Determination of the oligomeric number and intermolecular distances of membrane protein assemblies by anisotropic 1H-driven spin diffusion NMR spectroscopy

Determination of the high-resolution quaternary structure of oligomeric membrane proteins requires knowledge of both the oligomeric number and intermolecular distances. The centerband-only detection of exchange (CODEX) technique has been shown to enable the extraction of the oligomeric number through the equilibrium exchange intensity at long mixing times. To obtain quantitative distances, we now provide an analysis of the mixing-time-dependent CODEX intensities using the 1H-driven spin diffusion theory. The exchange curve is fit to a rate equation, where the rate constants are proportional to the square of the dipolar coupling and the spectral overlap integral between the exchanging spins. Using a number of 13C- and 19F-labeled crystalline model compounds with known intermolecular distances, we empirically determined the overlap integrals of 13C and 19F CODEX for specific spinning speeds and chemical shift anisotropies. These consensus overlap integral values can be applied to structurally unknown systems to determine distances. Applying the 19F CODEX experiment and analysis, we studied the transmembrane peptide of the M2 protein (M2TMP) of influenza A virus bound to 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine bilayers. The experiment proved for the first time that M2TMP associates as tetramers in lipid bilayers, similar to its oligomeric state in detergent micelles. Moreover, the nearest-neighbor interhelical F-F distance between (4-19F)Phe30 is 7.9-9.5 angstroms. This distance constrains the orientation and the packing of the helices in the tetrameric bundle and supports the structural model derived from previous solid-state NMR 15N orientational data. Thus, the CODEX technique presents a general method for determining the oligomeric number and intermolecular distances in the approximately 10 angstroms range in membrane proteins and other complex biological assemblies.     

Simultaneous quantification and identification of individual chemicals in metabolite mixtures by two-dimensional extrapolated time-zero (1)H-(13)C HSQC (HSQC(0))

Quantitative one-dimensional (1D) (1)H NMR spectroscopy is a useful tool for determining metabolite concentrations because of the direct proportionality of signal intensity to the quantity of analyte. However, severe signal overlap in 1D (1)H NMR spectra of complex metabolite mixtures hinders accurate quantification. Extension of 1D (1)H to 2D (1)H-(13)C HSQC leads to the dispersion of peaks along the (13)C dimension and greatly alleviates peak overlapping. Although peaks are better resolved in 2D (1)H-(13)C HSQC than in 1D (1)H NMR spectra, the simple proportionality of cross peaks to the quantity of individual metabolites is lost by resonance-specific signal attenuation during the coherence transfer periods. As a result, peaks for individual metabolites usually are quantified by reference to calibration data collected from samples of known concentration. We show here that data from a series of HSQC spectra acquired with incremented repetition times (the time between the end of the first (1)H excitation pulse to the beginning of data acquisition) can be extrapolated back to zero time to yield a time-zero 2D (1)H-(13)C HSQC spectrum (HSQC(0)) in which signal intensities are proportional to concentrations of individual metabolites. Relative concentrations determined from cross peak intensities can be converted to absolute concentrations by reference to an internal standard of known concentration. Clustering of the HSQC(0) cross peaks by their normalized intensities identifies those corresponding to metabolites present at a given concentration, and this information can assist in assigning these peaks to specific compounds. The concentration measurement for an individual metabolite can be improved by averaging the intensities of multiple, nonoverlapping cross peaks assigned to that metabolite.     

3D TEDOR NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly (13)C,(15)N-labeled solids

