In vivo enzymology: 13C NMR measurement of a kinetic isotope effect for methanol oxidation in Methylosinus trichosporium OB3b

The competitive oxidation of ¹³CH₃OH and ¹³CD₃OH has been observed using in vivo ¹³C NMR spectroscopy. Simultaneous ¹H and ²H decoupling gave isotopically shifted ¹³C singlets for the two methanol isotopomers. The measured enzymic isotope effect, kH/kD is approx. 1.8, indicating that CH bond cleavage is not rate-determining.     

Lysine methylation strategies for characterizing protein conformations by NMR

In the presence of formaldehyde and a mild reducing agent, reductive methylation is known to achieve near complete dimethylation of protein amino groups under non-denaturing conditions, in aqueous media. Amino methylation of proteins is employed in mass spectrometric, crystallographic, and NMR studies. Where biosynthetic labeling is prohibitive, amino ¹³C-methylation provides an attractive option for monitoring folding, kinetics, protein?Cprotein and protein-DNA interactions by NMR. Here, we demonstrate two improvements over traditional ¹³C-reductive methylation schemes: (1) By judicious choice of stoichiometry and pH, ??-aminos can be preferentially monomethylated. Monomethyl tags are less perturbing and generally exhibit improved resolution over dimethyllysines, and (2) By use of deuterated reducing agents and ¹³C-formaldehyde, amino groups can be labeled with ¹³CH₂D tags. Use of deutero-¹³C-formaldehyde affords either ¹³CHD₂, or ¹³CD₃ probes depending on choice of reducing agent. Making use of ¹³C?C²H scalar couplings, we demonstrate a filtering scheme that eliminates natural abundance ¹³C signal.     

Syntheses and structural characterization of mononuclear Rh-Cp* and Ir-Cp* complexes with η-phenanthrene, η-pyrene and η-triphenylene

Four novel mononuclear Rh-Cp* and Ir-Cp* complexes with polycyclic aromatic hydrocarbons (PAHs), [M(Cp*)(η⁶-PAHs)](BF₄)₂ (M = Rh and Ir; Cp* = η⁵-C₅Me₅; PAHs = phenanthrene (phn), pyrene (pyr) and triphenylene (triph)), were prepared by the reactions of the intermediate [M(Cp*)(Me₂CO)₃]²⁺ with appreciable PAHs. Their structures were characterized by a single crystal X-ray analysis, ¹H, ¹³C {¹H} NMR and 2D NMR techniques. The X-ray crystallographic studies showed that the [M(Cp*)]²⁺ fragment is η⁶-coordinated to one terminal benzene ring in each PAH. In particular, it is interesting to note that the partial π/π/π/π interaction was formed in the Ir-pyr complex [Ir(Cp*)(η⁶-pyr)](BF₄)₂. The 1D and 2D NMR studies described that the Rh-Cp* and Ir-Cp* complexes with PAHs gave unique ¹H and ¹³C {¹H} NMR spectra with positive coordination shifts (Δδ(¹H, ¹³C)) in (CD₃)₂CO at 23 °C, which are likely induced by the local effect and the non-local effect on the coordination of the [M(Cp*)]²⁺ fragment to PAHs. The decreasing of the coupling constants (³J_H-H) in the η⁶-coordinated benzene ring is also induced, with no changes in the uncoordinated benzene rings. The time-course of ¹H NMR spectra showed that Rh-Cp* and Ir-Cp* complexes with PAHs are partially dissociated to [M(Cp*)(Me₂CO)₃]²⁺ and metal-free PAHs in (CD₃)₂CO at 23 °C. It was demonstrated that their stabilities are in the order of Ir-triph, Ir-phn, Ir-pyr and Rh-triph complexes in (CD₃)₂CO.     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

Proton magnetic resonance study of rotation about carbon-nitrogen bonds in pentakis(1,1-dimethylurea)dioxouranium(VI) and its 1,3-dimethylurea analogue

