REDOR: an assessment of the efficacy of dipolar recoupling with adiabatic inversion pulses

A numerical assessment of the efficacy of REDOR recoupling of heteronuclear dipolar interactions employing adiabatic dephasing pulses has been carried out by considering an isolated dipolar coupled spin 1/2 I-S system. At moderate magic angle spinning frequencies in the range of 3–6 kHz and when the CSA of the dephased spins is large, it is shown that efficient broadband heteronuclear dipolar recoupling and reliable distance estimates can be achieved even under conditions where a significant fraction of the rotor period is occupied by the adiabatic pulse. The efficacy of REDOR with adiabatic inversion pulses has been demonstrated experimentally in two model ¹⁵N-¹³C spin systems, (¹³C,¹⁵N) Aib-(¹⁵N) Aib-NH₂ (Aib = -aminoisobutyric acid) and (1-¹³C,¹⁵N) glycine.     

Off-resonance 13C–2H REDOR NMR for site-resolved studies of molecular motion

We introduce a ¹³C–²H Rotational Echo DOuble Resonance (REDOR) technique that uses the difference between on-resonance and off-resonance ²H irradiation to detect dynamic segments in deuterated molecules. By selectively inverting specific regions of the ²H magic-angle spinning (MAS) sideband manifold to recouple some of the deuterons to nearby carbons, we distinguish dynamic and rigid residues in 1D and 2D ¹³C spectra. We demonstrate this approach on deuterated GB1, H/D exchanged GB1, and perdeuterated bacterial cellulose. Numerical simulations reproduce the measured mixing-time and ²H carrier-frequency dependence of the REDOR dephasing of bacterial cellulose. Combining numerical simulations with experiments thus allow the extraction of motionally averaged quadrupolar couplings from REDOR dephasing values.

     

Redor in IS1S2 systems

An approach to the determination of the 2-¹³C chemical shift (CS) tensor orientation in pyrimidine bases via heteronuclear MAS NMR spectroscopy is presented. Considering a dipolar coupled spin 1/2 network of the type S₁-I-S₂ consisting of directly bonded heteronuclear spins, we have carried out numerical simulations to assess the sensitivity of I-S REDOR spinning sidebands to the Euler angles defining the orientation of the I-S₁ and I-S₂ dipolar vectors in the I spin CS tensor principal axes system. Our investigations clearly demonstrate the potential of I-S REDOR studies in IS₁S₂ systems for obtaining with high reliability and accuracy the I spin chemical shift tensor orientation in the molecular frame spanned by the two internuclear vectors I-S₁ and I-S₂. The significant contribution to the observed REDOR sideband intensities from anti-phase operator terms which are present at the start of the data acquisition is illustrated. The procedure for the recording and analysis of the I-S REDOR spectra in IS₁S₂ systems is presented and the measurement of the 2-¹³C CS tensor orientation in a polycrystalline sample of [1,3-¹⁵N₂, 2-¹³C] uracil, which is one of the four bases in RNA, is experimentally demonstrated.     

Selectively dispersed isotope labeling for protein structure determination by magic angle spinning NMR

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-¹³C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-¹³C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra.     

Possible conformation of amphotericin B dimer in membrane-bound assembly as deduced from solid-state NMR

Aiming for structural analysis of amphotericin B (AmB) ion-channel assemblies in membrane, a covalent dimer was synthesized between ¹³C-labled AmB methyl ester and ¹⁹F-labled AmB. The dimer showed slightly weaker but significant biological activities against fungi and red blood cells compared with those of monomeric AmB. Then the dimer was subjected to ¹³C{¹⁹F}REDOR (Rotational-Echo Double Resonance) experiments in hydrated lipid bilayers. The obtained REDOR dephasing effects were explained by two components; a short ¹³C/¹⁹F distance (6.9 Å) accounting for 23% of the REDOR dephasing, and a longer one (14 Å) comprising the rest of the dephasing. The shorter distance is likely to reflect the formation of barrel-stave ion channel.     

Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins

A simple spectral editing procedure is described that generates separate subspectra for the methyl ¹³C-¹H₃ multiplet components of ¹H-¹³C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields ¹H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of ¹H-¹³C J and dipolar couplings. The editing procedure is illustrated for a Ca²⁺-calmodulin sample in isotropic and liquid crystalline phases.     

Location of fluorotryptophan sequestered in an amphiphilic nanoparticle by rotational-echo double-resonance NMR.

Rotational-echo double-resonance (REDOR) 13C NMR spectra (with 19F dephasing) have been obtained of 6-fluorotryptophan complexed by a polymeric amphiphilic nanosphere consisting of a polystyrene core covalently attached to a poly(acrylic acid)-polyacrylamide shell. The REDOR spectra show that aromatic carbons from the polystyrene core and oxygenated carbons in the poly(acrylic acid)-polyacrylamide shell are both proximate to the 19F of 6-fluorotryptophan. Molecular modeling restrained by distances inferred from the REDOR spectra suggests that all of the 6-fluorotryptophans are in the shell but within 10 A of the core-shell interface.     

Dominant formation of a single-length channel by amphotericin B in dimyristoylphosphatidylcholine membrane evidenced by 13C-31P rotational echo double resonance

(13)C-Labeled amphotericin B (AmB) was prepared by feeding the producing organism Streptomyces nodosus with [3-(13)C]propionate. The REDOR experiments for dimyristoylphosphatidylcholine (DMPC) membrane using the (13)C-labeled AmB showed the prominent dephasing effects between the phosphate group in PC and C41 carboxyl carbon in the polar head. In addition, C39/C40 methyl carbons also gave rise to the significant reduction of their (13)C NMR signals, implying that both terminal parts of AmB reside close to the surface of the DMPC membrane. Conversely, the same REDOR experiments with use of distearoylphosphatidylcholine (DSPC) showed no dephasing for the C39/C40 methyl signals while a marked reduction of the C41 carbonyl signal was again observed. These findings should be most reasonably accounted for by the notion that AmB can span across the DMPC membrane with a single-length interaction but cannot span the DSPC membrane due to its greater thickness. To our knowledge, the results provide the first direct spectroscopic evidence for the formation of a single-length channel across a biomembrane, which was previously suggested by channel current recording experiments.     

Microsecond motions probed by near-rotary-resonance <Emphasis Type="Italic">R</Emphasis><Subscript>1ρ</Subscript><Superscript>15</Superscript>N MAS NMR experiments: the model case of protein overall-rocking in crystals

F_oF₁-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H⁺ translocation in membranes. The F_o c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state ¹³C NMR (ssNMR) signals of the F_o c-subunit ring of thermophilic Bacillus PS3 (TF_o c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly ¹³C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional C^α(i+1)–C′C^α(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue ¹³C–¹³C chemical shift correlations, 97% of C^α, 97% of C^β and 92% of C′ signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TF_o c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TF_o c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.     

Editing and diagonal peak suppression in three-dimensional HCCH protein NMR correlation experiments

A novel three-dimensional (3D) HCCH NMR experiment is introduced. It involves ¹³C-¹³C COSY or TOCSY coherence transfer plus two independent editing steps according to the number of protons attached to the individual carbons before and after the ¹³C-¹³C homonuclear mixing. This double editing leads to simplification of HCCH protein side chain spectra that otherwise are prone to spectral overlap. Another interesting feature is amino acid selectivity, i.e. that the presence of certain correlations in a doubly edited HCCH subspectrum gives a clue as to assignment to a particular subgroup of amino acids or segments thereof. Finally, the selection of two different multiplicities in the two editing steps leads to diagonal peak suppression in the ¹H-¹H (3D spectrum recorded with two ¹H and one ¹³C dimension) or the ¹³C-¹³C (3D spectrum recorded with one ¹H and two ¹³C dimensions) two-dimensional projection. The new experiment is demonstrated using a ¹³C,¹⁵N-labeled protein sample, chymotrypsin inhibitor 2, at 500 MHz.     

