Optimal methyl labeling for studies of supra-molecular systems

Selective methyl labeling combined with HMQC spectroscopy that exploits a TROSY effect in ¹³CH₃ spin systems has significantly extended the utility of solution NMR spectroscopy in studies of high molecular weight particles. Herein we compare the utility of ¹³CH₃- versus ¹³CHD₂-labeling of Ile, Leu, Val probes in supra-molecular systems through quantification of relative signal-to-noise ratios in optimized spectra of highly deuterated, ¹³CH₃- and ¹³CHD₂-labeled samples of the half proteasome (α₇α₇, 360 kDa). It is shown that the sensitivity of spectra recorded on Ile, Leu, Val ¹³CH₃-labeled samples is between 1.5 and 2 fold higher than the corresponding data sets obtained on α₇α₇ with ¹³CHD₂ probes. Thus, labeling of supra-molecules with ¹³CH₃ isotopomers remains the method of choice, but in applications where ¹³CHD₂ moieties are required, sensitivity will in general not be limiting.     

Selective <Superscript>1</Superscript>H-<Superscript>13</Superscript>C NMR spectroscopy of methyl groups in residually protonated samples of large proteins

Methyl ¹³CHD₂ isotopomers of all methyl-containing amino-acids can be observed in residually protonated samples of large proteins obtained from [U-¹³C,¹H]-glucose/D₂O-based bacterial media, with sensitivity sufficient for a number of NMR applications. Selective detection of some subsets of methyl groups (Ala^β, Thr^γ2) is possible using simple ‘out-and-back’ NMR methodology. Such selective methyl-detected ‘out-and-back’ NMR experiments allow complete assignments of threonine γ2 methyls in residually protonated, [U-¹³C,¹H]-glucose/D₂O-derived samples of an 82-kDa enzyme Malate Synthase G. [U-¹³C,¹H]-glucose/D₂O-derived protein samples are relatively inexpensive and are usually available at very early stages of any NMR study of high-molecular-weight systems.     

Resolution-optimized NMR measurement of <Superscript>1</Superscript>D<Subscript>CH</Subscript>, <Superscript>1</Superscript>D<Subscript>CC</Subscript> and <Superscript>2</Superscript>D<Subscript>CH</Subscript> residual dipolar couplings in nucleic acid bases

New methods are described for accurate measurement of multiple residual dipolar couplings in nucleic acid bases. The methods use TROSY-type pulse sequences for optimizing resolution and sensitivity, and rely on the E.COSY principle to measure the relatively small two-bond ²D_CH couplings at high precision. Measurements are demonstrated for a 24-nt stem-loop RNA sequence, uniformly enriched in ¹³C, and aligned in Pf1. The recently described pseudo-3D method is used to provide homonuclear ¹H-¹H decoupling, which minimizes cross-correlation effects and optimizes resolution. Up to seven ¹H-¹³C and ¹³C-¹³C couplings are measured for pyrimidines (U and C), including ¹D_C5H5, ¹D_C6H6, ²D_C5H6, ²D_C6H5, ¹D_C5C4, ¹D_C5C6, and ²D_C4H5. For adenine, four base couplings (¹D_C2H2, ¹D_C8H8, ¹D_C4C5, and ¹D_C5C6) are readily measured whereas for guanine only three couplings are accessible at high relative accuracy (¹D_C8H8, ¹D_C4C5, and ¹D_C5C6). Only three dipolar couplings are linearly independent in planar structures such as nucleic acid bases, permitting cross validation of the data and evaluation of their accuracies. For the vast majority of dipolar couplings, the error is found to be less than ±3% of their possible range, indicating that the measurement accuracy is not limiting when using these couplings as restraints in structure calculations. Reported isotropic values of the one- and two-bond J couplings cluster very tightly for each type of nucleotide.     