In CD₃CN solutions the kinetic parameters characterising rotation about the CNMe₂ and CNH₂ bonds in [UO₂(1,1-DMU)₅]²⁺ (1,1-DMU = 1,1- dimethylurea) were determined as: k(265 K) = 39.1 ± 0.4 and 2960 ± 60 s^?1, ΔH3 = 49.1 ± 0.76 and 61.1 ±0.5 kJ mol^?1, ΔS2 = ?28.3 ± 2.7 and 53.1 ± 2.2 J K^?1 mol^?1 respectively from ¹H NMR studies. Resonances arising from the three isomeric 1,3-DMU (= 1,3-dimethylurea) ligands were observed for [UO₂(1,3-DMU)₅]²⁺ in CD₃CN solution and the kinetic parameters characterising their isomerisations were also determined. The three isomers of 1,3-DMU have not previously been detected in solution and it appears that coordination of 1,3-DMU to UO₂²⁺ increases the barrier to rotation about the carbon nitrogen bond, as is also shown to be the case for 1,1-DMU.     

NMR studies on UO22+ complexes with pyridoxal

The interaction of pyridoxal with dioxouranium(VI) acetate was studied by ¹H and ¹³C NMR measurements in D₂O and CD₃OD.The results indicate that the preferred bonding site is the C-3O^? donor, and the major species obtained under the experimental conditions used is the equimolar complex.     

An NMR study of the loss of carbon-20 in the biosynthesis of gibberellin A3 by Gibberella fujikuroi

Resuspension cultures of Gibberella fujikuroi, strain GF-1a, were shown to metabolise potassium [3′-¹³C] mevalonate to ¹³C-enriched C₁₉-gibberellins, plus ¹³CO₂ (derived from the loss of carbon-20). The formation of [¹³C]-gibberellins could be observed in vivo using ¹³C NMR; however that of ¹³CO₂ could not. In contrast, removal of the mycelium and concentration of the filtrate at pH 12 enabled the ¹³CO₂ produced to be observed using ¹³C NMR. During incubations of H¹⁴CO₂Na with this fungus, complete conversion to other radioactive products was observed, and the significance of these results in the light of previous work is discussed.     

Characterization of Acetate and Pyruvate Metabolism in Suspension Cultures of Zea mays by C NMR Spectroscopy

Carbon-13 nuclear magnetic resonance (NMR) spectroscopy has been applied to the direct observation of acetate and pyruvate metabolism in suspension cultures of Zea mays (var Black Mexican Sweet). Growth of the corn cells in the presence of 2 millimolar [2-¹³C]acetate resulted in a rapid uptake of the substrate from the medium and initial labeling (0-4 hours) of primarily the intracellular glutamate and malate pools. Further metabolism of these intermediates resulted in labeling of glutamine, aspartate, and alanine. With [1-¹³C]acetate as the substrate very little incorporation into intermediary metabolites was observed in the ¹³C NMR spectra due to loss of the label as ¹³CO₂. Uptake of [3-¹³C]pyruvate by the cells was considerably slower than with [2-¹³C]acetate; however, the labelling patterns were similar with the exception of increased [3-¹³C] alanine generation with pyruvate as the substrate. Growth of the cells for up to 96 hours with 2 millimolar [3-¹³C]pyruvate ultimately resulted in labeling of valine, leucine, isoleucine, threonine, and the polyamine putrescine.     

Multinuclear NMR of cis-dicarbonylbis(dithiocarbamato)iron(II) complexes in chloroform solution

The ¹³C and ¹⁵SN NMR spectra of eleven cis-Fe(S₂CNRR′)₂(CO)₂ complexes, where R and R′ are organic substituents, have been measured at ambient temperature in CDCl₃ (0.08–0.16 M). The ¹³C absorptions for the carbonyl ligands correlate well with the force constants for the CO stretching vibrations in CHCl₃ solution. Each of the parameters (¹³CO absorption and k_cis for CO) correlate well with the aqueous solution pK_a for⁺H₂NRR′, corrected for the phenyl-containing substituents, high pK_a values corresponding to high ¹³CO absorptions and low k_cis CO force constants. [p ]Evidence was found in the ¹³C NMR spectra for hindered rotation about the CN bond in S₂CNC₂ in complexes with higher pK_a(corr) values and in the ¹³C spectra of the corresponding thiuram disulfides. [p ]The ¹⁵N (natural abundance) NMR spectra for each of the complexes was determined. Each revealed a single sharp absorption in a region of the ¹⁵N NMR spectrum which indicates substantial CN double bond character, as one would expect for coordinated dithiocarbamate ligands.     

