Selective suppression of excipient signals in 2D <Superscript>1</Superscript>H–<Superscript>13</Superscript>C methyl spectra of biopharmaceutical products

While the use of ¹H–¹³C methyl correlated NMR spectroscopy at natural isotopic abundance has been demonstrated as feasible on protein therapeutics as large as monoclonal antibodies, spectral interference from aliphatic excipients remains a significant obstacle to its widespread application. These signals can cause large baseline artifacts, obscure protein resonances, and cause dynamic range suppression of weak peaks in non-uniform sampling applications, thus hampering both traditional peak-based spectral analyses as well as emerging chemometric methods of analysis. Here we detail modifications to the 2D ¹H–¹³C gradient-selected HSQC experiment that make use of selective pulsing techniques for targeted removal of interfering excipient signals in spectra of the NISTmAb prepared in several different formulations. This approach is demonstrated to selectively reduce interfering excipient signals while still yielding 2D spectra with only modest losses in protein signal. Furthermore, it is shown that spectral modeling based on the SMILE algorithm can be used to simulate and subtract any residual excipient signals and their attendant artifacts from the resulting 2D NMR spectra.     

<Superscript>2</Superscript>H–<Superscript>13</Superscript>C correlation solid-state NMR for investigating dynamics and water accessibilities of proteins and carbohydrates

Site-specific determination of molecular motion and water accessibility by indirect detection of ²H NMR spectra has advantages over dipolar-coupling based techniques due to the large quadrupolar couplings and the ensuing high angular resolution. Recently, a Rotor Echo Short Pulse IRrAdiaTION mediated cross polarization (^RESPIRATIONCP) technique was developed, which allowed efficient transfer of ²H magnetization to ¹³C at moderate ²H radiofrequency field strengths available on most commercial MAS probes. In this work, we investigate the ²H–¹³C magnetization transfer characteristics of one-bond perdeuterated CD _n spin systems and two-bond H/D exchanged C–(O)–D and C–(N)–D spin systems in carbohydrates and proteins. Our results show that multi-bond, broadband ²H–¹³C polarization transfer can be achieved using ²H radiofrequency fields of ~50 kHz, relatively short contact times of 1.3–1.7 ms, and with sufficiently high sensitivity to enable 2D ²H–¹³C correlation experiments with undistorted ²H spectra in the indirect dimension. To demonstrate the utility of this ²H–¹³C technique for studying molecular motion, we show ²H–¹³C correlation spectra of perdeuterated bacterial cellulose, whose surface glucan chains exhibit a motionally averaged C6 ²H quadrupolar coupling that indicates fast trans-gauche isomerization about the C5–C6 bond. In comparison, the interior chains in the microfibril core are fully immobilized. Application of the ²H–¹³C correlation experiment to H/D exchanged Arabidopsis primary cell walls show that the O–D quadrupolar spectra of the highest polysaccharide peaks can be fit to a two-component model, in which 74% of the spectral intensity, assigned to cellulose, has a near-rigid-limit coupling, while 26% of the intensity, assigned to matrix polysaccharides, has a weakened coupling of 50 kHz. The latter O–D quadrupolar order parameter of 0.22 is significantly smaller than previously reported C–D dipolar order parameters of 0.46–0.55 for pectins, suggesting that additional motions exist at the C–O bonds in the wall polysaccharides. ²H–¹³C polarization transfer profiles are also compared between statistically deuterated and H/D exchanged GB1.     

Carbonyl carbon label selective (CCLS) <Superscript>1</Superscript>H–<Superscript>15</Superscript>N HSQC experiment for improved detection of backbone <Superscript>13</Superscript>C–<Superscript>15</Superscript>N cross peaks in larger proteins

We present a highly sensitive pulse sequence, carbonyl carbon label selective ¹H–¹⁵N HSQC (CCLS-HSQC) for the detection of signals from ¹H–¹⁵N units involved in ¹³C′–¹⁵N linkages. The CCLS-HSQC pulse sequence utilizes a modified ¹⁵N CT evolution period equal to 1/( ) (∼33 ms) to select for ¹³C′–¹⁵N pairs. By collecting CCLS-HSQC and HNCO data for two proteins (8 kDa ubiquitin and 20 kDa HscB) at various temperatures (5–40°C) in order to vary correlation times, we demonstrate the superiority of the CCLS-HSQC pulse sequence for proteins with long correlation times (i.e. higher molecular weight). We then show that the CCLS-HSQC experiment yields assignments in the case of a 41 kDa protein incorporating pairs of ¹⁵N- and ¹³C′-labeled amino acids, where a TROSY 2D-HN(CO) had failed. Although the approach requires that the ¹H–¹⁵N HSQC cross peaks be observable, it does not require deuteration of the protein. The method is suitable for larger proteins and is less affected by conformational exchange than HNCO experiments, which require a longer period of transverse ¹⁵N magnetization. The method also is tolerant to the partial loss of signal from isotopic dilution (scrambling). This approach will be applicable to families of proteins that have been resistant to NMR structural and dynamic analysis, such as large enzymes, and partially folded or unfolded proteins.     

