Design of high-power,broadband 180° pulses and mixing sequences for fast MAS solid state chemical shift correlation NMR spectroscopy

An approach for the design of high-power, broadband 180° pulses and mixing sequences for generating dipolar and scalar coupling mediated ¹³C–¹³C chemical shift correlation spectra of isotopically labelled biological systems at fast magic-angle spinning frequencies without ¹H decoupling during mixing is presented. Considering RF field strengths in the range of 100–120 kHz, as typically available in MAS probes employed at high spinning speeds, and limited B ₁ field inhomogeneities, the Fourier coefficients defining the phase modulation profile of the RF pulses were optimised numerically to obtain broadband inversion and refocussing pulses and mixing sequences. Experimental measurements were carried out to assess the performance characteristics of the mixing sequences reported here.     

Sensitivity improvement for correlations involving arginine side-chain Nε/Hε resonances in multi-dimensional NMR experiments using broadband 15N 180° pulses

Due to practical limitations in available ¹⁵N rf field strength, imperfections in ¹⁵N 180° pulses arising from off-resonance effects can result in significant sensitivity loss, even if the chemical shift offset is relatively small. Indeed, in multi-dimensional NMR experiments optimized for protein backbone amide groups, cross-peaks arising from the Arg guanidino ¹⁵Nε (~85 ppm) are highly attenuated by the presence of multiple INEPT transfer steps. To improve the sensitivity for correlations involving Arg Nε–Hε groups, we have incorporated ¹⁵N broadband 180° pulses into 3D ¹⁵N-separated NOE-HSQC and HNCACB experiments. Two ¹⁵N-WURST pulses incorporated at the INEPT transfer steps of the 3D ¹⁵N-separated NOE-HSQC pulse sequence resulted in a ~1.5-fold increase in sensitivity for the Arg Nε–Hε signals at 800 MHz. For the 3D HNCACB experiment, five ¹⁵N Abramovich-Vega pulses were incorporated for broadband inversion and refocusing, and the sensitivity of Arg¹Hε-¹⁵Nε-¹³Cγ/¹³Cδ correlation peaks was enhanced by a factor of ~1.7 at 500 MHz. These experiments eliminate the necessity for additional experiments to assign Arg ¹Hε and ¹⁵Nε resonances. In addition, the increased sensitivity afforded for the detection of NOE cross-peaks involving correlations with the ¹⁵Nε/¹Hε of Arg in 3D ¹⁵N-separated NOE experiments should prove to be very useful for structural analysis of interactions involving Arg side-chains.     

An improved double-tuned and isotope-filtered pulse scheme based on a pulsed field gradient and a wide-band inversion shaped pulse

Summary We have developed an improved isotope-filtered pulse scheme in combination with a double-tuned filter, a hyperbolic secant inversion pulse, and a z-filter with a pulsed field gradient. These filtering pulse schemes have been incorporated into several one-, two-, and three-dimensional experiments, which were applied to the ¹³C/¹⁵N uniformly labeled N-terminal SH3 domain of Grb2 complexed with the unlabeled Sos-derived peptide. The proton resonances of the Sos-derived peptide were unambiguously assigned using isotope-filtered DQF-COSY, TOCSY and NOESY spectra. Furthermore, in the isotope-filtered, isotope-edited 3D NOESY spectrum, intermolecular NOEs between the labeled protein and the unlabeled peptide could be identified. Through these applications, we demonstrate the high filtering efficiency of the presented pulse scheme.     

Broadband homonuclear TOCSY with amplitude and phase-modulated RF mixing schemes

We have explored the design of broadband scalar coupling mediated ¹³C–¹³C and cross-relaxation suppressed ¹H–¹H TOCSY sequences employing phase/amplitude modulated inversion pulses. Considering a variety of supercycles, pulsewidths and a RF field strength of 10 kHz, the Fourier coefficients defining the amplitude and phase modulation profiles of the 180° pulses were optimised numerically so as to obtain efficient magnetisation transfer within the desired range of resonance offsets. The coherence transfer characteristics of the mixing schemes were assessed via numerical simulations and experimental measurements and were compared with commonly used sequences based on rectangular RF pulses. The efficacies of the clean ¹H–¹H TOCSY sequences were also examined via numerical simulations for application to weakly oriented systems and sequences with efficient, broadband and clean dipolar transfer characteristics were identified. In general, the amplitude and phase modulated TOCSY sequences presented here have moderately better performance characteristics than the sequences currently employed in biomolecular NMR spectroscopy.     

