Allosteric effects of atropine and carbachol on the activity of α<Subscript>2</Subscript>-adrenoceptors in rat brain cortex membranes

Activation and inhibition of muscarinic cholinoceptors by atropine and carbachol are shown to exert allosteric effects on the binding of specific nonselective α₂-adrenoceptor antagonist [³H]RX821002 in rat brain cortex membranes. The ligand-receptor interaction for α₂-adrenoceptors corresponded to the model suggesting the presence of one homogeneous pool of receptors with two specific binding sites. The parameters of the [³H]RX821002 binding were as follows: [³H]RX821002 -K _d = 1.94 ± 0.08 nM, B _max = 13.4 ± 1.8 fmol/mg protein, n = 2. The inhibition of muscarinic cholinoceptors by atropine induced an increase of affinity (K _d = 1.36 ± 0.12 nM) and a decrease of the α2-adrenoceptor density (B _max = 10.18 ± 0.48 fmol/mg protein). The muscarinic cholinoceptor agonist carbachol induced an increase of the affinity (K _d = 1.56 ± 0.05 nM) and quantity of binding sites (B _max = 16.61 ± 0.29 fmol/mg protein). As a result, under the influence of atropine and carbachol, the efficiency of binding (E = B _max/2K _d) increased from 3.50 ± 0.40 to 5.60 ± 0.79 and 6.86 ± 0.20 fmol/mg protein/nM, respectively. The data suggest that α₂-adrenoceptors exist in rat brain cortex as homodimers.     

<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.     

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.     

Modulation of Na<Superscript>+</Superscript>/Mg<Superscript>2+</Superscript> exchanger stoichiometry ratio by Cl<Superscript>−</Superscript> ions in basolateral rat liver plasma membrane vesicles

The Na⁺/Mg²⁺ exchanger represents the main Mg²⁺ extrusion mechanism operating in mammalian cells including hepatocytes. We have previously reported that this exchanger, located in the basolateral domain of the hepatocyte, promotes the extrusion of intravesicular trapped Mg²⁺ for extravesicular Na⁺ with ratio 1. This electrogenic exchange is supported by the accumulation of tetraphenyl-phosphonium within the vesicles at the time when Mg²⁺ efflux occurs. In this present study, the role of extra- and intra-vesicular Cl^? on the Na⁺/Mg²⁺ exchange ratio was investigated. The results reported here suggest that Cl^? ions are not required for the Na⁺ to Mg²⁺ exchange to occur, but the stoichiometry ratio of the exchanger switches from electrogenic (1Na _in ⁺ :1 Mg _out ²⁺ ) in the presence of intravesicular Cl^? to electroneutral (2Na _in ⁺ :1 Mg _out ²⁺ ) in their absence. In basolateral liver plasma membrane vesicles loaded with MgCl₂ labeled with ³⁶Cl^?, a small but significant Cl^? efflux (~30 nmol Cl^?/mg protein/1 min) is observed following addition of NaCl or Na-isethionate to the extravesicular medium. Both Cl^? and Mg²⁺ effluxes are inhibited by imipramine but not by amiloride, DIDS, niflumic acid, bumetanide, or furosemide. In vesicles loaded with Mg-gluconate and stimulated by Na-isethionate, an electroneutral Mg²⁺ extrusion is observed. Taken together, these results suggest that the Na⁺/Mg²⁺ exchanger can operate irrespective of the absence or the presence of Cl^? in the extracellular or intracellular environment. Changes in trans-cellular Cl^? content, however, can affect the modus operandi of the Na⁺/Mg²⁺ exchanger, and consequently impact cellular Na⁺ and Mg²⁺ homeostasis as well as the hepatocyte membrane potential.     

Impact of two-bond <Superscript>15</Superscript>N–<Superscript>15</Superscript>N scalar couplings on <Superscript>15</Superscript>N transverse relaxation measurements for arginine side chains of proteins

