Correlating fast and slow chemical shift spinning sideband patterns in solid-state NMR

An experiment is presented that enables the measurement of small chemical shift anisotropy tensors under fast magic-angle spinning (MAS). The two-dimensional spectra obtained give a fast MAS sideband pattern in the directly observed dimension with the spinning sideband intensities equivalent to the chemical shift anisotropy scaled by a factor of N, or equivalently the sample spinning frequency scaled by 1/N, in the indirectly observed dimension. The scaling factor may be arbitrarily varied by changing the number and timings of the rotor synchronized pi-pulses used. Desirable features of the experiment include a fixed length pulse sequence and efficient sampling of the indirectly observed dimension. In addition, neither quadrature detection in the indirect dimension nor storage periods are required, consequently no signal intensity is discarded by the pulse sequence. The experiment is demonstrated using (31)P NMR of sodium phosphate and (13)C NMR of fumaric acid monoethyl ester for which a scaling factor of N=10.2 was employed.     

Progress in correlation spectroscopy at ultra-fast magic-angle spinning: basic building blocks and complex experiments for the study of protein structure and dynamics

Recent progress in multi-dimensional solid-state NMR correlation spectroscopy at high static magnetic fields and ultra-fast magic-angle spinning is discussed. A focus of the review is on applications to protein resonance assignment and structure determination as well as on the characterization of protein dynamics in the solid state. First, the consequences of ultra-fast spinning on sensitivity and sample heating are considered. Recoupling and decoupling techniques at ultra-fast MAS are then presented, as well as more complex experiments assembled from these basic building blocks. Furthermore, we discuss new avenues in biomolecular solid-state NMR spectroscopy that become feasible in the ultra-fast spinning regime, such as sensitivity enhancement based on paramagnetic doping, and the prospect of direct proton detection.     

Practical Methods for Solid-State NMR Distance Measurements on Large Biomolecules: Constant-Time Rotational Resonance

Simple modifications of the rotational resonance experiment substantially reduce the total experimental time needed to measure weak homonuclear dipolar couplings, a critical factor for achieving routine internuclear distance measurements in large biomolecular systems. These modifications also address several problems cited in the literature. Here we introduce a constant-time rotational resonance experiment that eliminates the need for control spectra to correct for effects from variable RF heating, particularly critical for accurate long-distance measurements. This reduces the total number of experiments needed by as much as a factor of 2. Other improvements incorporated include achieving selective inversion with a delay rather than a weak pulse (P. R. Costa et al., J. Am. Chem. Soc. 119, 10487-10493, 1997), which we observe results in the elimination of oscillations in peak intensities for short mixing time points. This reduces the total experiment time in two ways. First, there is no longer a need to average different "zero"-time points (S. O. Smith et al., Biochemistry 33, 6334-6341, 1994) to correct for intensity variations. Second, short-mixing-time lineshape differences observed in large membrane-bound proteins only appear with the weak-pulse inversion and not when using the delay inversion. Consistent lineshapes between short and long mixing times permit the use of a single spectrum for subtraction of natural abundance background signals from all labeled-protein time points. Elimination of these effects improves the accuracy and efficiency of rotational resonance internuclear distance measurements.     

REDOR NMR on a hydrophobic peptide in oriented membranes

A method is presented for the calculation of REDOR dephasing for specifically labeled membrane-spanning peptides in uniformly aligned lipid bilayers under magic angle oriented sample spinning (MAOSS) conditions. Numerical simulations are performed for dephasing of (13)C signal by (15)N when the labels are placed in an alpha-helical peptide at the carbonyl of residue (i) and amide nitrogen of residue (i + 2) to show the dependency of REDOR echo intensity on the peptide tilt angle relative to the membrane normal. The approach was applied to the labeled transmembrane domain of phospholamban ([(15)N-Leu(37), (13)C-Leu(39)]PLBTM) incorporated into dimyristoylphosphatidylcholine bilayers. The dephasing observed for a random membrane dispersion showed that the peptide was alpha-helical in the region including the two labels, and dephasing in oriented membranes showed that the peptide helix was tilted by 25 degrees +/- 7 degrees relative to the bilayer normal. These results agree with those obtained by other spectroscopic methods.     