Global fold of human cannabinoid type 2 receptor probed by solid‐state 13C‐, 15N‐MAS NMR and molecular dynamics simulations

The global fold of human cannabinoid type 2 (CB₂) receptor in the agonist‐bound active state in lipid bilayers was investigated by solid‐state ¹³C‐ and ¹⁵N magic‐angle spinning (MAS) NMR, in combination with chemical‐shift prediction from a structural model of the receptor obtained by microsecond‐long molecular dynamics (MD) simulations. Uniformly ¹³C‐ and ¹⁵N‐labeled CB₂ receptor was expressed in milligram quantities by bacterial fermentation, purified, and functionally reconstituted into liposomes. ¹³C MAS NMR spectra were recorded without sensitivity enhancement for direct comparison of C_α, C_β, and C?O bands of superimposed resonances with predictions from protein structures generated by MD. The experimental NMR spectra matched the calculated spectra reasonably well indicating agreement of the global fold of the protein between experiment and simulations. In particular, the ¹³C chemical shift distribution of C_α resonances was shown to be very sensitive to both the primary amino acid sequence and the secondary structure of CB₂. Thus the shape of the C_α band can be used as an indicator of CB₂ global fold. The prediction from MD simulations indicated that upon receptor activation a rather limited number of amino acid residues, mainly located in the extracellular Loop 2 and the second half of intracellular Loop 3, change their chemical shifts significantly (≥1.5 ppm for carbons and ≥5.0 ppm for nitrogens). Simulated two‐dimensional ¹³C_α(i)? ¹³C?O(i) and ¹³C?O(i)? ¹⁵NH(i + 1) dipolar‐interaction correlation spectra provide guidance for selective amino acid labeling and signal assignment schemes to study the molecular mechanism of activation of CB₂ by solid‐state MAS NMR. Proteins 2014; 82:452–465. © 2013 Wiley Periodicals, Inc.     

13C spin relaxation measurements in RNA: Sensitivity and resolution improvement using spin-state selective correlation experiments

A set of new NMR pulse sequences has been designed for the measurement of ¹³C relaxation rate constants in RNA and DNA bases: the spin-lattice relaxation rate constant R(C_z), the spin-spin relaxation rate constant R(C₊), and the CSA-dipolar cross-correlated relaxation rate constant . The use of spin-state selective correlation techniques provides increased sensitivity and spectral resolution. Sensitivity optimised C-C filters are included in the pulse schemes for the suppression of signals originating from undesired carbon isotopomers. The experiments are applied to a 15% ¹³C-labelled 33-mer RNA–theophylline complex. The measured ratios indicate that ¹³C CSA tensors do not vary significantly for the same type of carbon (C₂, C₆, C₈), but that they differ from one type to another. In addition, conformational exchange effects in the RNA bases are detected as a change in the relaxation decay of the narrow ¹³C doublet component when varying the spacing of a CPMG pulse train. This new approach allows the detection of small exchange effects with a higher precision compared to conventional techniques.     

High resolution 13C NMR spectra on oriented lipid bilayers: from quantifying the various sources of line broadening to performing 2D experiments with 0.2-0.3 ppm resolution in the carbon dimension

¹³C NMR spectra routinely performed on oriented lipid bilayers display linewidth of 1–2 ppm, although T₂ measurements indicate that 0.1–0.2 ppm could be obtained. We have prepared a DMPC – ¹³C₄-cholesterol (7/3) sample, and oriented the lipid bilayers between glass plates so that the bilayer normal makes an angle of 90° (or of the magic angle) with B₀. We have measured T₂s, CSAs, and linewidths for the choline ¹³C--methyl, the cholesterol-C₄ carbons and the lipid head group phosphorus, at both angles and 313 K. The magnetic field distribution within the sample was calculated using the surface current formalism. The line shapes were simulated as a function of B₀ field inhomogeneities and sample mosaic spread. Both effects contribute to the experimental linewidth. Using three signals of different CSA, we have quantified both contributions and measured the mosaic spread accurately. Direct shimming on a sample signal is essential to obtain sharp resonances and ¹³C labelled choline methyl resonance of DMPC is a good candidate for this task. After optimisation of the important parameters (shimming on the choline resonance, mosaic spread of ±0.30° ), ¹³C linewidth of 0.2–0.3 ppm have been obtained. This newly achieved resolution on bilayers oriented at 90°, has allowed to perform two 2D experiments, with a good sensitivity: 2D PELF (correlation of carbon chemical shifts and C-H dipolar couplings) and 2D D-resolved experiment (correlation of carbon chemical shifts and C-C dipolar couplings). A C-C dipolar coupling of 35 ± 2 Hz between the choline methyl carbons was determined.     