NMR relaxation of arginine (Arg) ¹⁵N_ε nuclei is useful for studying side-chain dynamics of proteins. In this work, we studied the impact of two geminal ¹⁵N–¹⁵N scalar couplings on measurements of transverse relaxation rates (R ₂ ) for Arg side-chain ¹⁵N_ε nuclei. For 12 Arg side chains of the DNA-binding domain of the Antp protein, we measured the geminal ¹⁵N–¹⁵N couplings ( ² J _NN ) of the ¹⁵N_ε nuclei and found that the magnitudes of the ² J _NN coupling constants were virtually uniform with an average of 1.2 Hz. Our simulations, assuming ideal 180° rotations for all ¹⁵N nuclei, suggested that the two ² J _NN couplings of this magnitude could in principle cause significant modulation in signal intensities during the Carr–Purcell-Meiboom–Gill (CPMG) scheme for Arg ¹⁵N_ε R ₂ measurements. However, our experimental data show that the expected modulation via two ² J _NN couplings vanishes during the ¹⁵N CPMG scheme. This quenching of J modulation can be explained by the mechanism described in Dittmer and Bodenhausen (Chemphyschem 7:831–836, 2006). This effect allows for accurate measurements of R ₂ relaxation rates for Arg side-chain ¹⁵N_ε nuclei despite the presence of two ² J _NN couplings. Although the so-called recoupling conditions may cause overestimate of R ₂ rates for very mobile Arg side chains, such conditions can readily be avoided through appropriate experimental settings.     

Comparative characteristics of binding kinetics of specific antagonists by α<Subscript>1</Subscript>- and α<Subscript>2</Subscript>-adrenoceptors in rat cerebral cortex membranes

The binding of specific nonselective α₁- and α₂-adrenoceptor antagonists [³H]prazosine and [³H]RX821002 has been studied on rat cerebral cortex synaptosomal membranes. It is shown that for α₁-adrenoceptors the ligand-receptor interaction corresponds to the model assuming the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of [³H]prazosine binding to α₁-adrenoceptors were: K _d= 1.56 ± 0.17 nM, B _max = 30.25 ± 1.78 fmol/mg protein, n = 2. The parameters of [³H]RX821002 binding to α₂-adrenoceptors were: K _d = 1.94 ± 0.08 nM, B _max = 12.77 ± 3.17 fmol/mg protein, n = 2. For α₂ -adrenoceptors the ligand-receptor interaction corresponded to the same model. For α₁ - and α₂-adrenoceptor antagonists the dissociation constants (K _d) are approximately equal (1.56 ± 0.17 and 1.94 ± 0.08 nM, respectively), but the concentration of α₂-adrenoceptors is two times lower than that of α₁-adrenoceptors ( 12.77 ± 3.17 and 30.25 ± 1.78 fmol/mg protein, respectively). The efficiency (E = B _max/2K _d) of the ligand binding to α₁-adrenoceptors is 2.3 times higher than that to α₂-adrenoceptors (7.46 ± 1.32 and 3.29 ± 0.68 fmol/mg protein/nM, respectively. The data suggest that α₁- and α₂ -adrenoceptors in rat cerebral cortex exist as dimers.     

Comparative properties of sensitive to GABA<Subscript>A</Subscript>-ergic ligands Cl<Superscript>−</Superscript>, HCO<Subscript>3</Subscript><Superscript>−</Superscript>-activated Mg<Superscript>2+</Superscript>-ATPase from brain plasma membranes of fish and rats

Action of Cl^? + HCO₃ ^?1 ions on Mg²⁺-ATPase from brain plasma membranes of fish and rats has been studied. Maximal effect of the anions on the “basal” Mg²⁺-ATPase activity is revealed in the presence of 10 mM Cl^? and 3 mM HCO₃ ^?1 at physiological values of pH of incubation medium. The studied Cl^?, HCO₃ ^?-activated Mg²⁺-ATPases of both animal species, by their sensitivity to SH-reagents (5,5-dithio-bis-nitrobenzoic acid, N-ethylmaleimide), oligomycin, and orthovanadate, are similar to transport ATPase of the P-type, but differ from them by molecular properties and by sensitivity to ligands of GABA_A-receptors. It has been established that the sensitive to GABA_A-ergic ligands, Cl^?, HCO₃ ^?-activated Mg²⁺-ATPase from brain of the both animal species is protein of molecular mass around 300 kDa and of Stock’s radius 5.4 nm. In fish the enzyme is composed of one major unit of molecular mass approximately 56 kDa, while in rats-of three subunits of molecular masses about 57, 53, and 45 kDa. A functional and structural coupling of the ATP-hydrolyzing areas of the studied enzyme to sites of binding of GABA_A-receptor ligands is suggested.     