Sensitivity enhancement using paramagnetic relaxation in MAS solid-state NMR of perdeuterated proteins

Previously, Ishii et al., could show that chelated paramagnetic ions can be employed to significantly decrease the recycle delay of a MAS solid-state NMR experiment [N.P. Wickramasinghe, M. Kotecha, A. Samoson, J. Past, Y. Ishii, Sensitivity enhancement in C-13 solid-state NMR of protein microcrystals by use of paramagnetic metal ions for optimizing H-1 T-1 relaxation, J. Magn. Reson. 184 (2007) 350-356]. Application of the method is limited to very robust samples, for which sample stability is not compromised by RF induced heating. In addition, probe integrity might be perturbed in standard MAS PRE experiments due to the use of very short duty cycles. We show that these deleterious effects can be avoided if perdeuterated proteins are employed that have been re-crystallized from D(2)O:H(2)O=9:1 containing buffer solutions. The experiments are demonstrated using the SH3 domain of chicken alpha-spectrin as a model system. The labeling scheme allows to record proton detected (1)H, (15)N correlation spectra with very high resolution in the absence of heteronuclear dipolar decoupling. Cu-edta as a doping reagent yields a reduction of the recycle delay by up to a factor of 15. In particular, we find that the (1)H T(1) for the bulk H(N) magnetization is reduced from 4.4s to 0.3s if the Cu-edta concentration is increased from 0mM to 250 mM. Possible perturbations like chemical shift changes or line broadening due to the paramagnetic chelate complex are minimal. No degradation of our samples was observed in the course of the experiments.     

Perspectives of solution NMR spectroscopy for structural and functional studies of integral membrane proteins

This article discusses future perspectives of solution NMR spectroscopy to study structures and functions of integral membrane proteins at atomic resolution, based on a review of recent progress in this area. Several selected examples of structure determinations, as well as functional studies of integral membrane proteins are highlighted. We expect NMR spectroscopy to make future key scientific contributions to understanding membrane protein function, in particular for large membrane protein systems with known three-dimensional structure. Such situations can benefit from the fact that functional NMR studies have substantially less limitations by molecular size than a full de novo structure determination. Therefore, the general potential for NMR spectroscopy to solve biologic key questions associated with integral membrane proteins is very promising.     

Atomic refinement with correlated solid-state NMR restraints

The orientation data provided by solid-state NMR can provide a great deal of structural information about membrane proteins. The quality of the information provided is, however, somewhat degraded by sign degeneracies in measurements of the dipolar coupling tensor. This is reflected in the dipolar coupling penalty function used in atomic refinement, which is less capable of properly restraining atoms when dipolar sign degeneracies are present. In this report we generate simulated solid-state NMR data using a variety of procedures, including back-calculation from crystal structures of alpha-helical and beta-sheet membrane proteins. We demonstrate that a large fraction of the dipolar sign degeneracies are resolved if anisotropic dipolar coupling measurements are correlated with anisotropic chemical shift measurements, and that all sign degeneracies can be resolved if three data types are correlated. The advantages of correlating data are demonstrated with atomic refinement of two test membrane proteins. When refinement is performed using correlated dipolar couplings and chemical shifts, perturbed structures converge to conformations with a larger fraction of correct dipolar signs than when data are uncorrelated. In addition, the final structures are closer to the original unperturbed structures when correlated data are used in the refinement. Thus, refinement with correlated data leads to improved atomic structures. The software used to correlate dipolar coupling and chemical shift data and to set up energy functions and their derivatives for refinement, CNS-SS02, is available at our web site.     

Structure determination of aligned systems by solid-state NMR magic angle spinning methods

Single crystal rotational echo double resonance (REDOR) experiments can be used to determine the three-dimensional orientation of heteronuclear bond vectors in an amino acid, as well as the crystal's orientation relative to the rotor fixed frame (RFF). We also demonstrate that for samples uniaxially aligned along the rotor axis, the polar tilt angle of a bond vector relative to the RFF can be measured by use of an analytical expression that describes the REDOR curve for that system. These bond orientations were verified by X-ray indexing of the single crystal sample, and were shown to be as accurate as +/- 1 degrees .     

Solid-state variable-temperature H MAS NMR studies on deuterated polyethylene

Solid-state varible-temperature/magic angle spinning(VT/MAS) ¹H NMR measurements were carried out on deuterated polyethylene. From these experimental results it was found that the ¹H chemical shift induced by conformational and morphological changes of the polyethylene sample is within the linewidth of ≈ 0.5 ppm. Furthermore, from MAS/dipolar decoupling experiments it was found that the resonance frequency of the proton varies linearly with the inverse square of the deuterium decoupling power. This experimental finding is discussed theoretically.     

固体核磁共振中膜蛋白双交叉极化效率与动力学参数相关的定量分析

固体核磁共振（NMR）中双交叉极化（DCP）是用于膜蛋白信号指认的多维异核相关实验的基本技术模块.DCP的效率在很大程度上决定了多维异核相关实验的效率.本文分析了3种典型的膜环境中的膜蛋白（AQPZ、DAGK和EV71 2B）的DCP效率及其影响因素.结果显示,在相同的实验条件下,3种蛋白样品的DCP效率存在明显差异：其中AQPZ的DCP效率最高（31%）,DAGK的效率次之（23%）,EV71 2B的效率最低（14%）.通过测量它们在旋转坐标下的自旋-晶格弛豫时间（T_1ρ）和偶极耦合常数（D_HN）,发现膜蛋白的运动会明显缩短T_1ρ,但对D_HN的影响较小.在实验的基础上,建立了T_1ρ与DCP效率相关的模型,并基于DCP动力学的定量分析,证明了运动导致的T_1ρ缩短是降低DCP效率的主要原因.因此,可以通过定量分析未知样品的T_1ρ来预测其DCP的最优效率,为DCP实验的优化提供依据.     

