% in the efficiency for 15N to 13Calpha coherence transfer relative to the typically double-cross-polarization experiments.

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 alpha-helical and beta-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.

The conformation of the 20-residue antibiotic ionophore alamethicin in macroscopically oriented phospholipid bilayers has been studied using (15)N solid-state nuclear magnetic resonance (NMR) spectroscopy in combination with molecular modeling and molecular dynamics simulations. Differently (15)N-labeled variants of alamethicin and an analog with three of the alpha-amino-isobutyric acid residues replaced by alanines have been investigated to establish experimental structural constraints and determine the orientation of alamethicin in hydrated phospholipid (dimyristoylphosphatidylcholine) bilayers and to investigate the potential for a major kink in the region of the central Pro(14) residue. From the anisotropic (15)N chemical shifts and (1)H-(15)N dipolar couplings determined for alamethicin with (15)N-labeling on the Ala(6), Val(9), and Val(15) residues and incorporated into phospholipid bilayer with a peptide:lipid molar ratio of 1:8, we deduce that alamethicin has a largely linear alpha-helical structure spanning the membrane with the molecular axis tilted by 10-20 degrees relative to the bilayer normal. In particular, we find compatibility with a straight alpha-helix tilted by 17 degrees and a slightly kinked molecular dynamics structure tilted by 11 degrees relative to the bilayer normal. In contrast, the structural constraints derived by solid-state NMR appear not to be compatible with any of several model structures crossing the membrane with vanishing tilt angle or the earlier reported x-ray diffraction structure (Fox and Richards, Nature. 300:325-330, 1982). The solid-state NMR-compatible structures may support the formation of a left-handed and parallel multimeric ion channel.

The secondary structure and membrane-associated conformation of a synthetic peptide corresponding to the putative membrane-binding C-terminal 38 residues of the bovine milk component PP3 was determined using 1H NMR in methanol, CD in methanol and SDS micelles, and 15N solid-state NMR in planar phospholipid bilayers. The solution NMR and CD spectra reveal that the PP3 peptide in methanol and SDS predominantly adopts an alpha-helical conformation extending over its entire length with a potential bend around residue 19. 15N solid-state NMR of two PP3 peptides 15N-labelled at the Gly7 and Ala32 positions, respectively, and dissolved in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol phospholipid bilayers shows that the peptide is associated to the membrane surface with the amphipathic helix axis oriented parallel to the bilayer surface.