Measuring <Superscript>13</Superscript>C<Superscript>β</Superscript> chemical shifts of invisible excited states in proteins by relaxation dispersion NMR spectroscopy

A labeling scheme is introduced that facilitates the measurement of accurate ¹³C^β chemical shifts of invisible, excited states of proteins by relaxation dispersion NMR spectroscopy. The approach makes use of protein over-expression in a strain of E. coli in which the TCA cycle enzyme succinate dehydrogenase is knocked out, leading to the production of samples with high levels of ¹³C enrichment (30–40%) at C^β side-chain carbon positions for 15 of the amino acids with little ¹³C label at positions one bond removed (≈5%). A pair of samples are produced using [1-¹³C]-glucose/NaH¹²CO₃ or [2-¹³C]-glucose as carbon sources with isolated and enriched (>30%) ¹³C^β positions for 11 and 4 residues, respectively. The efficacy of the labeling procedure is established by NMR spectroscopy. The utility of such samples for measurement of ¹³C^β chemical shifts of invisible, excited states in exchange with visible, ground conformations is confirmed by relaxation dispersion studies of a protein–ligand binding exchange reaction in which the extracted chemical shift differences from dispersion profiles compare favorably with those obtained directly from measurements on ligand free and fully bound protein samples.     

The Role of K<Superscript>+</Superscript>-Cl<Superscript>−</Superscript>-Cotransporter-2 in Neuropathic Pain

The pain sensory system normally functions under a fine balance between excitation and inhibition. When this balance is perturbed for some reason, it leads to neuropathic pain. There is accumulating evidence that attributes this pain generation to specific dysfunctions of the inhibitory system in the spinal cord. One possible mechanism leading to the induction of these dysfunctions is the down-regulation of K⁺-Cl^?-cotransporter-2 (KCC2) expression. In fact, various neuropathic pain models indicate a decrease of KCC2 expression in the spinal cord. The alteration of KCC2 expression affects GABAergic and glycinergic neurotransmissions, because KCC2 is a potassium-chloride exporter and serves to maintain intracellular chloride concentration. When there is a low level of KCC2 expression, GABAergic and glycinergic neurotransmissions transform from inhibitory signals to excitatory signals. In this review, the hypothesis that an alteration of KCC2 expression has a crucial influence on the initiation/development or maintenance of neuropathic pain is discussed. In addition, it is suggested that the alteration of inhibitory signals is dependent on the time after peripheral nerve injury.     

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.     

Effects of combined administration ofl-tryptophan and tricyclic antidepressants on α2- and β-adrenoceptors and monoamine levels in rat brain

In order to test whether co-administration of a serotonin precursor with antidepressant drugs could potentiate the effects of the antidepressants on monoamines or adrenoceptors in rat brain,l-tryptophan (20 mg/kg) was administered to rats daily for 7 or 15 days, either alone or in combination with desipramine (10 mg/ kg) or amitriptyline (10 mg/kg). After treatment withl-tryptophan for 7 days, increases were observed in rat hypothalamic and frontal cortex 5-hydroxy-3-indoleacetic acid levels as well as in hypothalamic dopamine and nucleus accumbens 3,4-dihydroxyphenylacetic acid levels. After 15 days, hippocampal -adrenoceptor density was found to be decreased. There was no evidence of potentiation of desipramine or amitriptyline action whenl-tryptophan was administered in combination with the antidepressants. On the contrary, the antidepressants appeared to interact withl-tryptophan to reduce its effects.     

<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.     

Determination of the lifetime of D<Stack><Subscript>2</Subscript><Superscript>−</Superscript></Stack> and HD<Superscript>−</Superscript> ions

A method for determining the lifetime of unstable ions is described. The method is based on measuring the decrease in the ion beam current onto a fixed detector with increasing path length of the ion beam from the ion source to the detector. The measurements performed for D^? ₂ and HD^? molecular ions have shown that their lifetimes are 3.5 ± 0.1 and 4.4 ± 0.1 μs, respectively.     

