1H,13C-1H,1H Dipolar Cross-correlated Spin Relaxation in Methyl Groups

Relaxation in methyl groups is strongly influenced by cross-correlated interactions involving the methyl dipoles. One of the major interference effects results from intra-methyl (1)H-(13)C, (1)H-(1)H dipolar interactions, leading to significant differences in the relaxation of certain multiplet components that contribute to double- and zero-quantum (1)H-(13)C spectra. NMR experiments are presented for the measurement of this differential relaxation effect. It is shown that this difference in relaxation between double- and zero-quantum multiplet components can be used as a sensitive reporter of side chain dynamics and that accurate methyl axis order parameters can be measured in proteins that tumble with correlation times greater than approximately 5 ns.     

Measurement of Dipolar Cross-Correlation in Methylene Groups in Uniformly 13C-, 15N-Labeled Proteins

A CC(CO)NH TOCSY-based 3D pulse scheme is presented for measuring (1)H-(13)C dipole-dipole cross-correlated relaxation at CH(2) positions in uniformly (13)C-, (15)N-labeled proteins. Simulations based on magnetization evolution under relaxation and scalar coupling interactions show that cross-correlation rates between (1)H-(13)C dipoles in CH(2) groups can be simply obtained from the intensities of (13)C triplets. The normalized cross-correlation relaxation rates are related to cross-correlation order parameters for macromolecules undergoing isotropic motion, which reflect the degrees of spatial restriction of CH(2) groups. The study on human intestinal fatty acid binding protein (131 residues) in the presence of oleic acid demonstrates that side chain dynamics at most CH(2) positions can be characterized for proteins less than 15 kDa in size, with the proposed TOCSY-based approach.     

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

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

Multiplet component separation for measurement of methyl 13C-1H dipolar couplings in weakly aligned proteins

A simple spectral editing procedure is described that generates separate subspectra for the methyl ¹³C-¹H₃ multiplet components of ¹H-¹³C HSQC spectra. The editing procedure relies on co-addition of in-phase and antiphase spectra and yields ¹H-coupled constant-time HSQC subspectra for the methyl region that have the simplicity of the regular decoupled CT-HSQC spectrum. Resulting spectra permit rapid and reliable measurement of ¹H-¹³C J and dipolar couplings. The editing procedure is illustrated for a Ca²⁺-calmodulin sample in isotropic and liquid crystalline phases.     

Support of1H NMR assignments in proteins by biosynthetically directed fractional13C-labeling

Summary Biosynthetically directed fractional incorporation of¹³C into proteins results in nonrandom¹³C-labeling patterns that can be investigated by analysis of the¹³C–¹³C scalar coupling fine structures in heteronuclear¹³C–¹H or homonuclear¹³C–¹³C correlation experiments. Previously this approach was used for obtaining stereospecific¹H and¹³C assignments of the diastereotopic methyl groups of valine and leucine. In the present paper we investigate to what extent the labeling patterns are characteristic for other individual amino acids or groups of amino acids, and can thus be used to support the¹H spin-system identifications. Studies of the hydrolysates of fractionally¹³C-labeled proteins showed that the 59 aliphatic carbon positions in the 20 proteinogenic amino acids exhibit 16 different types of¹³C–¹³C coupling fine structures. These provide support for the assignment of the resonances of all methyl groups in a protein, which are otherwise often poorly resolved in homonuclear¹H NMR spectra. In particular, besides the individual methyl assignments in Val and Leu, unambiguous distinctions are obtained between the methyl groups of Ala and Thr, and between the - and -methyl groups of Ile. In addition to the methyl resonances, the CH₂ groups of Glu and Gln can be uniquely assigned because of the large coupling constant with the -carbon, and the identification of most of the other spin systems can be supported on the basis of coupling patterns that are common to small groups of amino acid residues.Abbreviations NOE nuclear Overhauser effect - fractional¹³C labeling biosynthetically directed fractional¹³C-labeling - TOCSY total correlation spectroscopy - ROESY rotating frame Overhauser enhancement spectroscopy - [¹³C,¹H]-COSY two-dimensional¹³C–¹H correlation spectroscopy - isotopomer isotope isomer - P22 c2 repressor c2 repressor of the salmonella phage P22 consisting of a polypeptide chain with 216 residues - P22 c2(1-76) N-terminal domain of the P22 c2 repressor with residues 1–76     

Improved labeling strategy for 13C relaxation measurements of methyl groups in proteins

Selective incorporation of ¹³C into the methyl groupsof protein side chains is described as a means for simplifying themeasurement and interpretation of ¹³C relaxation parameters.High incorporation (>90%) is accomplished by using pyruvate(3-¹³C, 99%) as the sole carbon source in the growthmedia for protein overexpression in E. coli. This improved labeling schemeincreases the sensitivity of the relaxation experiments by approximatelyfivefold when compared to randomly fractionally ¹³C-labeledprotein, allowing high-quality measurements on relatively dilute (<1 mM)protein samples at a relatively low cost.     

Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance

Flexible ligands pose challenges to standard structure-activity studies since they frequently reorganize their conformations upon protein binding and catalysis. Here, we demonstrate the utility of side chain ¹³C relaxation dispersion measurements to identify and quantify the conformational dynamics that drive this reorganization. The dispersion measurements probe methylene ¹³CH₂ and methyl ¹³CH₃ groups; the latter are highly prevalent side chain moieties in known drugs. Combining these side chain studies with existing backbone dispersion studies enables a comprehensive investigation of μs–ms conformational dynamics related to binding and catalysis. We perform these measurements at natural ¹³C abundance, in congruence with common pharmaceutical research settings. We illustrate these methods through a study of the interaction of a phosphopeptide ligand with the peptidyl-prolyl isomerase, Pin1. The results illuminate the side-chain moieties that undergo conformational readjustments upon complex formation. In particular, we find evidence that multiple exchange processes influence the side chain dispersion profiles. Collectively, our studies illustrate how side-chain relaxation dispersion can shed light on ligand conformational transitions required for activity, and thereby suggest strategies for its optimization.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

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.     

Side chain dynamics monitored by 13C-13C cross-relaxation

A method to measure (13)C-(13)C cross-relaxation rates in a fully (13)C labeled protein has been developed that can give information about the mobility of side chains in proteins. The method makes use of the (H)CCH-NOESY pulse sequence and includes a suppression scheme for zero-quantum (ZQ) coherences that allows the extraction of initial rates from NOE buildup curves.The method has been used to measure (13)C-(13)C cross-relaxation rates in the 269-residue serine-protease PB92. We focused on C(alpha)-C(beta) cross-relaxation rates, which could be extracted for 64% of all residues, discarding serine residues because of imperfect ZQ suppression, and methyl (13)C-(13)C cross-relaxation rates, which could be extracted for 47% of the methyl containing C-C pairs. The C(alpha)-C(beta) cross-relaxation rates are on average larger in secondary structure elements as compared to loop regions, in agreement with the expected higher rigidity in these elements. The cross-relaxation rates for methyl containing C-C pairs show a general decrease of rates further into the side chain, indicating more flexibility with increasing separation from the main chain. In the case of leucine residues also long-range C(beta)-C(delta) cross-peaks are observed. Surprisingly, for most of the leucines a cross-peak with only one of the methyl C(delta) carbons is observed, which correlates well with the chi(2) torsion-angle and can be explained by a difference in mobility for the two methyl groups due to an anisotropic side chain motion.     

3D 13C-15N-heteronuclear two-spin coherence spectroscopy for polypeptide backbone assignments in 13C-15N-double-labeled proteins

Summary The pulse sequence of a new constant-time 3D triple-resonance experiment, ct-HA[CAN]HN, is presented. This experiment delineates exclusively scalar connectivities and uses ¹³C–¹⁵N heteronuclear two-spin coherence to overlay the chemical shift evolution periods of the ¹³C and ¹⁵N nuclei, thereby providing the four resonance frequencies of the -proton, the -carbon, the amide nitrogen, and the amide proton of a given amino acid residue in three dimensions. This experiment promises to be a valid alternative to 4D experiments, providing the same information on intraresidue polypeptide backbone connectivities in ¹³C-¹⁵N-double-labeled proteins.Abbreviations 3D, 4D three-dimensional, four-dimensional - TPPI time-proportional phase incrementation - ct constant-time - rf radiofrequency - NOE nuclear Overhauser enhancement - NOESY two-dimensional nuclear Overhauser enhancement spectroscopy - glutaredoxin(C14S) mutant E. coli glutaredoxin with the cysteine at position 14 replaced by serine     

Off-resonance rotating-frame relaxation dispersion experiment for 13C in aromatic side chains using L-optimized TROSY-selection