Utilization of 13C-labeled amino acids to probe the α-helical local secondary structure of a membrane peptide using electron spin echo envelope modulation (ESEEM) spectroscopy

Electron spin echo envelope modulation (ESEEM) spectroscopy in combination with site-directed spin labeling (SDSL) has been established as a valuable biophysical technique to provide site-specific local secondary structure of membrane proteins. This pulsed electron paramagnetic resonance (EPR) method can successfully distinguish between α-helices, β-sheets, and 3₁₀-helices by strategically using ²H-labeled amino acids and SDSL. In this study, we have explored the use of ¹³C-labeled residues as the NMR active nuclei for this approach for the first time. ¹³C-labeled d₅-valine (Val) or ¹³C-labeled d₆-leucine (Leu) were substituted at a specific Val or Leu residue (i), and a nitroxide spin label was positioned 2 or 3 residues away (denoted i-2 and i-3) on the acetylcholine receptor M2δ (AChR M2δ) in a lipid bilayer. The ¹³C ESEEM peaks in the FT frequency domain data were observed for the i-3 samples, and no ¹³C peaks were observed in the i-2 samples. The resulting spectra were indicative of the α-helical local secondary structure of AChR M2δ in bicelles. This study provides more versatility and alternative options when using this ESEEM approach to study the more challenging recombinant membrane protein secondary structures.     

Novel bis(diethylenetriamine)thallium(III) complex. Synthesis and characterization in pyridine solution and in solid

A new complex of thallium(III) with the nitrogen donor ligand diethylenetriamine (dien) has been prepared and characterized by multinuclear NMR (¹H, ¹³C, ²⁰⁵Tl), infrared and Raman spectroscopy, and X-ray diffraction. In solution, the symmetric s-facial isomer of [Tl(dien)₂]³⁺ is formed. This is a fluxional molecule even at low temperature (235 K); therefore, the different rotamers cannot be observed separately. A complete characterization of the complex is given from its non-trivial NMR spectra. The crystal structure of [Tl(dien)₂](ClO₄)₃·H₂O shows u-facial geometry, where the coordination environment around thallium can be described as a distorted trigonal prism.     

13C-2H correlation NMR spectroscopy studies of the in vivo transformations of natural products from Artemisia annua

¹³C-²H correlation NMR spectroscopy (¹³C-²H COSY) permits the identification of ¹³C and ²H nuclei which are connected to one another by a single chemical bond via the sizeable ¹J_CD coupling constant. The practical development of this technique is described using a ¹³C-²H COSY pulse sequence which is derived from the classical ¹³C-¹H correlation experiment. An example is given of the application of ¹³C-²H COSY to the study of the biogenesis of natural products from the anti-malarial plant Artemisia annua, using a doubly-labelled precursor molecule. Although the biogenesis of artemisinin, the anti-malarial principle from this species, has been extensively studied over the past twenty years there is still no consensus as to the true biosynthetic route to this important natural product – indeed, some published experimental results are directly contradictory. One possible reason for this confusion may be the ease with which some of the metabolites from A. annua undergo spontaneous autoxidation, as exemplified by our recent in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid, and the application of ¹³C-²H COSY to this biosynthetic problem has been important in helping to mitigate against such processes. In this in vivo application of ¹³C-²H COSY, [15-¹³C²H₃]-dihydroartemisinic acid (the doubly-labelled analogue of the natural product from this species which was obtained through synthesis) was fed to A. annua plants and was shown to be converted into several natural products which have been described previously, including artemisinin. It is proposed that all of these transformations occurred via a tertiary hydroperoxide intermediate, which is derived from dihyroartemisinic acid. This intermediate was observed directly in this feeding experiment by the ¹³C-²H COSY technique; its observation by more traditional procedures (e.g., chromatographic separation, followed by spectroscopic analysis of the purified product) would have been difficult owing to the instability of the hydroperoxide group (as had been established previously by our in vitro studies of the spontaneous autoxidation of dihydroartemisinic acid). This same hydroperoxide has been reported as the initial product of the spontaneous autoxidation of dihydroartemisinic acid in our previous in vitro studies. Its observation in this feeding experiment by the ¹³C-²H COSY technique, a procedure which requires the minimum of sample manipulation in order to achieve a reliable identification of metabolites (based on both ¹³C and ²H chemical shifts at the 15-position), provides the best possible evidence for its status as a genuine biosynthetic intermediate, rather than merely as an artifact of the experimental procedure.     