Carbon-13 NMR studies of C2H5N13C binding to various hemoproteins

The addition of an excess of C₂H₅N¹³C to myoglobin and human adult and fetal hemoglobins, gives three characteristic NMR spectra with new ¹³C resonances respectively at δ = ?10,56 ppm, δ = ?7,03 and ?7,95 ppm and δ = ?6,28 and ?7,95 ppm (CH₃CO₂Na as external standard). These signals correspond to the C₂H₅N¹³C bound to the Fe(II) of the different heme units, according to CO exchange experiments. Characteristic resonances can be assigned to C₂H₅N¹³C bound to α, β and γ subunits. C₂H₅N¹³C appears as a more sensitive probe than ¹³CO for hemoprotein NMR studies.     

Residue-specific membrane location of peptides and proteins using specifically and extensively deuterated lipids and 13C–2H rotational-echo double-resonance solid-state NMR

Residue-specific location of peptides in the hydrophobic core of membranes was examined using ¹³C–²H REDOR and samples in which the lipids were selectively deuterated. The transmembrane topology of the KALP peptide was validated with this approach with substantial dephasing observed for deuteration in the bilayer center and reduced or no dephasing for deuteration closer to the headgroups. Insertion of β sheet HIV and helical and β sheet influenza virus fusion peptides into the hydrophobic core of the membrane was validated in samples with extensively deuterated lipids.     

Rapid measurement of long-range distances in proteins by multidimensional <Superscript>13</Superscript>C–<Superscript>19</Superscript>F REDOR NMR under fast magic-angle spinning

The ability to simultaneously measure many long-range distances is critical to efficient and accurate determination of protein structures by solid-state NMR (SSNMR). So far, the most common distance constraints for proteins are ¹³C–¹⁵N distances, which are usually measured using the rotational-echo double-resonance (REDOR) technique. However, these measurements are restricted to distances of up to ~?5 Å due to the low gyromagnetic ratios of ¹⁵N and ¹³C. Here we present a robust 2D ¹³C–¹⁹F REDOR experiment to measure multiple distances to ~?10 Å. The technique targets proteins that contain a small number of recombinantly or synthetically incorporated fluorines. The ¹³C–¹⁹F REDOR sequence is combined with 2D ¹³C–¹³C correlation to resolve multiple distances in highly ¹³C-labeled proteins. We show that, at the high magnetic fields which are important for obtaining well resolved ¹³C spectra, the deleterious effect of the large ¹⁹F chemical shift anisotropy for REDOR is ameliorated by fast magic-angle spinning and is further taken into account in numerical simulations. We demonstrate this 2D ¹³C–¹³C resolved ¹³C–¹⁹F REDOR technique on ¹³C, ¹⁵N-labeled GB1. A 5-¹⁹F-Trp tagged GB1 sample shows the extraction of distances to a single fluorine atom, while a 3-¹⁹F-Tyr labeled GB1 sample allows us to evaluate the effects of multi-spin coupling and statistical ¹⁹F labeling on distance measurement. Finally, we apply this 2D REDOR experiment to membrane-bound influenza B M2 transmembrane peptide, and show that the distance between the proton-selective histidine residue and the gating tryptophan residue differs from the distances in the solution NMR structure of detergent-bound BM2. This 2D ¹³C–¹⁹F REDOR technique should facilitate SSNMR-based protein structure determination by increasing the measurable distances to the ~?10 Å range.     