Measurement of imino <Superscript>1</Superscript>H–<Superscript>1</Superscript>H residual dipolar couplings in RNA

Imino ¹H–¹⁵N residual dipolar couplings (RDCs) provide additional structural information that complements standard ¹H–¹H NOEs leading to improvements in both the local and global structure of RNAs. Here, we report measurement of imino ¹H–¹H RDCs for the Iron Responsive Element (IRE) RNA and native E. coli tRNA^Val using a BEST-Jcomp-HMQC2 experiment. ¹H–¹H RDCs are observed between the imino protons in G–U wobble base pairs and between imino protons on neighboring base pairs in both RNAs. These imino ¹H–¹H RDCs complement standard ¹H–¹⁵N RDCs because the ¹H–¹H vectors generally point along the helical axis, roughly perpendicular to ¹H–¹⁵N RDCs. The use of longitudinal relaxation enhancement increased the signal-to-noise of the spectra by ~3.5-fold over the standard experiment. The ability to measure imino ¹H–¹H RDCs offers a new restraint, which can be used in NMR domain orientation and structural studies of RNAs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Major groove width variations in RNA structures determined by NMR and impact of <Superscript>13</Superscript>C residual chemical shift anisotropy and <Superscript>1</Superscript>H–<Superscript>13</Superscript>C residual dipolar coupling on refinement

Ribonucleic acid structure determination by NMR spectroscopy relies primarily on local structural restraints provided by ¹H– ¹H NOEs and J-couplings. When employed loosely, these restraints are broadly compatible with A- and B-like helical geometries and give rise to calculated structures that are highly sensitive to the force fields employed during refinement. A survey of recently reported NMR structures reveals significant variations in helical parameters, particularly the major groove width. Although helical parameters observed in high-resolution X-ray crystal structures of isolated A-form RNA helices are sensitive to crystal packing effects, variations among the published X-ray structures are significantly smaller than those observed in NMR structures. Here we show that restraints derived from aromatic ¹H– ¹³C residual dipolar couplings (RDCs) and residual chemical shift anisotropies (RCSAs) can overcome NMR restraint and force field deficiencies and afford structures with helical properties similar to those observed in high-resolution X-ray structures.     

Sensitive <Superscript>13</Superscript>C–<Superscript>13</Superscript>C correlation spectra of amyloid fibrils at very high spinning frequencies and magnetic fields

Sensitive 2D solid-state ¹³C–¹³C correlation spectra of amyloid β fibrils have been recorded at very fast spinning frequencies and very high magnetic fields. It is demonstrated that PARIS-xy recoupling using moderate rf amplitudes can provide structural information by promoting efficient magnetization transfer even under such challenging experimental conditions. Furthermore, it has been shown both experimentally and by numerical simulations that the method is not very sensitive to dipolar truncation effects and can reveal direct transfer across distances of about 3.5–4Å.     

A simple strategy for <Superscript>13</Superscript>C,<Superscript>1</Superscript>H labeling at the Ile-γ2 methyl position in highly deuterated proteins

A straightforward approach for the production of highly deuterated proteins labeled with ¹³C and ¹H at Ile-γ2 methyl positions is described. The utility of the methodology is illustrated with an application involving the half proteasome (360 kDa). High quality 2D Ile ¹³C^γ2,¹H^γ2 HMQC data sets, exploiting the methyl-TROSY principle, are recorded with excellent sensitivity and resolution, that compare favorably with Ile ¹³C^δ1,¹H^δ1 spectra. This labeling scheme adds to a growing list of different approaches that are significantly impacting the utility of solution NMR spectroscopy in studies of supra-molecular systems.     

<Superscript>1</Superscript>H, <Superscript>13</Superscript>C,and <Superscript>15</Superscript>N resonance assignments for the pro-inflammatory cytokine interleukin-36α

Interleukin-36α (IL-36α) is a recently characterised member of the interleukin-1 superfamily. It is involved in the pathogenesis of inflammatory arthritis in one third of psoriasis patients. By binding of IL-36α to its receptor IL-36R via the NF-κB pathway other cytokines involved in inflammatory and apoptotic cascade are activated. The efficacy of complex formation is controlled by N-terminal processing. To obtain a more detailed view on the structure function relationship we performed a heteronuclear multidimensional NMR investigation and here report the ¹H, ¹³C, and ¹⁵N resonance assignments for the backbone and side chain nuclei of the pro-inflammatory cytokine interleukin-36α.     