<Superscript>15</Superscript>N and <Superscript>13</Superscript>C- SOFAST-HMQC editing enhances 3D-NOESY sensitivity in highly deuterated,selectively [<Superscript>1</Superscript>H,<Superscript>13</Superscript>C]-labeled proteins

The ongoing NMR method development effort strives for high quality multidimensional data with reduced collection time. Here, we apply ‘SOFAST-HMQC’ to frequency editing in 3D NOESY experiments and demonstrate the sensitivity benefits using highly deuterated and ¹⁵N, methyl labeled samples in H₂O. The experiments benefit from a combination of selective T ₁ relaxation (or L-optimized effect), from Ernst angle optimization and, in certain types of experiments, from using the mixing time for both NOE buildup and magnetization recovery. This effect enhances sensitivity by up to 2.4× at fast pulsing versus reference HMQC sequences of same overall length and water suppression characteristics. Representative experiments designed to address interesting protein NMR challenges are detailed. Editing capabilities are exploited with heteronuclear ¹⁵N,¹³C-edited, or with diagonal-free ¹³C aromatic/methyl-resolved 3D-SOFAST-HMQC–NOESY–HMQC. The latter experiment is used here to elucidate the methyl-aromatic NOE network in the hydrophobic core of the 19 kDa FliT-FliJ flagellar protein complex. Incorporation of fast pulsing to reference experiments such as 3D-NOESY–HMQC boosts digital resolution, simplifies the process of NOE assignment and helps to automate protein structure determination.     

Optimized labeling of 13CHD2 methyl isotopomers in perdeuterated proteins: Potential advantages for 13C relaxation studies of methyl dynamics of larger proteins

¹³CHD₂ methyl isotopomers are particularly useful to study methyl dynamics in proteins because, as compared with other methyl isotopomers, the ¹³C relaxation mechanism for this isotopomer is straightforward. However, in the case of proteins, where ()² 1, the refocused INEPT pulse sequence does not completely suppress unwanted ¹³CH₃ signals. The presence of weak ¹³CH₃ peaks is usually not a serious problem for smaller proteins because there are relatively few methyl signals and they are sharp; however, signal overlap becomes more common as the size of the protein increases. We overcome this problem by preparing a protein using a 98% D₂O cell culture medium containing 3-¹³C pyruvic acid, 50–60% deuterated at the 3-position, and 4-¹³C 2-ketobutyric acid, 98% and 62% deuterated at the 3- and 4-positions, respectively. This approach significantly reduces the population of the CH₃ isotopomer while optimizing the production of ¹³CHD₂, the isotopomer desired for ¹³C relaxation measurements. In larger proteins where the deuterium T₂ may be too short to measure accurately, we also suggest the alternative measurement of the proton T₂ of the ¹³CH₂D methyl isotopomer, because these protons are well-isolated from other protons in these highly deuterated samples.     

Structure determination of proteins in <Superscript>2</Superscript>H<Subscript>2</Subscript>O solution aided by a deuterium-decoupled 3D HCA(N)CO experiment

We developed an NMR pulse sequence, 3D HCA(N)CO, to correlate the chemical shifts of protein backbone ¹Hα and ¹³Cα to those of ¹³C′ in the preceding residue. By applying ²H decoupling, the experiment was accomplished with high sensitivity comparable to that of HCA(CO)N. When combined with HCACO, HCAN and HCA(CO)N, the HCA(N)CO sequence allows the sequential assignment using backbone ¹³C′ and amide ¹⁵N chemical shifts without resort to backbone amide protons. This assignment strategy was demonstrated for ¹³C/¹⁵N-labeled GB1 dissolved in ²H₂O. The quality of the GB1 structure determined in ²H₂O was similar to that determined in H₂O in spite of significantly smaller number of NOE correlations. Thus this strategy enables the determination of protein structures in ²H₂O or H₂O at high pH values.     

Optimizing <Superscript>19</Superscript>F NMR protein spectroscopy by fractional biosynthetic labeling

In protein NMR experiments which employ nonnative labeling, incomplete enrichment is often associated with inhomogeneous line broadening due to the presence of multiple labeled species. We investigate the merits of fractional enrichment strategies using a monofluorinated phenylalanine species, where resolution is dramatically improved over that achieved by complete enrichment. In NMR studies of calmodulin, a 148 residue calcium binding protein, ¹⁹F and ¹H-¹⁵N HSQC spectra reveal a significant extent of line broadening and the appearance of minor conformers in the presence of complete (>95%) 3-fluorophenylalanine labeling. The effects of varying levels of enrichment of 3-fluorophenylalanine (i.e. between 3 and >95%) were further studied by ¹⁹F and ¹H-¹⁵N HSQC spectra,¹⁵N T₁ and T₂ relaxation measurements, ¹⁹F T₂ relaxation, translational diffusion and heat denaturation experiments via circular dichroism. Our results show that while several properties, including translational diffusion and thermal stability show little variation between non-fluorinated and >95% ¹⁹F labeled samples, ¹⁹F and ¹H-¹⁵N HSQC spectra show significant improvements in line widths and resolution at or below 76% enrichment. Moreover, high levels of fluorination (>80%) appear to increase protein disorder as evidenced by backbone ¹⁵N dynamics. In this study, reasonable signal to noise can be achieved between 60–76% ¹⁹F enrichment, without any detectable perturbations from labeling.     