We describe three-dimensional magic-angle-spinning NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly (13)C,(15)N-labeled solids. The approaches employ transferred echo double resonance (TEDOR) for (13)C-(15)N coherence transfer and (15)N and (13)C frequency labeling for site-specific resolution, and build on several previous 3D TEDOR techniques. The novel feature of the 3D TEDOR pulse sequences presented here is that they are specifically designed to circumvent the detrimental effects of homonuclear (13)C-(13)C J-couplings on the measurement of weak (13)C-(15)N dipolar couplings. In particular, homonuclear J-couplings lead to two undesirable effects: (i) they generate anti-phase and multiple-quantum (MQ) spin coherences, which lead to spurious cross-peaks and phase-twisted lines in the 2D (15)N-(13)C correlation spectra, and thus degrade the spectral resolution and prohibit the extraction of reliable cross-peak intensities, and (ii) they significantly reduce cross-peak intensities for strongly J-coupled (13)C sites (e.g., CO and C(alpha)). The first experiment employs z-filter periods to suppress the anti-phase and MQ coherences and generates 2D spectra with purely absorptive peaks for all TEDOR mixing times. The second approach uses band-selective (13)C pulses to refocus J-couplings between (13)C spins within the selective pulse bandwidth and (13)C spins outside the bandwidth. The internuclear distances are extracted by using a simple analytical model, which accounts explicitly for multiple spin-spin couplings contributing to cross-peak buildup. The experiments are demonstrated in two U-(13)C,(15)N-labeled peptides, N-acetyl-L-Val-L-Leu (N-ac-VL) and N-formyl-L-Met-L-Leu-L-Phe (N-f-MLF), where 20 and 26 (13)C-(15)N distances up to approximately 5-6 A were measured, respectively. Of the measured distances, 10 in N-ac-VL and 13 in N-f-MLF are greater than 3 A and provide valuable structural constraints.     

Centerband-Only Detection of Exchange NMR with Natural-Abundance Correction Reveals an Expanded Unit Cell in Phenylalanine Crystals

The NMR pulse sequence CODEX (centerband-only detection of exchange) is a widely used method to report on the number of magnetically inequivalent spins that exchange magnetization via spin diffusion. For crystals, this rules out certain symmetries, and the rate of equilibration is sensitive to distances. Here we show that for ¹³C CODEX, consideration of natural abundance spins is necessary for crystals of high complexity, demonstrated here with the amino acid phenylalanine. The NMR data rule out the C₂ space group that was originally reported for phenylalanine, and are only consistent with a larger unit cell containing eight magnetically inequivalent molecules. Such an expanded cell was recently described based on single crystal data. The large unit cell dictates the use of long spin diffusion times of more than 200 seconds, in order to equilibrate over the entire unit cell volume of 1622 ų.     

Application of HMQC spectroscopy to the observation of the metal resonance in polymetallic compounds: the effect of multiple-metal spin transitions

Multiple-metal spin transitions which distort the HMQC spectra of rhodium carbonyl clusters are discussed. These effects are seen whenever the detector nucleus, e.g. 13C or 31P, couples to more than one metal spin and are not restricted to detector ligands occupying edge- or face-bridging sites. These effects are illustrated in, but not limited to, the 13C-{103Rh} and 31P-{103Rh} HMQC spectra of [Rh6(CO)15L], (where L = P(4-F-C6H4)3), [Rh4(CO)11{P(OPh)3}], [Rh6C(CO)15]2- and [Rh2(carboxylate)2PPh3]. The effect is to modulate the intensity and position of the correlations in the metal dimension; cross peaks are displaced from the true chemical shift, additional cross peaks are seen and the intensity of the coherences varies as a function of the preparation delay, d2, and coupling constant, and may go to zero at the conventional value of 1/(2J). Analyses of the relevant spin systems are given together with experimental strategies to overcome these effects.     

Frequency selective heteronuclear dipolar recoupling in rotating solids: accurate (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled peptides

We describe a magic-angle spinning NMR experiment for selective (13)C-(15)N distance measurements in uniformly (13)C,(15)N-labeled solids, where multiple (13)C-(15)N and (13)C-(13)C interactions complicate the accurate measurement of structurally interesting, weak (13)C-(15)N dipolar couplings. The new experiment, termed FSR (frequency selective REDOR), combines the REDOR pulse sequence with a frequency selective spin-echo to recouple a single (13)C-(15)N dipolar interaction in a multiple spin system. Concurrently the remaining (13)C-(15)N dipolar couplings and all (13)C-(13)C scalar couplings to the selected (13)C are suppressed. The (13)C-(15)N coupling of interest is extracted by a least-squares fit of the experimentally observed modulation of the (13)C spin-echo intensity to the analytical expression describing the dipolar dephasing in an isolated heteronuclear spin pair under conventional REDOR. The experiment is demonstrated in three uniformly (13)C,(15)N-labeled model systems: asparagine, N-acetyl-L-Val-L-Leu and N-formyl-L-Met-L-Leu-L-Phe; in N-formyl-[U-(13)C,(15)N]L-Met-L-Leu-L-Phe we have determined a total of 16 internuclear distances in the 2.5-6 A range.     