Protein dynamics on the microsecond–millisecond time scales often play a critical role in biological function. NMR relaxation dispersion experiments are powerful approaches for investigating biologically relevant dynamics with site-specific resolution, as shown by a growing number of publications on enzyme catalysis, protein folding, ligand binding, and allostery. To date, the majority of studies has probed the backbone amides or side-chain methyl groups, while experiments targeting other sites have been used more sparingly. Aromatic side chains are useful probes of protein dynamics, because they are over-represented in protein binding interfaces, have important catalytic roles in enzymes, and form a sizable part of the protein interior. Here we present an off-resonance R _1ρ experiment for measuring microsecond to millisecond conformational exchange of aromatic side chains in selectively ¹³C labeled proteins by means of longitudinal- and transverse-relaxation optimization. Using selective excitation and inversion of the narrow component of the ¹³C doublet, the experiment achieves significant sensitivity enhancement in terms of both signal intensity and the fractional contribution from exchange to transverse relaxation; additional signal enhancement is achieved by optimizing the longitudinal relaxation recovery of the covalently attached ¹H spins. We validated the L-TROSY-selected R _1ρ experiment by measuring exchange parameters for Y23 in bovine pancreatic trypsin inhibitor at a temperature of 328 K, where the ring flip is in the fast exchange regime with a mean waiting time between flips of 320 μs. The determined chemical shift difference matches perfectly with that measured from the NMR spectrum at lower temperatures, where separate peaks are observed for the two sites. We further show that potentially complicating effects of strong scalar coupling between protons (Weininger et al. in J Phys Chem B 117: 9241–9247, 2013b) can be accounted for using a simple expression, and provide recommendations for data acquisition when the studied system exhibits this behavior. The present method extends the repertoire of relaxation methods tailored for aromatic side chains by enabling studies of faster processes and improved control over artifacts due to strong coupling.     

Effect of deuteration on some structural parameters of methyl groups in proteins as evaluated by residual dipolar couplings

One bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar and residual dipolar couplings have been measured at sites in an ¹⁵N, ¹³C, 50% ²H labeled sample of the B1 immunoglobulin binding domain of peptostreptococcal protein L to investigate changes in the structure of methyl groups in response to deuterium substitution. Both one bond methyl ¹H-¹³C and ¹³C_methyl–¹³C scalar coupling constants have been found to decrease slightly with increasing deuterium content. Previous studies have shown that ¹H-¹³C couplings in methyl groups are exquisitely sensitive to electronic structure, with decreases in coupling values as a function of deuteration consistent with a slight lengthening of the remaining H-C bonds. Changes in the H_methylC_methylC angle are found to be small, with average differences on the order of 0.3 ± 0.1° and 0.4 ± 0.2° between CH₃, CH₂D and CH₃, CHD₂ isotopomers, respectively. Knowledge of methyl geometry is a prerequisite for the extraction of accurate dynamics parameters from spin relaxation studies involving these groups.     

Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA

Heteronuclear NMR spin relaxation studies of conformational dynamics are coming into increasing use to help understand the functions of ribozymes and other RNAs. Due to strong magnetic interactions within the ribose ring, however, these studies have thus far largely been limited to ¹³C and ¹⁵N resonances on the nucleotide base side chains. We report here the application of the alternate-site ¹³C isotopic labeling scheme, pioneered by LeMaster for relaxation studies of amino acid side chains, to nucleic acid systems. We have used different strains of E. coli to prepare mononucleotides containing ¹³C label in one of two patterns: Either C1′ or C2′ in addition to C4′, termed (1′/2′,4′) labeling, or nearly complete labeling at the C2′ and C4′ sites only, termed (2′,4′) labeling. These patterns provide isolated H spin systems on the labeled carbon atoms and thus allow spin relaxation studies without interference from scalar or dipolar coupling. Using relaxation studies of AMP dissolved in glycerol at varying temperature to produce systems with correlation times characteristic of different size RNAs, we demonstrate the removal of errors due to interaction in T ₁ measurements of larger nucleic acids and in T _1ρ measurements in RNA molecules. By extending the applicability of spin relaxation measurements to backbone ribose groups, this technology should greatly improve the flexibility and completeness of NMR analyses of conformational dynamics in RNA.     