Rotor-synchronized dipolar-filter sequence at fast MAS in solid-state NMR

Dipolar filters are of considerable importance for eliminating the ¹H NMR signal of the rigid components of heterogeneous compounds while selecting the signal of their mobile parts. On the basis of such filters, structural and dynamical information of these compounds can often be acquired through further manipulations (e.g. spin diffusion) on the spin systems. To overcome the destructive interferences between the magic angle spinning (MAS) speed and the cycle-time of the widely-used Rotor-Asynchronized Dipolar Filter (RADF) sequence, we introduce a new method called Rotor-Synchronized Dipolar Filter (RSDF). This communication shows that this sequence does not present any interference with the spinning speed and is more compatible than RADF with high MAS frequencies (ν_R > 12 kHz). This new pulse sequence will potentially contribute to future researches on heterogeneous materials, such as multiphase polymer and membrane systems.     

High-resolution solid-state 119Sn and 195Pt NMR studies of MPtSn semiconductors (M = Ti, Zr, Hf, Th)

Solid-state 119Sn and 195Pt magic-angle spinning (MAS) NMR spectra are reported on a series of MPtSn compounds (M = Ti, Zr, Hf, Th). In favorable cases (TiPtSn and ZrPtSn) the spectra reveal expected J-coupling patterns originating from indirect spin coupling between Pt and Sn nuclei. MAS has no effect on the broad and asymmetric spectra of either 119Sn and 195Pt nuclei in HfPtSn.     

Spectral Editing in C MAS NMR under Moderately Fast Spinning Conditions

Novel procedures for the spectral assignment of peaks in high-resolution solid-state ¹³C NMR are discussed and demonstrated. These methods are based on the observation that at moderate and already widely available rates of magic-angle spinning (10–14 kHz MAS), CH and CH₂ moieties behave to a large extent as if they were effectively isolated from the surrounding proton reservoir. Dipolar-based analogs of editing techniques that are commonly used in liquid-state NMR such as APT and INEPT can then be derived, while avoiding the need for periods of homonuclear ¹H–¹H multipulse decoupling. The resulting experiments end up being very simple, essentially tuning-free, and capable of establishing unambiguous distinctions among CH, CH₂, and carbon sites. The principles underlying such sequences were explored using both numerical calculations and experimental measurements, and once validated their editing applications were illustrated on a number of compounds.     

Interference of homonuclear decoupling and exchange in the solid-state NMR of perfluorocyclohexane

We observe an interference between RF irradiation used for homonuclear decoupling of 19F and conformational exchange in the 13C spectrum of perfluorocyclohexane. We show that these effects can be readily reproduced in simulation, and characterise their dependence on the various NMR and experimental parameters. Their application to observing exchange rates on the kHz timescale is evaluated with respect to T(1rho) measurements and the connections between the two approaches established. The implications for experiments that use homonuclear decoupling of 1H to resolve 1J(CH)couplings in the solid-state are also evaluated in detail.     

Fast acquisition of multi-dimensional spectra in solid-state NMR enabled by ultra-fast MAS

The advantages offered by ultra-fast (>60 kHz) magic angle spinning (MAS) rotation for the study of biological samples, notably containing paramagnetic centers are explored.It is shown that optimal conditions for performing solid-state ¹³C NMR under 60 kHz MAS are obtained with low-power CW ¹H decoupling, as well as after a low-power ¹H,¹³C cross-polarization step at a double-quantum matching condition. Acquisition with low-power decoupling highlights the existence of rotational decoupling sidebands. The sideband intensities and the existence of first and second rotary conditions are explained in the framework of the Floquet–van Vleck theory.As a result, optimal ¹³C spectra of the oxidized, paramagnetic form of human copper zinc superoxide dismutase (SOD) can be obtained employing rf-fields which do not exceed 40 kHz during the whole experiment. This enables the removal of unwanted heating which can lead to deterioration of the sample. Furthermore, combined with the short ¹H T₁s, this allows the repetition rate of the experiments to be shortened from 3 s to 500 ms, thus compensating for the sensitivity loss due to the smaller sample volume in a 1.3 mm rotor. The result is that 2D ¹³C–¹³C correlation could be acquired in about 24 h on less than 1 mg of SOD sample.     

Same spectral signature in liquid-state and solid-state 1H photo-CIDNP NMR spectra of cyclohexanone

ABSTRACT

For the first time, a ¹H solid-state photo-CIDNP effect is reported. The effect is observed in frozen solution of cyclohexanone in 1,4-dioxane-d₈ under magic-angle spinning and continuous irradiation by the full spectrum of a xenon arc lamp. The spectral features show close similarity with those observed by ¹H liquid-state photo-CIDNP NMR.