Improved accuracy of <Superscript>15</Superscript>N–<Superscript>1</Superscript>H scalar and residual dipolar couplings from gradient-enhanced IPAP-HSQC experiments on protonated proteins

The presence of dipole-dipole cross-correlated relaxation as well as unresolved E.COSY effects adversely impacts the accuracy of ¹ J _NH splittings measured from gradient-enhanced IPAP-HSQC spectra. For isotropic samples, the size of the systematic errors caused by these effects depends on the values of ² J _NHα , ³ J _NHβ and ³ J _HNHα . Insertion of band-selective ¹H decoupling pulses in the IPAP-HSQC experiment eliminates these systematic errors and for the protein GB3 yields ¹ J _NH splittings that agree to within a root-mean-square difference of 0.04 Hz with values measured for perdeuterated GB3. Accuracy of the method is also highlighted by a good fit to the GB3 structure of the ¹H-¹⁵N RDCs extracted from the minute differences in ¹J_NH splitting measured at 500 and 750 MHz ¹H frequencies, resulting from magnetic susceptibility anisotropy. A nearly complete set of ² J _NHα couplings was measured in GB3 in order to evaluate whether the impact of cross-correlated relaxation is dominated by the ¹⁵N–¹H ^α or ¹⁵N–¹H ^β dipolar interaction. As expected, we find that ² J _NHα  ≤ 2 Hz, with values in the α-helix (0.86 ± 0.52 Hz) slightly larger than in β-sheet (0.66 ± 0.26 Hz). Results indicate that under isotropic conditions, N–H^N/N–H ^β cross-correlated relaxation often dominates. Unresolved E.COSY effects under isotropic conditions involve ³ J _HNHα and J _NHα , but when weakly aligned any aliphatic proton proximate to both N and H^N can contribute. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The role of Ca<Superscript>2+</Superscript>, H<Superscript>+</Superscript>, and Cl<Superscript>−</Superscript> ions in generation of variation potential in pumpkin plants

The contributions of Ca²⁺, H⁺, and Cl⁻ in generation of variation potentials (VP) in 3- to 4-week-old pumpkin (Cucurbita pepo L., cv. Mozoleevskaya) plants were assessed. During VP generation, transient alkalinization of the medium around the stem was recorded with a potentiometric method. The pH changes were kinetically similar to the electric potential changes and were apparently due to temporal suppression of the plasma-membrane electrogenic H⁺ pump. These data and the observed inhibition of VP in the stem zone treated locally with a metabolic inhibitor (NaN₃) indicate that the VP generation is related to the reversible suppression of the H⁺-pump. The anion channel blocker (ethacrynic acid) decelerated significantly the front slope of VP and reduced the VP amplitude. A short-term increase in external Cl⁻ concentration around the stem was observed during potential transients representing the VP front slope and the pulses integrated into VP. The removal of Ca²⁺ from extracellular medium inhibited the VP generation. It is proposed that Ca²⁺ plays a role in activation of anion channels and in the H⁺-pump inactivation. The VP generation is probably determined by a complex mechanism, with contributions from passive ion fluxes (Ca²⁺, Cl⁻) moving along the electrochemical gradients and from changes in the electrogenic pump activity.     

Estimation of denitrification potential in a karst aquifer using the <Superscript>15</Superscript>N and <Superscript>18</Superscript>O isotopes of NO<Stack><Subscript>3</Subscript><Superscript>−</Superscript></Stack>

A confined aquifer in the Malm Karst of the Franconian Alb, South Germany was investigated in order to understand the role of the vadose zone in denitrifiaction processes. The concentrations of chemical tracers Sr²⁺ and Cl^– and concentrations of stable isotope ¹⁸O were measured in spring water and precipitation during storm events. Based on these measurements a conceptual model for runoff was constructed. The results indicate that pre-event water, already stored in the system at the beginning of the event, flows downslope on vertical and lateral preferential flow paths. Chemical tracers used in a mixing model for hydrograph separation have shown that the pre-event water contribution is up to 30%. Applying this information to a conceptual runoff generation model, the values of ¹⁵N and ¹⁸O in nitrate could be calculated. Field observations showed the occurence of significant microbial denitrification processes above the soil/bedrock interface before nitrate percolates through to the deeper horizon of the vadose zone. The source of nitrate could be determined and denitrification processes were calculated. Assuming that the nitrate reduction follows a Rayleigh process one could approximate a nitrate input concentration of about 170 mg/l and a residual nitrate concentration of only about 15%. The results of the chemical and isotopic tracers postulate fertilizers as nitrate source with some influence of atmospheric nitrate. The combined application of hydrograph separation and determination of isotope values in ¹⁵N and ¹⁸O of nitrate lead to an improved understanding of microbial processes (nitrification, denitrification) in dynamic systems.     

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.     

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.