Robust refocusing of 13C magnetization in multidimensional NMR experiments by adiabatic fast passage pulses

We show that adiabatic fast passage (AFP) pulses are robust refocusing elements of transverse ¹³C magnetization in multidimensional NMR experiments. A pair of identical AFP pulses can refocus selected parts or a complete ¹³ C chemical shift range in ¹³C spectra. In the constant time ¹³C-¹H HSQC, replacement of attenuated rectangular pulses by selective AFP pulses results in a sensitivity enhancement of up to a factor of 1.8. In the 3D CBCA(CO)NH the signal-to-noise ratio is increased by a factor of up to 1.6.     

Improved pulse sequences for measuring coupling constants in 13C, 15N-labeled proteins

Summary NMR pulse sequences for measuring coupling constants in ¹³C, ¹⁵N-labeled proteins are presented. These pulse sequences represent improvements over earlier experiments with respect to resolution and number of radiofrequency pulses. The experiments are useful for measuring J_NH , J_NCO, J_NC , J_H ^N _CO and J_H ^N _H . Applications to chymotrypsin inhibitor 2 (CI-2) are shown.     

Improved NMR experiments with 13C-isotropic mixing for assignment of aromatic and aliphatic side chains in labeled proteins

Three improved ¹³C-spinlock experiments for side chain assignments of isotope labelled proteins in liquid state are presented. These are based on wide bandwidth spinlock techniques that have become possible with contemporary cryogenic probes. The first application, the H(C^aliC^aro)H-TOCSY, is an HCCH-TOCSY in which all CH_n moieties of a protein are detected in a single experiment, including the aromatic ones. This enables unambiguous assignment of aromatic and aliphatic amino acids in a single, highly sensitive experiment. In the second application, the ¹³C-detected C^all-TOCSY, magnetization transfer comprises all carbons—aliphatic, aromatic as well as the carbonyl carbons—making the complete carbon assignment possible using one spectrum only. Thirdly, the frequently used HC(CCO)NH experiment was redesigned by replacing the long C-carbonyl refocused INEPT transfer step by direct ¹³C–¹³C-TOCSY magnetization transfer from side chain carbons to the backbone carbonyls. The resulting HC(CCO)NH experiment minimizes relaxation losses because it is shorter and represents a more sensitive alternative particularly for larger proteins. The performance of the experiments is demonstrated on isotope labeled proteins up to the size of 43 kDa.     

Estimates of methyl 13C and 1H CSA values (Δσ) in proteins from cross-correlated spin relaxation

Simple pulse schemes are presented for the measurement of methyl ¹³C and ¹H CSA values from ¹H–¹³C dipole/¹³C CSA and ¹H–¹³C dipole/¹H CSA cross-correlated relaxation. The methodology is applied to protein L and malate synthase G. Average ¹³C CSA values are considerably smaller for Ile than Leu/Val (17 vs 25 ppm) and are in good agreement with previous solid state NMR studies of powders of amino acids and dipeptides and in reasonable agreement with quantum-chemical DFT calculations of methyl carbon CSA values in peptide fragments. Small averaged ¹H CSA values on the order of 1 ppm are measured, consistent with a solid state NMR determination of the methyl group ¹H CSA in dimethylmalonic acid.     

Three-dimensional 1H-TOCSY-relayed ct-[13C,1H]-HMQC for aromatic spin system identification in uniformly 13C-labeled proteins

Summary Three-dimensional ¹H-TOCSY-relayed ct-[¹³C,¹H]-HMQC is a novel experiment for aromatic spin system identification in uniformly ¹³C-labeled proteins, which is implemented so that it correlates the chemical shift of a given aromatic proton with those of the directly attached carbon and all vicinal protons. The ct-HMQC scheme is used both for overlay of the indirect ¹H and ¹³C chemical shift evolution periods and for the generation of ¹H-¹H antiphase magnetization to accelerate the ¹H-TOCSY magnetization transfer at short mixing times. As an illustration, data recorded for the 18 kDa protein cyclophilin A are presented. Since transverse relaxation of ¹³C-¹H zero-quantum and double-quantum coherences is to first order insensitive to ¹³C-¹H heteronuclear dipolar relaxation, the new experiment should work also for proteins with molecular weights above 20 kDa.     