A 1H and 13C NMR Study on the Role of Salt-Bridges in the Formation of a Type I β-Turn in N-Acetyl-L-Asp-L-Glu-L-Lys-L-Ser-NH2

Abstract

The conformation of the tetrapeptide N-Acetyl-Asp⁷-Glu⁸-Lys⁹-Ser¹⁰-NH₂, a fragment of the type I collagen α-1 chain N-telopeptide, has been studied by ¹H and ¹³C NMR and circular dichroism spectroscopy. The spectroscopic evidence, based on two-dimensional, phase-sensitive NMR techniques such as COSY, ROESY, proton-carbon shift correlation and selective COLOC, indicates a strong dependence of the conformation on the experimental conditions. In CD₃OH/H₂O (60/40) at ca. neutral pH the tetrapeptide forms a β-turn, stabilized by a hydrogen bond between NH(S¹⁰) and CO(D⁷) and a strong salt-bridge between COO^?(E⁸) and NH₃ ⁺(K⁹). The β-turn is type I and appears to coexist with a non-hydrogen-bonded structure. The coexistence of these two conformers is proven by proton NMR data such as NH-NH ROEs, reduced NH-H_α(E⁸) coupling constant NH(E⁸) low-field shift and the temperature coefficient of NH(S¹⁰), whereas the conclusion regarding the salt-bridge is based on ¹³C results. In the same solvent, at a pH below the pKa of the carboxyl groups, no evidence for a conformation other than extended can be found. In aqueous solution at approximately neutral pH, evidence for the E⁸-K⁹ charge interaction is observed, but not for a hydrogen bond anywhere in the molecule.     

1H, 13C and 199Hg NMR characteristics of new amine-mercuric chloride complexes

Reduction of some N-alkylimines has been achieved with NaBH₄ to give the corresponding secondary amines with high yields (85–95%). These amines were characterized on the bases of their ¹H and ¹³C NMR spectra. The reaction of these amines with mercuric chloride to afford the corresponding complexes was found to occur through a weak dative bond between the nitrogen lone pair of electrons and the mercury atom to form HgCl₂L₂ complexes. The ¹H, ¹³C and ¹⁹⁹Hg NMR chemical shifts have been obtained as well as ¹J(¹³CH) and ²J(¹³CH) coupling constants. Labelling with nitrogen-15 revealed that there is a weak coupling between the nitrogen and the ¹⁹⁹Hg.     

The prevalence of carbon-13 in respiratory carbon dioxide as an indicator of the types of endogenous substrate. The change from lipid to carbohydrate during the respiratory rise in potato slices

Isotope discrimination is a common feature of biosynthesis in nature, with the result that different classes of carbon compounds frequently display different ¹³C/¹²C ratios. The ¹³C/¹²C ratio of lipid in potato tuber tissue is considerably lower than that for starch or protein. We have collected respiratory CO₂ from potato discs in successive periods through 24 hr from the time of cutting—an interval in which the respiration rate rises 3–5-fold. The ¹³C/¹²C ratio of the evolved CO₂ was determined for each period, and compared with the ¹³C/¹²C ratios of the major tissue metabolites. In the first hours the carbon isotope ratio of the CO₂ matches that of lipid. With time, the ratio approaches that typical of starch or protein. An estimation has been made of the contribution of lipid and carbohydrate to the total respiration at each juncture. In connection with additional observations, it was deduced that the basal, or initial, respiration represents lipid metabolism—possibly the α-oxidation of long chain fatty acids—while the developed repiration represents conventional tricarboxylic acid cycle oxidation of the products of carbohydrate glycolysis. The true isotopic composition of the respiratory CO₂ may be obscured by fractionation attending the refixation of CO₂ during respiration, and by CO₂ arising from dissolved CO₂ and bicarbonate preexisting in the tuber. Means are described for coping with both pitfalls.     