Accurate measurement of <Superscript>15</Superscript>N–<Superscript>13</Superscript>C residual dipolar couplings in nucleic acids

New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ ¹H–¹³C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H₁–C₁–N_1/9 (MQ-HCN-QJ) or H_6/8–C_6/8–N_1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and 1/(2J_NC) (attenuated spectrum). The different types of ¹⁵N–¹³C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time ¹⁵N evolution period and the offset of two frequency-selective ¹³C pulses. The methods are demonstrated for a uniformly ¹³C, ¹⁵N-enriched 24-nucleotide stem-loop RNA sequence, helix-35, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35 sample result in an estimated precision of ca. 0.07 Hz for ¹D_NC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of ¹⁵N_1/9–¹³C₁, ¹⁵N_1/9–¹³C_6/8, and ¹⁵N_1/9–¹³C_2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35, refined using independently measured observables, including ¹³C–¹H, ¹³C–¹³C and ¹H–¹H dipolar couplings.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-0646-2.     

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.     

<Superscript>1</Superscript>H–<Superscript>15</Superscript>N correlation spectroscopy of nanocrystalline proteins

The limits of resolution that can be obtained in ¹H–¹⁵N 2D NMR spectroscopy of isotopically enriched nanocrystalline proteins are explored. Combinations of frequency switched Lee–Goldburg (FSLG) decoupling, fast magic angle sample spinning (MAS), and isotopic dilution via deuteration are investigated as methods for narrowing the amide ¹H resonances. Heteronuclear decoupling of ¹⁵N from the ¹H resonances is also studied. Using human ubiquitin as a model system, the best resolution is most easily obtained with uniformly ²H and ¹⁵N enriched protein where the amides have been exchanged in normal water, MAS at 20 kHz, and WALTZ-16 decoupling of the ¹⁵N nuclei. The combination of these techniques results in average ¹H lines of only 0.26 ppm full width at half maximum. Techniques for optimizing instrument stability and ¹⁵N decoupling are described for achieving the best possible performance in these experiments.     

CACA-TOCSY with alternate <Superscript>13</Superscript>C–<Superscript>12</Superscript>C labeling: a <Superscript>13</Superscript>C<Superscript>α</Superscript> direct detection experiment for mainchain resonance assignment,dihedral angle information,and amino acid type identification

We present a ¹³C direct detection CACA-TOCSY experiment for samples with alternate ¹³C–¹²C labeling. It provides inter-residue correlations between ¹³C^α resonances of residue i and adjacent C^αs at positions i − 1 and i + 1. Furthermore, longer mixing times yield correlations to C^α nuclei separated by more than one residue. The experiment also provides C^α-to-sidechain correlations, some amino acid type identifications and estimates for ψ dihedral angles. The power of the experiment derives from the alternate ¹³C–¹²C labeling with [1,3-¹³C] glycerol or [2-¹³C] glycerol, which allows utilizing the small scalar ³J_CC couplings that are masked by strong ¹J_CC couplings in uniformly ¹³C labeled samples.     

Projected [<Superscript>1</Superscript>H,<Superscript>15</Superscript>N]-HMQC-[<Superscript>1</Superscript>H,<Superscript>1</Superscript>H]-NOESY for large molecular systems: application to a 121 kDa protein-DNA complex

We present a projected [¹H,¹⁵N]-HMQC-[¹H,¹H]-NOESY experiment for observation of NOE interactions between amide protons with degenerate ¹⁵N chemical shifts in large molecular systems. The projection is achieved by simultaneous evolution of the multiple quantum coherence of the nitrogen spin and the attached proton spin. In this way NOE signals can be separated from direct-correlation peaks also in spectra with low resolution by fully exploiting both ¹H and ¹⁵N frequency differences, such that sensitivity can be increased by using short maximum evolution times. The sensitivity of the experiment is not dependent on the projection angle for projections up to 45° and no additional pulses or delays are required as compared to the conventional 2D [¹H,¹⁵N]-HMQC-NOESY. The experiment provides two distinct 2D spectra corresponding to the positive and negative angle projections, respectively. With a linear combination of 1D cross-sections from the two projections the unavoidable sensitivity loss in projection spectra can be compensated for each particular NOE interaction. We demonstrate the application of the novel projection experiment for the observation of an NOE interaction between two sequential glycines with degenerate ¹⁵N chemical shifts in a 121.3 kDa complex of the linker H1 histone protein with a 152 bp linear DNA.     