The effect of cold-induced increased metabolic rate on the rate of <Superscript>13</Superscript>C and <Superscript>15</Superscript>N incorporation in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>)

Animals with high metabolic rates are believed to have high rates of carbon and nitrogen isotopic incorporation. We hypothesized that (1) chronic exposure to cold, and hence an increase in metabolic rate, would increase the rate of isotopic incorporation of both ¹³C and ¹⁵N into red blood cells; and (2) that the rate of isotopic incorporation into red blood cells would be allometrically related to body mass. Two groups of sparrows were chronically exposed to either 5 or 22°C and switched from a ¹³C-depleted C₃-plant diet to a more ¹³C-enriched C₄-plant one. We used respirometry to estimate the resting metabolic rate of birds exposed chronically to our two experimental temperatures. The allometric relationship between the rate of ¹³C incorporation into blood and body mass was determined from published data. The of birds at 5°C was 1.9 times higher than that of birds at 22°C. Chronic exposure to a low temperature did not have an effect on the rate of isotopic incorporation of ¹⁵N save for a very small effect on the incorporation of ¹³C. The isotopic incorporation rate of ¹³C was 1.5 times faster than that of ¹⁵N. The fractional rate of ¹³C incorporation into avian blood was allometrically related to body mass with an exponent similar to −1/4. We conclude that the relationship between metabolic rate and the rate of isotopic incorporation into an animal’s tissues is indirect. It is probably mediated by protein turnover and thus more complex than previous studies have assumed.     

Numerical design of RN<Stack><Subscript><Emphasis Type="Italic">n</Emphasis></Subscript><Superscript>ν</Superscript></Stack> symmetry-based RF pulse schemes for recoupling and decoupling of nuclear spin interactions at high MAS frequencies

An approach for the efficient implementation of RN _n ^ν symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RN _n ^ν symmetry involve the repeated application of the pulse sandwich {R _ϕ R _−ϕ}, corresponding to a propagator U _RF = exp(−i4ϕI _z), where ϕ = πν/N and R is typically a pulse that rotates the nuclear spins through 180° about the x-axis. In this study, broadband, phase-modulated 180° pulses of constant amplitude were employed as the initial ‘R’ element and the phase-modulation profile of this ‘R’ element was numerically optimised for generating RN _n ^ν symmetry-based pulse schemes with satisfactory magnetisation transfer characteristics. At representative MAS frequencies, RF pulse sequences were implemented for achieving ¹³C–¹³C double-quantum dipolar recoupling and through bond scalar coupling mediated chemical shift correlation and evaluated via numerical simulations and experimental measurements. The results from these investigations are presented here. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Translocation and conservation of organic nitrogen within the coral-zooxanthella symbiotic system of Acropora pulchra, as demonstrated by dual isotope-labeling techniques

Carbon (C) and nitrogen (N) metabolism of the hermatypic coral Acropora pulchra and its symbiotic algae (zooxanthellae) was investigated using ¹³C and ¹⁵N isotope tracers. A. pulchra was incubated in seawater containing ¹³C-labeled bicarbonate and ¹⁵N-labeled nitrate (NO₃⁻) for 24 h (pulse period), and subsequently ¹³C and ¹⁵N isotopic ratios of the host coral and the zooxanthellae were followed in ¹³C- and ¹⁵N-free seawater for 2 weeks (chase period). Under our experimental condition of NO₃⁻ (12 μM), C and N were absorbed by the coral-algal symbiotic system with the C:N ratio of 23 during the pulse period. Taking account of concentration dependence of NO₃⁻ uptake rates determined by a separate experiment, C:N uptake ratios under supposed in situ NO₃⁻ conditions (< 1.0 μM) would be > 3.0 times higher, if the photosynthetic rate did not change. During the pulse period, more than half of the absorbed ¹³C and ¹⁵N appeared in the host fraction in organic forms. ¹³C:¹⁵N ratio at the end of the pulse period was similar between the host and the algal fraction, suggesting that algal photosynthetic products were translocated to the host. It is also implied that C:N ratios of the translocated products change depending on N availability for the zooxanthellae. During the chase period, atom % excess (APE) ¹⁵N of the zooxanthellae constantly declined, while that of the host slightly increased. Consequently, APE ¹⁵N of the both fractions appeared to approach a common steady state value, suggesting that ¹⁵N was recycled within the coral-algal symbiotic system. As for C, > 86% of C photosynthetically fixed by the zooxanthellae accumulated in the host at the end of the pulse period, and had a turnover time of ca. 20 days for the host C pool during the following chase period. C:N ratios of organic matter newly synthesized with NO₃⁻ exponentially declined and converged into 5.7 and 4.5 for the host and the zooxanthellae, respectively. This suggests that organic compounds of high C:N ratios such as lipids and carbohydrates were selectively consumed more rapidly than those of low C:N ratios such as proteins and nucleic acids.     