Characterization of slow conformational dynamics in solids: dipolar CODEX

A solid state NMR experiment is introduced for probing relatively slow conformational exchange, based on dephasing and refocusing dipolar couplings. The method is closely related to the previously described Centerband-Only Detection of Exchange or CODEX experiment. The use of dipolar couplings for this application is advantageous because their values are known a priori from molecular structures, and their orientations and reorientations relate in a simple way to molecular geometry and motion. Furthermore the use of dipolar couplings in conjunction with selective isotopic enrichment schemes is consistent with selection for unique sites in complex biopolymers. We used this experiment to probe the correlation time for the motion of ¹³C, ¹⁵N enriched urea molecules within their crystalline lattice.     

C4′/H4′ selective,non-uniformly sampled 4D HC(P)CH experiment for sequential assignments of 13C-labeled RNAs

A through bond, C4′/H4′ selective, “out and stay” type 4D HC(P)CH experiment is introduced which provides sequential connectivity via H4′_(i)–C4′_(i)–C4′_(i?1)–H4′_(i?1) correlations. The ³¹P dimension (used in the conventional 3D HCP experiment) is replaced with evolution of better dispersed C4′ dimension. The experiment fully utilizes ¹³C-labeling of RNA by inclusion of two C4′ evolution periods. An additional evolution of H4′ is included to further enhance peak resolution. Band selective ¹³C inversion pulses are used to achieve selectivity and prevent signal dephasing due to the of C4′–C3′ and C4′–C5′ homonuclear couplings. For reasonable resolution, non-uniform sampling is employed in all indirect dimensions. To reduce sensitivity losses, multiple quantum coherences are preserved during shared-time evolution and coherence transfer delays. In the experiment the intra-nucleotide peaks are suppressed whereas inter-nucleotide peaks are enhanced to reduce the ambiguities. The performance of the experiment is verified on a fully ¹³C, ¹⁵N-labeled 34-nt hairpin RNA comprising typical structure elements.     

C(6)-[benzene ring]-indole-3-acetic Acid: a new internal standard for quantitative mass spectral analysis of indole-3-acetic Acid in plants

Indole-3-acetic acid (IAA) labeled with ¹³C in the six carbons of the benzene ring is described for use as an internal standard for quantitative mass spectral analysis of IAA by gas chromatography/selected ion monitoring. [¹³C₆]IAA was compared to the available deuterium labeled compounds and shown to offer the advantages of nonexchangeability of the isotope label, high isotopic enrichment, and chromatographic properties identical to that of the unlabeled compound. The utility of [¹³C₆]IAA for measurement of endogenous IAA levels was demonstrated by analysis of IAA in Lemna gibba G-3.     

Carbon-13 spin-lattice relaxation studies of intramolecular motion in lysine and a series of oligolysines

¹³C Spin-lattice relaxation times T₁ for individual carbon nuclei have been measured in a series of oligo-l-lysines, as well as lysine and glycine monomers. Anomalous behavior of profiles of T₁ versus pD occurs for lysine and glycine; the T₁ values of the C_α and CO groups are maximal at pH values corresponding to zwitterionic structures. This is interpreted in terms of the hindered intramolecular rotation around the carbonyl-C_α bond at acidic and basic pD values. Lysine monomer manifests a much less pronounced increase in T₁ values from the α- to the ?-carbon than does lysine in an oligomer or polymer. The rate of reorientation of the C_α and C_β carbons of N-terminal groups are faster than those of the central and C-terminal residues, especially at pD greater than 10 for tri-l-lysine hydrochloride and penta-l-lysine acetate. This is interpreted in terms of interaction between the ?-amino groups and the negatively charged carboxyl groups at pD < 10. Segmental motion is shown to make a significant contribution to relaxation of side-chain carbons, making them less sensitive to molecular size than the carbonyl carbons.