15N spin diffusion rate in solid-state NMR of totally enriched proteins: The magic angle spinning frequency effect (original) (raw)
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Journal of Biomolecular Nmr - J BIOMOL NMR, 1997
A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE = Double INEPT Edited), is proposed. It is based on the selectiveinversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sortsout the spin diffusion paths according to the principal mediators, either the CH/CH3 or theCH2 protons. This is useful in the structure refinement process, as it enables proper alignmentof the aliphatic protons in the vicinity of NH protons.
Journal of biomolecular NMR, 2002
The simultaneous interpretation of a suite of dipole-dipole and dipole-CSA cross-correlation rates involving the backbone nuclei 13Calpha, 1Halpha, 13CO, 15N and 1HN can be used to resolve the ambiguities associated with each individual cross-correlation rate. The method is based on the transformation of experimental cross-correlation rates via calculated values based on standard peptide plane geometry and solid-state 13CO CSA parameters into a dihedral angle probability surface. Triple resonance NMR experiments with improved sensitivity have been devised for the quantification of relaxation interference between 1Halpha(i)-13Calpha(i)/15N(i)-1HN(i) and 1Halpha(i-1)-13Calpha(i-1)/15N(i)-1HN(i) dipole-dipole mechanisms in 15N, 13C-labeled proteins. The approach is illustrated with an application to 13C, 15N-labeled ubiquitin.
The Journal of Physical Chemistry B, 2016
Transverse relaxation rate measurements in MAS solid-state NMR provide information about molecular motions occurring on nanoseconds-to-milliseconds (ns-ms) time scales. The measurement of heteronuclear (13 C , 15 N) relaxation rate constants in the presence of a spin-lock radio-frequency field (R 1ρ relaxation) provides access to such motions, and an increasing number of studies involving R 1ρ relaxation in proteins has been reported. However, two factors that influence the observed relaxation rate constants have so far been neglected, namely (i) the role of CSA/dipolar cross-correlated relaxation (CCR), and (ii) the impact of fast proton spin flips (i.e. proton spin diffusion and relaxation). We show that CSA/D CCR in R 1ρ experiments is measurable, and that this cross-correlated relaxation rate constant depends on ns-ms motions, and can thus itself provide insight into dynamics. We find that proton spin-diffusion attenuates this cross-correlated relaxation, due to its decoupling effect on the doublet components. For measurements of dynamics, the use of R 1ρ rate constants has practical advantages over the use of CCR rates, and the present manuscript reveals factors that have so far been disregarded and which are important for accurate measurements and interpretation.
Biophysical Journal, 2014
The ability to detect nanosecond backbone dynamics with site-directed spin labeling (SDSL) in soluble proteins has been well established. However, for membrane proteins, the nitroxide appears to have more interactions with the protein surface, potentially hindering the sensitivity to backbone motions. To determine whether membrane protein backbone dynamics could be mapped with SDSL, a nitroxide was introduced at 55 independent sites in a model polytopic membrane protein, TM0026. Electron paramagnetic resonance spectral parameters were compared with NMR 15 N-relaxation data. Sequential scans revealed backbone dynamics with the same trends observed for the R 1 relaxation rate, suggesting that nitroxide dynamics remain coupled to the backbone on membrane proteins.
Journal of The American Chemical Society, 2007
Remarkable progress in solid-state NMR has enabled complete structure determination of uniformly labeled proteins in the size range of 5-10 kDa. Expanding these applications to larger or masslimited systems requires further improvements in spectral sensitivity, for which inverse detection of 13 C and 15 N signals with 1 H is one promising approach. Proton detection has previously been demonstrated to offer sensitivity benefits in the limit of sparse protonation or with ∼30 kHz magic-angle spinning (MAS).
Rapid Proton-Detected NMR Assignment for Proteins with Fast Magic Angle Spinning
Journal of the American Chemical Society, 2014
Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5−30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.
Angewandte Chemie International Edition, 2007
Advances over the last decade in magic-angle spinning solid-state NMR (MAS SSNMR) have enabled the complete structure determination of several small proteins.[1] In principle, SSNMR is not limited by molecular size, which is one major advantage over solution NMR in challenging applications such as membrane protein complexes and high molecular weight protein aggregates. However, solid-state structure determination of larger proteins is typically hindered by the low sensitivity and relatively short measurable distances imposed by the observation of nuclei with low gyromagnetic ratios (γ), such as 13 C and 15 N. The large γ of 1 H, while providing high detection sensitivity and NOE distance restraints for solution NMR, [2] results in large dipolar couplings in the solid state, which may degrade both spectral resolution and sensitivity.[3] Recent studies by Reif and Zilm and their respective coworkers have demonstrated that these challenges in resolution and sensitivity can be overcome by using spin dilution, replacing all non-exchangeable protons with deuterons.[3] In combination with ** This research was supported by the National Institutes of Health (R01 GM-75937 to C.M.R). We thank Philippe Nadaud and Prof.
Journal of the American Chemical Society, 1998
We present a new solid-state NMR approach, based on 1 H spin diffusion with X-nucleus (15 N, 13 C, 31 P) detection, for investigating the structure of membrane proteins. For any segment with a resolvable signal in the X-nucleus spectrum, the depth of insertion into the lipid bilayer can be determined. The technique represents the adaptation of the Goldman-Shen 1 H spin-diffusion experiment with X-nucleus detection to proteins in hydrated lipid bilayers (>25% water by weight) in the gel state at 240 K. The experiments are demonstrated on the 21-kDa channel-forming domain of the toxin-like colicin E1 molecule incorporated into lipid vesicles. More than 32% of the protons in our sample are in mobile H 2 O molecules, which can be selected efficiently by the 1 H T 2 filter in the Goldman-Shen sequence. The transfer of 1 H magnetization from mobile H 2 O to the colicin E1 channel domain is 80% complete within only 5 ms. This transfer to the protein, probed by the amide 15 N signals, is faster than the transfer to the rigid protons on average, proving that most of the protein is preferentially located between the water and the lipid bilayer. From the spindiffusion and dipolar-dephasing data, 60% of the 24 lysine side groups are shown to be highly mobile. Quantitative depth profiling is demonstrated using the 31 P in the lipid phosphate head groups and the 13 C nuclei in the lipid acyl chains as distance markers for the spin diffusion.
NMR Detection of Protein 15 N Spins near Paramagnetic Lanthanide Ions
Journal of the American Chemical Society, 2007
. Scheme of magnetization transfer pathways in the out-and-back N Z -exchange experiment. The letters ijk denote the path with state i at the beginning of the experiment, state j during t 1 evolution, and k during detection (t 2 and t 3 ). The numbers below denote the amount of N Z magnetization of each path as fraction of the total N Z magnetization (M tot ) present at the beginning of τ m1 . Relaxation during INEPT and frequency evolution periods is neglected, and R 1 is assumed to be the same in the diamagnetic and paramagnetic state. It is further assumed that τ m1 = τ m2 = τ m . . Pulse scheme of an N Z -exchange experiment starting from 15 N magnetization. All parameters are identical to those of , except that the receiver phase is φ rec (2x,2(-x)), and G 1 is set to G 5 +G 4 -G 3 to yield water flip-back for even numbers of N.