2D—HOHAHA和ROESY方法归属复杂天然有机化合物的NMR谱线

用同核化学位移相关谱及旋转坐标系中的同核化学位移相关谱和旋转坐标系中的同核NOE实验等新的二维核磁共振方法对首次从西洋参叶中分离出的Ocotillol型皂甙(OTS)的~1H化学位移进行了完全归属,为OTS分子溶液中的三维空间结构研究提供了可靠的结构参数.     

13C-13C NOESY: an attractive alternative for studying large macromolecules

13C direct detection provides a valuable alternative to 1H detection to overcome fast relaxation because of its smaller magnetic moment. 13C-13C NOESY spectra were acquired for a dimeric protein of molecular mass 32 000 and for a monomeric analogue. With increasing molecular mass, the quality of 13C-13C NOESY spectra improves while the scalar-based experiments become less sensitive, as predicted by the increase in the molecular mass. 13C-13C NOESY spectra of the dimer were acquired with different mixing times. The mixing time can be tuned to detect mainly one-bond correlations, or it can be increased to also detect correlations between nuclei at longer distances. It is proposed that 13C-13C dipolar-based experiments provide a promising tool for signal detection and assignment in large macromolecules, such as multimeric species and macromolecular complexes, for which scalar-based experiments become less effective.     

Measurement of multiple psi torsion angles in uniformly 13C,15N-labeled alpha-spectrin SH3 domain using 3D 15N-13C-13C-15N MAS dipolar-chemical shift correlation spectroscopy

We demonstrate the simultaneous measurement of several backbone torsion angles psi in the uniformly (13)C,(15)N-labeled alpha-Spectrin SH3 domain using two different 3D 15N-13C-13C-15N dipolar-chemical shift magic-angle spinning (MAS) NMR experiments. The first NCCN experiment utilizes double quantum (DQ) spectroscopy combined with the INADEQUATE type 13C-13C chemical shift correlation. The decay of the DQ coherences formed between 13C'(i) and 13C(alphai) spin pairs is determined by the "correlated" dipolar field due to 15N(i)-13C(alphai) and 13C'(i)-15N(i+1) dipolar couplings and is particularly sensitive to variations of the torsion angle in the regime |psi| > 140 degrees. However, the ability of this experiment to constrain multiple psi-torsion angles is limited by the resolution of the 13C(alpha)-(13)CO correlation spectrum. This problem is partially addressed in the second approach described here, which is an NCOCA NCCN experiment. In this case the resolution is enhanced by the superior spectral dispersion of the 15N resonances present in the 15N(i+1)-13C(alphai) part of the NCOCA chemical shift correlation spectrum. For the case of the 62-residue alpha-spectrin SH3 domain, we determined 13 psi angle constraints with the INADEQUATE NCCN experiment and 22 psi constraints were measured in the NCOCA NCCN experiment.     

13C-13C rotational resonance width distance measurements in uniformly 13C-labeled peptides

The rotational resonance width (R2W) experiment is a constant-time version of the rotational resonance (R2) experiment, in which the magnetization exchange is measured as a function of sample spinning frequency rather than the mixing time. The significant advantage of this experiment over conventional R2 is that both the dipolar coupling and the relaxation parameters can be independently and unambiguously extracted from the magnetization exchange profile. In this paper, we combine R2W with two-dimensional 13C-13C chemical shift correlation spectroscopy and demonstrate the utility of this technique for the site-specific measurement of multiple 13C-13C distances in uniformly labeled solids. The dipolar truncation effects, usually associated with distance measurements in uniformly labeled solids, are considerably attenuated in R2W experiments. Thus, R2W experiments are applicable to uniformly labeled biological systems. To validate this statement, multiple 13C-13C distances (in the range of 3-6 A) were determined in N-acetyl-[U-13C,15N]l-Val-l-Leu with an average precision of +/-0.5 A. Furthermore, the distance constraints extracted using a two-spin model agree well with the X-ray crystallographic data.     