Selective backbone labeling of proteins using {1,2-<Superscript>13</Superscript>C<Subscript>2</Subscript>}-pyruvate as carbon source

A simple isotope labeling approach for selective ¹³C/¹⁵N backbone labeling of proteins is described. Using {1,2-¹³C₂}-pyruvate as the sole carbon source in bacterial growth media, selective incorporation of ¹³C^α-¹³CO spin-pairs into the backbones of protein molecules with medium-to-high levels of ¹³C-enrichment is possible for a subset of 12 amino acids. The isotope labeling scheme has been tested on a pair of proteins—a 7-kDa immunoglobulin binding domain B1 of streptococcal protein G and an 82-kDa enzyme malate synthase G. A number of protein NMR applications are expected to benefit from the {1,2-¹³C₂}-pyruvate based protein production.     

Asymmetry of <Superscript>13</Superscript>C labeled 3-pyruvate affords improved site specific labeling of RNA for NMR spectroscopy

Selective isotopic labeling provides an unparalleled window within which to study the structure and dynamics of RNAs by high resolution NMR spectroscopy. Unlike commonly used carbon sources, the asymmetry of ¹³C-labeled pyruvate provides selective labeling in both the ribose and base moieties of nucleotides using E. coli variants, that until now were not feasible. Here we show that an E. coli mutant strain that lacks succinate and malate dehydrogenases (DL323) and grown on [3-¹³C]-pyruvate affords ribonucleotides with site specific labeling at C5′ (~95%) and C1′ (~42%) and minimal enrichment elsewhere in the ribose ring. Enrichment is also achieved at purine C2 and C8 (~95%) and pyrimidine C5 (~100%) positions with minimal labeling at pyrimidine C6 and purine C5 positions. These labeling patterns contrast with those obtained with DL323 E. coli grown on [1, 3-¹³C]-glycerol for which the ribose ring is labeled in all but the C4′ carbon position, leading to multiplet splitting of the C1′, C2′ and C3′ carbon atoms. The usefulness of these labeling patterns is demonstrated with a 27-nt RNA fragment derived from the 30S ribosomal subunit. Removal of the strong magnetic coupling within the ribose and base leads to increased sensitivity, substantial simplification of NMR spectra, and more precise and accurate dynamic parameters derived from NMR relaxation measurements. Thus these new labels offer valuable probes for characterizing the structure and dynamics of RNA that were previously limited by the constraint of uniformly labeled nucleotides.     

An intraresidual i(HCA)CO(CA)NH experiment for the assignment of main-chain resonances in <Superscript>15</Superscript>N, <Superscript>13</Superscript>C labeled proteins

An improved pulse sequence, intraresidual i(HCA)CO(CA)NH, is described for establishing solely ¹³C′(i), ¹⁵N(i), ¹H^N(i) connectivities in uniformly ¹⁵N/¹³C-labeled proteins. In comparison to the “out-and-back” style intra-HN(CA)CO experiment, the new pulse sequence offers at least two-fold higher experimental resolution in the ¹³C′ dimension and on average 1.6 times higher sensitivity especially for residues in α-helices. Performance of the new experiment was tested on a small globular protein ubiquitin and an intrinsically unfolded 110-residue cancer/testis antigen CT16/PAGE5. Use of intraresidual i(HCA)CO(CA)NH experiment in combination with the established HNCO experiment was crucial for the assignment of highly disordered CT16.     

Conformational dependence of <Superscript>13</Superscript>C shielding and coupling constants for methionine methyl groups