Effects of atmospheric CO2 enrichment on δ 13C, δ 15N values and turnover times of soil organic matter pools isolated by thermal techniques

CO₂ applied for Free-Air CO₂ Enrichment (FACE) experiments is strongly depleted in ¹³C and thus provides an opportunity to study C turnover in soil organic matter (SOM) based on its δ ¹³C value. Simultaneous use of ¹⁵N labeled fertilizers allows N turnover to be studied. Various SOM fractionation approaches (fractionation by density, particle size, chemical extractability etc.) have been applied to estimate C and N turnover rates in SOM pools. The thermal stability of SOM coupled with C and N isotopic analyses has never been studied in experiments with FACE. We tested the hypothesis that the mean residence time (MRT) of SOM pools is inversely proportional to its thermal stability. Soil samples from FACE plots under ambient (380 ppm) and elevated CO₂ (540 ppm; for 3 years) treatments were analyzed by thermogravimetry coupled with differential scanning calorimetry (TG-DSC). Based on differential weight losses (TG) and energy release or consumption (DSC), five SOM pools were distinguished. Soil samples were heated up to the respective temperature and the remaining soil was analyzed for δ ¹³C and δ ¹⁵N by IRMS. Energy consumption and mass losses in the temperature range 20–200°C were mainly connected with water volatilization. The maximum weight losses occurred from 200–310°C. This pool contained the largest amount of carbon: 61% of the total soil organic carbon in soil under ambient treatment and 63% in soil under elevated CO₂, respectively. δ ¹³C values of SOM pools under elevated CO₂ treatment showed an increase from −34.3‰ of the pool decomposed between 20–200°C to −18.1‰ above 480°C. The incorporation of new C and N into SOM pools was not inversely proportional to its thermal stability. SOM pools that decomposed between 20–200 and 200–310°C contained 2 and 3% of the new C, with a MRT of 149 and 92 years, respectively. The pool decomposed between 310–400°C contained the largest proportion of new C (22%), with a MRT of 12 years. The amount of fertilizer-derived N after 2 years of application in ambient and elevated CO₂ treatments was not significantly different in SOM pools decomposed up to 480°C having MRT of about 60 years. In contrast, the pool decomposed above 480°C contained only 0.5% of new N, with a MRT of more than 400 years in soils under both treatments. Thus, the separation of SOM based on its thermal stability was not sufficient to reveal pools with contrasting turnover rates of C and N. Responsible Editor: Bernard Nicolardot.     

Effect of C3–C4 Vegetation Change on δ13C and δ15N Values of Soil Organic Matter Fractions Separated by Thermal Stability

Carbon isotopic composition of soils subjected to C₃–C₄ vegetation change can be used to estimate C turnover in bulk soil and in soil organic matter (SOM) pools with fast and intermediate turnover rates. We hypothesized that the biological availability of SOM pools is inversely proportional to their thermal stability, so that thermogravimetry can be used to separate SOM pools with contrasting turnover rates. Soil samples from a field plot cultivated for 10.5 years with the perennial C₄ plant Miscanthus×gigantheus were analyzed by thermogravimetry coupled with differential scanning calorimetry (DSC). Three SOM fractions were distinguished according to the differential weight losses and exothermic or endothermic reactions measured by DSC. The δ¹³C and δ¹⁵N values of these three fractions obtained by gradual soil heating were measured by IRMS. The weight losses up to 190 °C mainly reflected water evaporation because no significant C and N losses were detected and δ¹³C and δ¹⁵N values of the residual SOM remained unchanged. The δ¹³C values (−16.4‰) of SOM fraction decomposed between 190 and 390 °C (containing 79% of total soil C) were slightly closer to that of the Miscanthus plant tissues (δ¹³C = −11.8‰) compared to the δ¹³C values (−16.8‰) of SOM fraction decomposed above 390 °C containing the residual 21% of SOM. Thus, the C turnover in the thermally labile fraction was faster than that in thermally stable fractions, but the differences were not very strong. Therefore, in this first study combining TG-DSC with isotopic analysis, we conclude that the thermal stability of SOM was not very strongly related to biological availability of SOM fractions. In contrast to δ¹³C, the δ¹⁵N values strongly differed between SOM fractions, suggesting that N turnover in the soil was different from C turnover. More detailed fractionation of SOM by thermal analysis with subsequent isotopic analysis may improve the resolution for δ¹³C.     