Structural changes in a binary mixed phospholipid bilayer of DOPG and DOPS upon saposin C interaction at acidic pH utilizing P and H solid-state NMR spectroscopy

Saposin C (Sap C) is known to stimulate the catalytic activity of the lysosomal enzyme glucosylceramidase (GCase) that facilitates the hydrolysis of glucosylceramide to ceramide and glucose. Both Sap C and acidic phospholipids are required for full activity of GCase. In order to better understand this interaction, mixed bilayer samples prepared from dioleoylphosphatidylglycerol (DOPG) and dioleoylphosphatidylserine (DOPS) (5:3 ratio) and Sap C were investigated using ²H and ³¹P solid-state NMR spectroscopy at temperatures ranging from 25 to 50 °C at pH 4.7. The Sap C concentrations used to carry out these experiments were 0 mol%, 1 mol% and 3 mol% with respect to the phospholipids. The molecular order parameters (S_CD) were calculated from the dePaked ²H solid-state NMR spectra of Distearoyl-d70-phosphatidylglycerol (DSPG-d70) incorporated with DOPG and DOPS binary mixed bilayers. The S_CD profiles indicate that the addition of Sap C to the negatively charged phospholipids is concentration dependent. S_CD profiles of 1 mol% of the Sap C protein show only a very slight decrease in the acyl chain order. However, the S_CD profiles of the 3 mol% of Sap C protein indicate that the interaction is predominantly increasing the disorder in the first half of the acyl chain near the head group (C1-C8) indicating that the amino and the carboxyl termini of Sap C are not inserting deep into the DOPG and DOPS mixed bilayers. The ³¹P solid-state NMR spectra show that the chemical shift anisotropy (CSA) for both phospholipids decrease and the spectral broadening increases upon addition of Sap C to the mixed bilayers. The data indicate that Sap C interacts similarly with the head groups of both acidic phospholipids and that Sap C has no preference to DOPS over DOPG. Moreover, our solid-state NMR spectroscopic data agree with the structural model previously proposed in the literature [X. Qi, G.A. Grabowski, Differential membrane interactions of saposins A and C. Implication for the functional specificity, J. Biol. Chem. 276 (2001) 27010-27017] [1].     

Reductive methylation andpKa determination of the lysine side chains in calbindin D9k

The Lys residues in the 75-residue Ca²⁺-binding protein calbindin D_9k were reductively methylated with¹³C-enriched formaldehyde. The possible structural effects resulting from the chemical modification were critically investigated by comparing two-dimensional NMR spectra and the exchange rates of some of the amide protons of the native and the modified protein. Our results show that the protein retains its structure even though 10 Lys out of a total of 75 amino acid residues were modified. In the Ca²⁺- and apo-forms of the protein, the¹³C-methylated Lys residues can be detected with high sensitivity and resolution using two-dimensional (¹H,¹³C)-heteronuclear multiple quantum coherence (HMQC) NMR spectroscopy. ThepKa values of the individual Lys residues in Ca²⁺-calbindin D_9k and apo-calbindin D_9k were obtained by combiningpH titration experiments and (¹H,¹³C)-HMQC NMR spectroscopy. Each Lys residue in the Ca²⁺- and apo-forms of calbindin D_9k has a uniquepKa value. The LyspKa values in the calcium protein range from 9.3 to 10.9, while those in the apo-protein vary between 9.7 and 10.7. Although apo-calbindin D_9k has a very similar structure compared to Ca²⁺-calbindin D_9k, the removal of two Ca²⁺ ions from the protein leads to an increase of thepKa values of the Lys residues.     

Reactions of Tp–Os nitrido complexes with the nucleophiles hydroxide and thiosulfate