Methionine residues fulfill a broad range of roles in protein function related to conformational plasticity, ligand binding, and sensing/mediating the effects of oxidative stress. A high degree of internal mobility, intrinsic detection sensitivity of the methyl group, and low copy number have made methionine labeling a popular approach for NMR investigation of selectively labeled protein macromolecules. However, selective labeling approaches are subject to more limited information content. In order to optimize the information available from such studies, we have performed DFT calculations on model systems to evaluate the conformational dependence of ³ J _CSCC, ³ J _CSCH, and the isotropic shielding, σ_iso. Results have been compared with experimental data reported in the literature, as well as data obtained on [methyl-¹³C]methionine and on model compounds. These studies indicate that relative to oxygen, the presence of the sulfur atom in the coupling pathway results in a significantly smaller coupling constant, ³ J _CSCC/³ J _COCC ~ 0.7. It is further demonstrated that the ³ J _CSCH coupling constant depends primarily on the subtended CSCH dihedral angle, and secondarily on the CSCC dihedral angle. Comparison of theoretical shielding calculations with the experimental shift range of the methyl group for methionine residues in proteins supports the conclusion that the intra-residue conformationally-dependent shift perturbation is the dominant determinant of δ¹³Cε. Analysis of calmodulin data based on these calculations indicates that several residues adopt non-standard rotamers characterized by very large ~100° χ³ values. The utility of the δ¹³Cε as a basis for estimating the gauche/trans ratio for χ³ is evaluated, and physical and technical factors that limit the accuracy of both the NMR and crystallographic analyses are discussed.     

Broadband <Superscript>15</Superscript>N–<Superscript>13</Superscript>C dipolar recoupling via symmetry-based RF pulse schemes at high MAS frequencies

An approach for generating efficient RN_n^{n_S, n_k} {\rm{RN}}_{n}^{\nu_{\rm{S}}, {\nu_{\rm{k}}}} symmetry-based dual channel RF pulse schemes for γ-encoded broadband ¹⁵N–¹³C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic “R” element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic “R” element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and ¹⁵N–¹³C chemical shift correlation experiments. Employing such ¹³C–¹⁵N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated.     

Simulation and optical spectroscopy of a DC discharge in a CH<Subscript>4</Subscript>/H<Subscript>2</Subscript>/N<Subscript>2</Subscript> mixture during deposition of nanostructured carbon films

Two-dimensional numerical simulations of a dc discharge in a CH₄/H₂/N₂ mixture in the regime of deposition of nanostructured carbon films are carried out with account of the cathode electron beam effects. The distributions of the gas temperature and species number densities are calculated, and the main plasmachemical kinetic processes governing the distribution of methyl radicals above the substrate are analyzed. It is shown that the number density of methyl radicals above the substrate is several orders of magnitude higher than the number densities of other hydrocarbon radicals, which indicates that the former play a dominant role in the growth of nanostructured carbon films. The model is verified by comparing the measured optical emission profiles of the H(n ≡ 3), C ₂ ^* , CH*, and CN* species and the calculated number densities of excited species, as well as the measured and calculated values of the discharge voltage and heat fluxes onto the electrodes and reactor walls. The key role of ion–electron recombination and dissociative excitation of H₂, C₂H₂, CH₄, and HCN molecules in the generation of emitting species (first of all, in the cold regions adjacent to the electrodes) is revealed.     

HNCO-based measurement of one-bond amide <Superscript>15</Superscript>N-<Superscript>1</Superscript>H couplings with optimized precision

A pair of 3D HNCO-based experiments have been developed with the aim of optimizing the precision of measurement of ¹J_NH couplings. Both pulse sequences record ¹J_NH coupling evolution during the entire constant time interval that ¹⁵N magnetization is dephasing or rephasing with respect to the directly bonded ¹³C′ nucleus, with ¹⁵N¹³C′ multiple quantum coherence maintained during the ¹³C′ evolution period. The first experiment, designed for smaller proteins, produces an apparent doubling of the ¹J_NH coupling without any accompanying increases in line width. The second experiment is a J-scaled TROSY-HNCO experiment in which the ¹J_NH coupling is measured by frequency difference between resonances offset symmetrically about the position of the downfield component of the ¹⁵N doublet (i.e. the TROSY resonance). This experiment delivers significant gains in precision of ¹J_NH coupling measurement compared to existing J-scaled TROSY-HNCO experiments. With the proper choice of acquisition parameters and sufficient sensitivity to acquire a 3D TROSY-HNCO experiment, it is shown that ¹J_NH couplings can be measured with a precision which approaches or exceeds the precision of measurement with which the frequency of the TROSY resonance itself can be determined.     

REGIOSELECTIVE SYNTHESIS OF 1,3,5-13C3 AND 2,4-13C2-LABELED 2-DEOXYRIBONOLACTONES

The synthesis of 1,3,5-¹³C₃- and 2,4-¹³C₂-labeled 5-O-bromobenzyl-2-deoxyribonolactones 2, precursors to ¹³C-enriched nucleoside phosphoramidites for solid-phase synthesis of DNA oligonucletides, is described. An equimolar combination of these two multiply labeled lactones affords a “population-labeled” mixture of isotopomers which exhibits an approximately 50-fold increase in the sensitivity of ¹³C-NMR compared to natural abundance measurements. The ¹³C-¹³C 2-bond and 4-bond coupling constants are reported for the lactones; all are < 2 Hz, confirming that this labeling scheme should be especially useful for NMR-relaxation measurements.     