Biosynthetically directed fractional 13C labeling facilitates identification of Phe and Tyr aromatic signals in proteins

Analysis of 2D [¹³C,¹H]-HSQC spectra of biosynthetic fractionally ¹³C labeled proteins is a reliable, straightforward means to obtain stereospecific assignments of Val and Leu methyl sites in proteins. Herein we show that the same fractionally labeled protein sample facilitates observation and identification of Phe and Tyr aromatic signals. This is the case, in part, because the fractional ¹³C labeling yields aromatic rings in which some of the ¹³C-¹³C J-couplings, present in uniformly labeled samples, are absent. Also, the number of homonuclear J-coupling partners differs for the -, - and -carbons. This enabled us to vary their signal intensities in distinctly different ways by appropriately setting the ¹³C constant-time period in 2D [¹³C,¹H]-HSQC spectra. We illustrate the application of this approach to an 18 kDa protein, c-VIAF, a modulator of apoptosis. In addition, we show that cancellation of the aromatic ¹³C CSA and ¹³C-¹H dipolar interactions can be fruitfully utilized in the case of the fractionally labeled sample to obtain high resolution ¹³C constant-time spectra with good sensitivity.     

Minimum pulses of stable and radioactive carbon isotopes to detect belowground carbon transfer between plants

Methodological problems, such as unwanted shifts in plant carbon allocation patterns following large isotopic labeling pulses, have hindered accurate quantification of belowground carbon movement in plant–soil systems. These problems must be addressed before we can understand the factors regulating carbon movement between plants and soils and the importance of this movement to the global carbon cycle. We studied the effects of pulse-label size on carbon allocation and transfer between ectomycorrhizal paper birch (Betula papyrifera Marsh.) and Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco) seedlings using increasing pulse levels of either ¹³C or ¹⁴C in two separate laboratory experiments. Our specific objectives were: (1) to determine the minimum pulse of ¹³CO₂ or ¹⁴CO₂ for detecting carbon movement between plants through belowground transfer pathways, (2) to determine whether carbon allocation patterns within these plants change when exposed to short pulses of elevated carbon dioxide and, (3) to determine whether carbon allocation patterns are similar when using two different carbon isotopes. We detected carbon movement between plants at each ¹³C and ¹⁴C pulse level. There was a tendency for the amount of interplant carbon transfer to increase with increasing ¹³C pulse level, but the same amount of transfer occurred at all ¹⁴C pulses between 0.19 and 0.56 MBq. Carbon allocation patterns did not change with pulse level but they were affected by the choice of carbon isotope. We conclude that at least 8 ml of ¹³C or 0.19 MBq of ¹⁴C is sufficient to detect belowground carbon transfer in small seedlings growing in close proximity.     

Determination of 13Cα relaxation times in uniformly 13C/15N-enriched proteins

Summary Relaxation times of ¹³C carbons of uniformly ¹³C/¹⁵N-enriched probes have been investigated. The relaxation behaviour was analyzed in terms of a multispin system. Pulse sequences for the determination of T₁, T₂ and the heteronuclear NOE of ¹³C in uniformly ¹³C/¹⁵N-enriched ribonuclease T1 are presented. The experiments performed in order to obtain T₁ and the heteronuclear NOE were similar to those of the corresponding ¹⁵N experiments published previously. The determination of T₂ for the C-carbon in a completely labeled protein is more complicated, since the magnetization transfer during the T₂ evolution period owing to the scalar coupling of C–C must be suppressed. Various different pulse sequences for the T₂ evolution period were simulated in order to optimize the bandwidth for which reliable T₂ relaxation times can be obtained. A proof for the quality of these pulse sequences is given by fitting the intensity decay of individual ¹H–¹³C cross peaks, in a series of (¹H, ¹³C)-ct-HSQC spectra with a modified CPMG sequence as well as a T_1p sequence for the transverse relaxation time, to a single exponential using a simplex algorithm.     