Investigation of labeled amino acid side-chain motion in collagen using 13C nuclear magnetic resonance

¹³C¹H double magnetic resonance was used to study the interactions and mobility of certain amino acid side-chains of collagen. Samples of collagen, labeled with [3-¹³C]alanine (a small hydrophobic amino acid), [methyl-¹³C]-methionine (a large hydrophobic), [6-¹³C]lysine (positively charged at physiological pH), and [5-¹³C]glutamic acid (negatively charged), were prepared via chick calvaria culture. ¹³C linewidths, lineshapes, NOE² values, and T₁ values were measured for each sample as fibrils and as native (helical) material in solution.The measured T₁ and NOE values for [3-¹³C]alanine-labeled collagen in solution, in conjunction with an ellipsoid model for collagen, indicate that the methyl rotation rate is 2 × 10¹⁰ s^?1 and that the overall rate of diffusion about the long axis is 4× 10⁶ s^?1. These values agree with values for model compounds which undergo internal methyl rotation (Lyerla & Horikawa, 1976) and with previous n.m.r. measurements of the rate of rotational diffusion of backbone ([1-¹³C]- and [2-¹³C]glycine)-labeled collagen (Jelinski & Torchia, 1979). In addition, the n.m.r. data indicate that the terminal carbons of lysine, methionine and glutamic acid in labeled collagen (both in solution and as fibrils) are characterized by reorientation rates of approximately 10⁹ to 10¹⁰ s^?1.Taken together, the n.m.r. data provide strong evidence that the contact regions between the helices in collagen fibrils are fluid and that there is not a unique set of interactions between amino acid side-chains. In this respect, these n.m.r. results support current concepts of globular protein structure which suggest that a variety of conformations, in dynamic equilibrium, are responsible for the structure and function of proteins.     

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

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

Differences in lysine pKa values may be used to improve NMR signal dispersion in reductively methylated proteins

Reductive methylation of lysine residues in proteins offers a way to introduce ¹³C methyl groups into otherwise unlabeled molecules. The ¹³C methyl groups on lysines possess favorable relaxation properties that allow highly sensitive NMR signal detection. One of the major limitations in the use of reductive methylation in NMR is the signal overlap of ¹³C methyl groups in NMR spectra. Here we show that the uniform influence of the solvent on chemical shifts of exposed lysine methyl groups could be overcome by adjusting the pH of the buffering solution closer to the pKa of lysine side chains. Under these conditions, due to variable pKa values of individual lysine side chains in the protein of interest different levels of lysine protonation are observed. These differences are reflected in the chemical shift differences of methyl groups in reductively methylated lysines. We show that this approach is successful in four different proteins including Ca²⁺-bound Calmodulin, Lysozyme, Ca²⁺-bound Troponin C, and Glutathione S-Transferase. In all cases significant improvement in NMR spectral resolution of methyl signals in reductively methylated proteins was obtained. The increased spectral resolution helps with more precise characterization of protein structural rearrangements caused by ligand binding as shown by studying binding of Calmodulin antagonist trifluoperazine to Calmodulin. Thus, this approach may be used to increase resolution in NMR spectra of ¹³C methyl groups on lysine residues in reductively methylated proteins that enhances the accuracy of protein structural assessment. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

An (H)C(CO)NH-TOCSY pulse scheme for sequential assignment of protonated methyl groups in otherwise deuterated 15N, 13C-labeled proteins

Summary A biosynthetic strategy has recently been developed for the production of ¹⁵N, ¹³C, ²H-labeled proteins using ¹H₃C-pyruvate as the sole carbon source and D₂O as the solvent. The methyl groups of Ala, Val, Leu and Ile (2 only) remain highly protonated, while the remaining positions in the molecule are largely deuterated. An (H)C(CO)NH-TOCSY experiment is presented for the sequential assignment of the protonated methyl groups. A high-sensitivity spectrum is recorded on a ¹⁵N, ¹³C, ²H, ¹H₃C-labeled SH2 domain at 3°C (correlation time 18.8 ns), demonstrating the utility of the method for proteins in the 30–40 kDa molecular weight range.     

Cross-correlation suppressed T1 and NOE experiments for protein side-chain 13CH2 groups

Relaxation measurements of side-chain ¹³CH₂-groups of uniformly ¹³C labeled human ubiquitin were performed at 600 MHz and 800 MHz magnetic field strength at 30°C. Dipole-dipole cross-correlated relaxation effects in T₁ experiments were suppressed by the combination of radio-frequency pulses and pulsed field gradients during the relaxation delay leading to monoexponential relaxation decays that allow a more accurate extraction of the ¹³C T₁ relaxation times. Heteronuclear ¹H-¹³C NOEs obtained by using different proton saturation schemes indicate that the influence of cross-correlation is small. The experimental T₁ and NOE data were interpreted in a model-free way in terms of a generalized order parameter and an internal correlation time.