13C13C spin coupling constants in phenanthrene derivatives

The synthesis of six 1-X- (X = OH, OCH₃, OCOCH₃, CH₃, CHO and CN) phenanthrene derivatives with a ¹³C label at C-1 is described. An analysis of the ¹³C¹³C spin coupling constants shows the importance of π-interaction for the coupling constant transmission. Small ¹³C¹³C spin coupling constants over 6 bonds are reported.     

High-resolution 13C NMR study of free and metal-complexed ionophores in the solid state: Conformation and dynamics of the macrocyclic ring and the effect of intermolecular short contact of methyl groups on spin–lattice relaxation times and displacements of chemical shifts

High-resolution ¹³C NMR spectra of 15 samples of uncomplexed and metal-complexed tetranactin and nonactin were recorded in the solid state, revealing characteristic displacements of peaks due to complex formation and the effect of crystalline packing on the ¹³C chemical shifts and spin–lattice relaxation times of the methyl groups. The C-1 ¹³C chemical shifts of uncomplexed and complexed tetranaction and nonactin are well related to the variation of nearby torsion angles characteristic of the macrocyclic conformation, as determined by x-ray diffraction. The existence of short intermolecular contact of methyl groups (<3.8 Å) at the surface of the molecules results in either prolonged ¹³C spin–lattice relaxation times in the laboratory frame (T₁^C) or substantial upfield displacement of peaks (up to 6 ppm). In addition, significantly reduced T₁^C values in uncomplexed nonactin (one order of magnitude smaller than those of other compounds) was ascribed to the presence of a puckering motion of the tetrahydrofuran ring and fluctuation of the macrocyclic ring in the solid state (with a time scale of 10⁻⁸ _s). Finally, how the conformations of these compounds in the solid are retained in chloroform solution was examined in view of the differences in the ¹³C chemical shifts between the solid and solution.     

13C/15N distance determination by CPMAS NMR in uniformly 13C labeled molecules

The REDOR and CPMAS techniques are applied for measuring 13C-15N dipolar coupling constants in glycine. It is shown that the selective CP or SPECIFIC CP technique removes the coherent evolution of the spin system under homonuclear 13C-13C J couplings. While the large coupling constant (approximately 900 Hz) is readily determined because of the presence of large oscillations in the CPMAS dynamics, their absence precludes the measurement of the small coupling constant (approximately 200 Hz). The experimental results and numerical simulations demonstrate that the determination of 13C-15N coupling constants of medium size (<1 kHz) by the CPMAS technique is mainly limited by the strength of the 1H decoupling field and the size of the 13C and 15N chemical shift anisotropies.     

Large structure rearrangement of colicin ia channel domain after membrane binding from 2D 13C spin diffusion NMR

One of the main mechanisms of membrane protein folding is by spontaneous insertion into the lipid bilayer from the aqueous environment. The bacterial toxin, colicin Ia, is one such protein. To shed light on the conformational changes involved in this dramatic transfer from the polar to the hydrophobic milieu, we carried out 2D magic-angle spinning (13)C NMR experiments on the water-soluble and membrane-bound states of the channel-forming domain of colicin Ia. Proton-driven (13)C spin diffusion spectra of selectively (13)C-labeled protein show unequivocal attenuation of cross-peaks after membrane binding. This attenuation can be assigned to distance increases but not reduction of the diffusion coefficient. Analysis of the statistics of the interhelical and intrahelical (13)C-(13)C distances in the soluble protein structure indicates that the observed cross-peak reduction is well correlated with a high percentage of short interhelical contacts in the soluble protein. This suggests that colicin Ia channel domain becomes open and extended upon membrane binding, thus lengthening interhelical distances. In comparison, cross-peaks with similar intensities between the two states are dominated by intrahelical contacts in the soluble state. This suggests that the membrane-bound structure of colicin Ia channel domain may be described as a "molten globule", in which the helical secondary structure is retained while the tertiary structure is unfolded. This study demonstrates that (13)C spin diffusion NMR is a valuable tool for obtaining qualitative long-range distance constraints on membrane protein folding.     