Assimilate partitioning affects 13C fractionation of recently assimilated carbon in maize

Coupling ¹³C natural abundance and ¹⁴C pulse labelling enabled us to investigate the dependence of ¹³C fractionation on assimilate partitioning between shoots, roots, exudates, and CO₂ respired by maize roots. The amount of recently assimilated C in these four pools was controlled by three levels of nutrient supply: full nutrient supply (NS), 10 times diluted nutrient supply (DNS), and deionised water (DW). After pulse labelling of maize shoots in a ¹⁴CO₂ atmosphere, ¹⁴C was traced to determine the amounts of recently assimilated C in the four pools and the δ¹³C values of the four pools were measured. Increasing amounts of recently assimilated C in the roots (from 8% to 10% of recovered ¹⁴C in NS and DNS treatments) led to a 0.3‰ ¹³C enrichment from NS to DNS treatments. A further increase of C allocation in the roots (from 10% to 13% of recovered ¹⁴C in DNS and DW treatments) resulted in an additional enrichment of the roots from DNS to DW treatments by 0.3‰. These findings support the hypothesis that ¹³C enrichment in a pool increases with an increasing amount of C transferred into that pool. δ¹³C of CO₂ evolved by root respiration was similar to that of the roots in DNS and DW treatments. However, if the amount of recently assimilated C in root respiration was reduced (NS treatment), the respired CO₂ became 0.7‰ ¹³C depleted compared to roots. Increasing amounts of recently assimilated C in the CO₂ from NS via DNS to DW treatments resulted in a 1.6‰ δ¹³C increase of root respired CO₂ from NS to DW treatments. Thus, for both pools, i.e. roots and root respiration, increasing amounts of recently assimilated C in the pool led to a δ¹³C increase. In DW and DNS plants there was no ¹³C fractionation between roots and exudates. However, high nutrient supply decreased the amount of recently assimilated C in exudates compared to the other two treatments and led to a 5.3‰ ¹³C enrichment in exudates compared to roots. We conclude that ¹³C discrimination between plant pools and within processes such as exudation and root respiration is not constant but strongly depends on the amount of C in the respective pool and on partitioning of recently assimilated C between plant pools. Section Editor: H. Lambers     

HNCAN pulse sequences for sequential backbone resonance assignment across proline residues in perdeuterated proteins

A TROSY-based triple-resonance pulse scheme is described which correlates backbone ¹H and ¹⁵N chemical shifts of an amino acid residue with the ¹⁵N chemical shifts of both the sequentially preceding and following residues. The sequence employs ¹ J _NC and ² J _NC couplings in two sequential magnetization transfer steps in an `out-and-back' manner. As a result, N,N connectivities are obtained irrespective of whether the neighbouring amide nitrogens are protonated or not, which makes the experiment suitable for the assignment of proline resonances. Two different three-dimensional variants of the pulse sequence are presented which differ in sensitivity and resolution to be achieved in one of the nitrogen dimensions. The new method is demonstrated with two uniformly ²H/¹³C/¹⁵N-labelled proteins in the 30-kDa range.     

Optimization of <Superscript>1</Superscript>H decoupling eliminates sideband artifacts in 3D TROSY-based triple resonance experiments

TROSY-based triple resonance experiments are essential for protein backbone assignment of large biomolecular systems by solution NMR spectroscopy. In a survey of the current Bruker pulse sequence library for TROSY-based experiments we found that several sequences were plagued by artifacts that affect spectral quality and hamper data analysis. Specifically, these experiments produce sidebands in the ¹³C(t ₁) dimension with inverted phase corresponding to ¹H_N resonance frequencies, with approximately 5% intensity of the parent ¹³C crosspeaks. These artifacts originate from the modulation of the ¹H_N frequency onto the resonance frequency of ¹³Cα and/or ¹³Cβ and are due to 180° pulses imperfections used for ¹H decoupling during the ¹³C(t ₁) evolution period. These sidebands can become severe for CA_i, CA_i?1 and/or CB_i, CB_i?1 correlation experiments such as TROSY-HNCACB. Here, we implement three alternative decoupling strategies that suppress these artifacts and, depending on the scheme employed, boost the sensitivity up to 14% on Bruker spectrometers. A class of comparable Agilent/Varian pulse sequences that use WALTZ16 ¹H decoupling can also be improved by this method resulting in up to 60–80% increase in sensitivity.