The reaction between TpOs(N)Cl₂ (1) [Tp = hydrotris(1-pyrazolyl)borate] and aqueous (ⁿBu₄N)(OH) in THF-d₈ forms the nitrosyl complex TpOs(NO)Cl₂ (5) among other products, suggesting an initial hydroxide attack at the nitrido ligand. In contrast, the reaction of the acetate complex TpOs(N)(OAc)₂ (2) with NaOH in Me₂CO/H₂O yields the osmium bis-hydroxide complex TpOs(N)(OH)₂ (3), which has been structurally characterized by single-crystal X-ray diffraction. Acetate for hydroxide exchange could occur by ligand substitution or by nucleophilic attack at the carbonyl carbon of the acetate ligands (saponification). Reacting 2 with Na¹⁸OH in H₂¹⁸O/CD₃CN yields predominantly doubly ¹⁸O-labeled TpOs(N)(¹⁸OH)₂ (3-¹⁸O₂) and unlabeled acetate, by ESI/MS and ¹³C{¹H} NMR. This indicates that hydroxide reacts by substitution rather than by attack at the ligand. The reaction of 2 with the softer nucleophile thiosulfate occurs at the nitrido ligand, giving the thionitrosyl complex TpOs(NS)(OAc)₂ (4). Reacting 4 with NaOH in (CD₃)₂CO/D₂O also generates the bis-hydroxide complex 3.     

Multinuclear magnetic resonance study of Pt(II) compounds with sulfoxide ligands and crystal structures of complexes of the types [Pt(R2SO)X3] and Pt(R2SO)2Cl2

Pt(II) complexes of the types K[Pt(R₂SO)X₃], NR₄[Pt(R₂SO)X₃] and Pt(R₂SO)₂Cl₂ (where X = Cl or Br) were characterized by multinuclear magnetic resonance spectroscopy (¹⁹⁵Pt, ¹H and ¹³C). In ¹⁹⁵Pt NMR, the chloro ionic compounds have shown signals between −2979 and −3106 ppm, while the cis disubstituted complexes were observed at higher fields, between −3450 and −3546 ppm. The signal of the compound trans-Pt(DPrSO)₂Cl₂ was found at higher field (−3666 ppm) than its cis analogue (−3517 ppm), since π-back-donation is considerably less effective in the trans geometry. In ¹H NMR, a single signal was observed for the sulfoxide in [Pt(DMSO)Cl₃]⁻, but for the other more sterically hindered ligands, two series of resonances were observed for the protons in α and β positions. The coupling constant ³J(¹⁹⁵Pt-¹H) are between 15 and 33 Hz. The ¹³C NMR results were interpreted in relation to the concept of inversed polarization of the π sulfoxide bond. The ²J(¹⁹⁵Pt-¹³C) values vary between 35 and 66 Hz, while a few ³J(¹⁹⁵Pt-¹³C) couplings were observed (13-26 Hz). The crystal structures of five monosubstituted ionic compounds N(n-Bu)₄[Pt(TMSO)Cl₃], N(Me)₄[Pt(DPrSO)Cl₃], K[Pt(EMSO)Cl₃], K[Pt(TMSO)Br₃] · H₂O and N(Et)₄[Pt(DPrSO)Br₃] and one disubstituted complex cis-Pt(DBuSO)₂Cl₂ were determined. The trans influence of the different ligands is discussed.     

3D-TROSY-based backbone and ILV-methyl resonance assignments of a 319-residue homodimer from a single protein sample

The feasibility of practically complete backbone and ILV methyl chemical shift assignments from a single [U-²H,¹⁵N,¹³C; Ile??1-{¹³CH₃}; Leu,Val-{¹³CH₃/¹²CD₃}]-labeled protein sample of the truncated form of ligand-free Bst-Tyrosyl tRNA Synthetase (Bst-??YRS), a 319-residue predominantly helical homodimer, is established. Protonation of ILV residues at methyl positions does not appreciably detract from the quality of TROSY triple resonance data. The assignments are performed at 40?°C to improve the sensitivity of the measurements and alleviate the overlap of ¹H?C¹⁵N correlations in the abundant ??-helical segments of the protein. A number of auxiliary approaches are used to assist in the assignment process: (1) selection of ¹H?C¹⁵N amide correlations of certain residue types (Ala, Thr/Ser) that simplifies 2D ¹H?C¹⁵N TROSY spectra, (2) straightforward identification of ILV residue types from the methyl-detected ??out-and-back?? HMCM(CG)CBCA experiment, and (3) strong sequential HN?CHN NOE connectivities in the helical regions. The two subunits of Bst-YRS were predicted earlier to exist in two different conformations in the absence of ligands. In agreement with our earlier findings (Godoy-Ruiz in J Am Chem Soc 133:19578?C195781, 2011), no evidence of dimer asymmetry has been observed in either amide- or methyl-detected experiments.