Facile measurement of <Superscript>1</Superscript>H–<Superscript>15</Superscript>N residual dipolar couplings in larger perdeuterated proteins

We present a simple method, ARTSY, for extracting ¹J_NH couplings and ¹H–¹⁵N RDCs from an interleaved set of two-dimensional ¹H–¹⁵N TROSY-HSQC spectra, based on the principle of quantitative J correlation. The primary advantage of the ARTSY method over other methods is the ability to measure couplings without scaling peak positions or altering the narrow line widths characteristic of TROSY spectra. Accuracy of the method is demonstrated for the model system GB3. Application to the catalytic core domain of HIV integrase, a 36 kDa homodimer with unfavorable spectral characteristics, demonstrates its practical utility. Precision of the RDC measurement is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC spectrum, and is approximately given by 30/(S/N) Hz.     

Spatial variation in flux, δ<Superscript>13</Superscript>C and δ<Superscript>2</Superscript>H of methane in a small Arctic lake with fringing wetland in western Greenland

Small lakes in northern latitudes represent a significant source of CH₄ to the atmosphere that is predicted to increase with warming in the Arctic. Yet, whole-lake CH₄ budgets are lacking as are measurements of δ¹³C-CH₄ and δ²H-CH₄. In this study, we quantify spatial variability of diffusive and ebullitive fluxes of CH₄ and corresponding δ¹³C-CH₄ and δ²H-CH₄ in a small, Arctic lake system with fringing wetland in southwestern Greenland during summer. Net CH₄ flux was highly variable, ranging from an average flux of 7 mg CH₄ m^?2 d^?1 in the deep-water zone to 154 mg CH₄ m^?2 d^?1 along the lake margin. Diffusive flux accounted for ~8.5 % of mean net CH₄ flux, with plant-mediated and ebullitive flux accounting for the balance of the total net flux. Methane content of emitted ebullition was low (mean ± SD 10 ± 17 %) compared to previous studies from boreal lakes and wetlands. Isotopic composition of net CH₄ emissions varied widely throughout the system, with δ¹³C-CH₄ ranging from ?66.2 to ?55.5 ‰, and δ²H-CH₄ ranging from ?345 to ?258 ‰. Carbon isotope composition of CH₄ in ebullitive flux showed wider variation compared to net flux, ranging from ?69.2 to ?49.2 ‰. Dissolved CH₄ concentrations were highest in the sediment and decreased up the water column. Higher concentrations of CH₄ in the hypoxic deep water coincided with decreasing dissolved O₂ concentrations, while methanotrophic oxidation dominated in the epilimnion based upon decreasing concentrations and increasing values of δ¹³C-CH₄ and δ²H-CH₄. The most depleted ¹³C- and ²H-isotopic values were observed in profundal bottom waters and in subsurface profundal sediments. Based upon paired δ¹³C and δ²H observations of CH₄, acetate fermentation was likely the dominant production pathway throughout the system. However, isotopic ratios of CH₄ in deeper sediments were consistent with mixing/transition between CH₄ production pathways, indicating a higher contribution of the CO₂ reduction pathway. The large spatial variability in fluxes of CH₄ and in isotopic composition of CH₄ throughout a single lake system indicates that the underlying mechanisms controlling CH₄ cycling (production, consumption and transport) are spatially heterogeneous. Net flux along the lake margin dominated whole-lake flux, suggesting the nearshore littoral area dominates CH₄ emissions in these systems. Future studies of whole-lake CH₄ budgets should consider this significant spatial heterogeneity.     

The use of <Superscript>1</Superscript>H–<Superscript>31</Superscript>P GHMBC and covariance NMR to unambiguously determine phosphate ester linkages in complex polysaccharide mixtures

Poly- and oligo-saccharides are commonly employed as antigens in many vaccines. These antigens contain phosphoester structural elements that are crucial to the antigenicity, and hence the effectiveness of the vaccine. Nuclear Magnetic Resonance (NMR) is a powerful tool for the site-specific identification of phosphoesters in saccharides. We describe here two advances in the characterization of phosphoesters in saccharides: (1) the use of ¹H–³¹P GHMBC to determine the site-specific identity of phosphoester moieties in heterogeneous mixtures and (2) the use of Unsymmetrical/Generalized Indirect Covariance (U/GIC) to calculate a carbon-phosphorus 2D spectrum. The sensitivity of the ¹H–³¹P GHMBC is far greater than the “standard” ¹H–³¹P GHSQC and allows long-range ^3–5J_HP couplings to be readily detected. This is the first example to be reported of using U/GIC to calculate a carbon-phosphorus spectrum. The U/GIC processing affords, in many cases, a fivefold to tenfold or greater increase in signal-to-noise ratios in the calculated spectrum. When coupled with the high sensitivity of ¹H–³¹P HMBC, U/GIC processing allows the complete and unambiguous assignments of phosphoester moieties present in heterogeneous samples at levels of ~5% (or less) of the total sample, expanding the breadth of samples that NMR can be used to analyze. This new analytical technique is generally applicable to any NMR-observable phosphoester.