How to measure absolute P3HT crystallinity via 13C CPMAS NMR

We outline the details of acquiring quantitative ¹³C cross‐polarization magic angle spinning (CPMAS) nuclear magnetic resonance on the most ubiquitous polymer for organic electronic applications, poly(3‐hexylthiophene) (P3HT), despite other groups' claims that CPMAS of P3HT is strictly nonquantitative. We lay out the optimal experimental conditions for measuring crystallinity in P3HT, which is a parameter that has proven to be critical in the electrical performance of P3HT‐containing organic photovoltaics but remains difficult to measure by scattering/diffraction and optical methods despite considerable efforts. Herein, we overview the spectral acquisition conditions of the two P3HT films with different crystallinities (0.47 and 0.55) and point out that because of the chemical similarity of P3HT to other alkyl side chain, highly conjugated main chain polymers, our protocol could straightforwardly be extended to other organic electronic materials. Variable temperature ¹H NMR results are shown as well, which (i) yield insight into the molecular dynamics of P3HT, (ii) add context for spectral editing techniques as applied to quantifying crystallinity, and (iii) show why T_1ρ^H, the ¹H spin–lattice relaxation time in the rotating frame, is a more optimal relaxation filter for distinguishing between crystalline and noncrystalline phases of highly conjugated alkyl side‐chain polymers than other relaxation times such as the ¹H spin–spin relaxation time, T₂^H, and the spin–lattice relaxation time in the toggling frame, T_1xz^H. A 7 ms T_1ρ^H spin lock filter, prior to CPMAS, allows for spectroscopic separation of crystalline and noncrystalline ¹³C nuclear magnetic resonance signals. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.     

Protein structure determination from 13C spin-diffusion solid-state NMR spectroscopy

Proton-driven 13C spin diffusion (PDSD) is a simple and robust two-dimensional NMR experiment. It leads to spectra with a high signal-to-noise ratio in which cross-peaks contain information about internuclear distances. We show that the total information content is sufficient to determine the atomic-resolution structure of a small protein from a single, uniformly 13C-, 15N-labeled microcrystalline sample. For the example of ubiquitin, the structure was determined by a manual procedure followed by an automatic optimization of the manual structure as well as by a fully automated structure determination approach. The relationship between internuclear distances and cross-peak intensities in the spectra is investigated.     

Two-dimensional homonuclear chemical shift correlation established by the cross-relaxation driven spin diffusion in solids

A new type of spin diffusion, cross-relaxation driven spin diffusion (CRDSD), is investigated using (15)N NMR on a N-acetyl-L-valyl-L-leucine (NAVL) single crystal under stationary condition. A two-dimensional (2D) pulse sequence that correlates the chemical shifts of (15)N nuclei, with a radio-frequency spin lock on the (15)N channel during the mixing time, is used to observe CRDSD. Experimental results obtained using CRDSD, rf-driven spin diffusion, and proton driven spin diffusion approaches on the NAVL single crystal are compared. Our experimental results suggest that the (15)N spin diffusion rate can be enhanced by about 1000 times using CRDSD than by the normal proton driven spin diffusion. Interestingly, the required spin-locking rf field strength for CRDSD is much lower than that used for the rf-driven spin diffusion experiments. The cross-peak patterns observed in 2D (15)N-(15)N correlation spectra using CRDSD and RFDSD are very different as they arise from different spin-spin interactions. A detailed theory describing CRDSD and RFDSD processes is also presented using a thermodynamic model. The speedy spin diffusion process rendered by the CRDSD approach will be useful to assign resonances from a uniformly (15)N or (13)C labeled proteins and peptides, particularly in aligned samples.     