HNCA-TOCSY-CANH experiments with alternate <Superscript>13</Superscript>C-<Superscript>12</Superscript>C labeling: a set of 3D experiment with unique supra-sequential information for mainchain resonance assignment

Described here is a set of three-dimensional (3D) NMR experiments that rely on CACA-TOCSY magnetization transfer via the weak ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} coupling. These pulse sequences, which resemble recently described ¹³C detected CACA-TOCSY (Takeuchi et al. 2010) experiments, are recorded in ¹H₂O, and use ¹H excitation and detection. These experiments require alternate ¹³C-¹²C labeling together with perdeuteration, which allows utilizing the small ³ \textJ_{\textCa\textCa} ^{ 3} {\text{J}}_{{{\text{C}}\alpha {\text{C}}\alpha }} scalar coupling that is otherwise masked by the stronger ¹J_CC couplings in uniformly ¹³C labeled samples. These new experiments provide a unique assignment ladder-mark that yields bidirectional supra-sequential information and can readily straddle proline residues. Unlike the conventional HNCA experiment, which contains only sequential information to the ^{1 3} \textC^a ^{ 1 3} {\text{C}}^{\alpha } of the preceding residue, the 3D hnCA-TOCSY-caNH experiment can yield sequential correlations to alpha carbons in positions i−1, i + 1 and i−2. Furthermore, the 3D hNca-TOCSY-caNH and Hnca-TOCSY-caNH experiments, which share the same magnetization pathway but use a different chemical shift encoding, directly couple the ¹⁵N-¹H spin pair of residue i to adjacent amide protons and nitrogens at positions i−2, i−1, i + 1 and i + 2, respectively. These new experimental features make protein backbone assignments more robust by reducing the degeneracy problem associated with the conventional 3D NMR experiments.     

Refocusing revisited: An optimized,gradient-enhanced refocused HSQC and its applications in 2D and 3D NMR and in deuterium exchange experiments

Summary 2D ¹⁵N-¹H correlation spectra are ideal for measuring backbone amide populations to determine amide exchange protection factors in studies of protein folding or other structural features. Most protein NMR spectroscopists use HSQC, which has been shown to be generally superior to HMQC in both resolution and sensitivity. The refocused HSQC experiment is intrinsically less sensitive than the regular HSQC, due to T₂ relaxation during the refocusing delays. However, we show here that, when high ¹⁵N resolution is needed, an optimized refocused HSQC sequence that utilizes a semi-constant time evolution period and pulsed field gradients has better signal-to-noise ratio and resolution, and integrates more accurately, than a similar HSQC. The differences are demonstrated on a 20 kDa protein. The technique can also be applied to 3D NOESY experiments to eliminate strong NH₂ geminal peaks and their truncation artefacts at a modest cost in sensitivity.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Pressure-dependent <Superscript>13</Superscript>C chemical shifts in proteins: origins and applications

Pressure-dependent ¹³C chemical shifts have been measured for aliphatic carbons in barnase and Protein G. Up to 200 MPa (2 kbar), most shift changes are linear, demonstrating pressure-independent compressibilities. CH₃, CH₂ and CH carbon shifts change on average by +0.23, −0.09 and −0.18 ppm, respectively, due to a combination of bond shortening and changes in bond angles, the latter matching one explanation for the γ-gauche effect. In addition, there is a residue-specific component, arising from both local compression and conformational change. To assess the relative magnitudes of these effects, residue-specific shift changes for protein G were converted into structural restraints and used to calculate the change in structure with pressure, using a genetic algorithm to convert shift changes into dihedral angle restraints. The results demonstrate that residual ¹³Cα shifts are dominated by dihedral angle changes and can be used to calculate structural change, whereas ¹³Cβ shifts retain significant dependence on local compression, making them less useful as structural restraints.     

Optimization of amino acid type-specific <Superscript>13</Superscript>C and <Superscript>15</Superscript>N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with ¹³C or/and ¹⁵N such that cross peaks between ¹³CO(i – 1) and ¹⁵NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with ¹³C and the second with ¹⁵N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B₂R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.