Evidence for spin diffusion in a H,H-NOESY study of imidazolium tetrafluoroborate ionic liquids

The ionic liquids 1-ethyl-3-methylimidazolium tetrafluoroborate ([EMIM][BF4]) and 1-methyl-3-propylimidazolium tetrafluoroborate ([PMIM][BF4]) were studied by H,H-NOESY NMR using a cross-relaxation matrix analysis. Cross-peak intensities are seen to increase with increasing mixing time. Experimental and theoretical hydrogen-hydrogen distances are in agreement at short mixing times (50 ms). Mixing times longer than 50 ms result in an increasing contribution of spin diffusion that produces unrealistically short hydrogen-hydrogen distances. Gas-phase ab initio molecular structures are obtained using Hartree-Fock (HF) and density functional theory (B3LYP) methods at the 6311 + G(2d,p) basis set level. The hydrogen-hydrogen distances obtained from the theoretical structures are in reasonable agreement with those calculated from the cross-relaxation matrices.     

Slow motion in [ring‐fluoro]polycarbonate by CODEX

Chain dynamics in [ring‐fluoro]polycarbonate (an A‐B alternating copolymer that has a single fluorine substituent on every fourth main chain ring) have been characterized by centerband only detection of exchange (CODEX) and rotating‐frame ¹³C spin‐lattice relaxation. The slow motions detected by CODEX are facilitated by a mechanically active lattice reorganization that permits a flip of the fluorinated ring about its C₂ axis. Nonfluorinated rings undergo small‐amplitude reorientations and C₂ flips, both of which are fast and not CODEX active. © 2008 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 46: 1062–1066, 2008     

Proton spin diffusion as a tool for characterizing polymer blends

Proton spin diffusion studies for characterizing minimum domain dimensions have been carried out on three blends: a 40/60 blend of nylon 6,6 with the aromatic rigid rod polymer poly(benzo[a,d]dithiazol-2,6-diyl-1,4-phenylene⁾, PBZT, a 47/53 blend of an amorphous nylon with PBZT, and a 50/50 blend of a polyetherimide (PEI) and a polybenzimidazole (PBI). Polarization gradients necessary for these experiments were generated using both chemical shift differences (via multiple pulse techniques) and linewidth differences. Polarization readout techniques included proton lineshape deconvolution, multiple pulse proton lineshapes and ¹³C CPMAS spectra utilizing short CP times. The two nylon/PBZT blends are expected to phase separate from thermodynamic arguments; however, kinetic considerations, more than thermodynamics, determine domain size. In the 40/60 blend, the minimum domain dimensions of each of the nylon and the PBZT phases were about 4 nm with some scattered larger crystals of nylon. In the 47/53 blend, mixing in some regions indicated domain dimensions similar to the 40/60 blend. In contrast to the 40/60 blend, however, the 47/53 blend was still far from internal spin polarization equilibrium after spin diffusion times of 140 ms. The implication is that the sample-average composition is not found over dimensions like 40 nm; the problem is that the possible morphological explanations are manifold. By investigating the proton rotating frame relaxation, T_1p, the possibility that some of the PBZT domains are isolated from the nylon, in a spin diffusion sense, was eliminated. It is more likely that about half of the nylon protons are isolated by spin diffusion from the PBZT protons on a 140 ms timescale. The PEI/PBI blend is a compatible blend of two aromatic polymers where mixing on a molecular scale is expected. We were interested in a measurement of the lower limit of domain size using proton spin diffusion. This lower limit turned out to be about 2.5 nm based on low temperature T_1p measurements as opposed to room temperature multiple pulse methods. The latter measurements monitored the disappearance of a polarization gradient between the PEI methyl protons and all the remaining protons. The superiority of the T_1p measurements over the multiple pulse method for establishing the smaller minimum domain dimension is not a general result and reasons are discussed. Finally, some general remarks about characterizing polymer blends by solid state NMR, particularly blends which have undergone spinodal phase separation, are included.