Identification of solvent-exposed regions of an FK-506 analog,ascomycin, bound to FKBP using a paramagnetic probe

Summary The solvent-exposed regions of [U-¹³C]ascomycin when bound to its putative target protein; FKBP, have been identified based on the different proton longitudinal relaxation rates (R₁ = 1/T₁) measured in the absence and presence of the paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethyl-piperidinyl-I-oxy (HyTEMPO). The proton T₁s of bound ascomycin were determined using a pulse sequence (T₁-HMQC) which consists of a 180° proton pulse and a variable delay () followed by a heteronuclear multiple quantum correlation (HMQC) experiment. The solvent-exposed regions of ascomycin determined by these experiments are compared to NOE data in which ascomycin/FKBP contacts were identified and to the X-ray structure of the FK-506/FKBP complex.     

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.     

Perspective: revisiting the field dependence of TROSY sensitivity

The discovery of the TROSY effect (Pervushin et al. in Proc Natl Acad Sci USA 94:12366–12371, 1997) for reducing transverse relaxation and line sharpening through selecting pathways in which dipole–dipole and CSA Hamiltonians partially cancel each other had a tremendous impact on solution NMR studies of macromolecules. Together with the methyl TROSY (Tugarinov and Kay in J Biomol NMR 28:165–172, 2004) it enabled structural and functional studies of significantly larger systems. The optimal field strengths for TROSY have been estimated to be on spectrometers operating around 900 MHz (21.14 T) for the ¹H_N TROSY (Pervushin et al. in Proc Natl Acad Sci USA 94:12366–12371, 1997) while the aromatic ¹³C (¹³C_aro) TROSY is posited to be optimal at around 600 MHz (14.09 T) (Pervushin et al. in J Am Chem Soc 120:6394–6400, 1998b; Pervushin in Q Rev Biophys 33:161–197, 2000). The initial rational was based on the consideration of where the quadratic B₀ field dependences of the TROSY relaxation rates reach a minimum. For sensitivity consideration, however, it is interesting to estimate which field strengths yield the tallest peaks. Recent studies of ¹⁵N-detected TROSYs suggested that maximal peak heights are expected at 1.15 GHz (27.01 T) although the slowest relaxation rates or longest transverse relaxation times T₂ are indeed expected around 900 MHz (21.14 T) (Takeuchi in J Biomol NMR 63:323–331, 2015; Takeuchi et al. in J Biomol NMR 64:143–151, 2016). This was based on the fact that the heights of Lorentzian lines are proportional to B _o ^3/2 * T₂ (B_o). Thus, multiplying the parabolic T₂(B_o) dependence with the increasing function of B _o ^3/2 shifts the maxima of peak-height field dependence from the T₂ maximum at 900 MHz to higher fields. Moreover, besides shifting the peak height maximum for ¹⁵N TROSY, this analysis yields estimates for optimal peak heights for ¹H_N detected TROSY to 1.5 GHz, and to 900 MHz for ¹³C-detected ¹³C_aroTROSY as is detailed below. To our knowledge, this aspect of field dependence of TROSY sensitivity has not been in the attention of the NMR community but may affect perspectives of NMR at ultra-high fields.     

Anisotropic rotational diffusion of perdeuterated HIV protease from 15N NMR relaxation measurements at two magnetic fields

Summary ¹⁵N NMR relaxation times in perdeuterated HIV-1 protease, complexed with the sub-nanomolar inhibitor DMP323, have been measured at 600 and 360 MHz ¹H frequency. The relative magnitudes of the principal components of the inertia tensor, calculated from the X-ray coordinates of the protein-drug complex, are 1.0:0.85:0.44. The relation between the T₁/T₂ ratios observed for the individual backbone amides and their N-H orientation within the 3D structure of the protease dimer yields a rotational diffusion tensor oriented nearly collinear to the inertia tensor. The relative magnitudes of its principal components (1.00:1.11:1.42) are also in good agreement with hydrodynamic modeling results. The orientation and magnitude of the diffusion tensors derived from relaxation data obtained at 360 and 600 MHz are nearly identical. The anisotropic nature of the rotational diffusion has little influence on the order parameters derived from the ¹⁵N T₁ and T₂ relaxation times; however, if anisotropy is ignored, this can result in erroneous identification of either exchange broadening or internal motions on a nanosecond time scale. The average ratio of the T₁ values measured at 360 and 600 MHz is 0.50±0.015, which is slightly larger than the value of 0.466 expected for an isotropic rigid rotor with _c = 10.7 ns. The average ratio of the T₂ values measured at 360 and 600 MHz is 1.14±0.04, which is also slightly larger than the expected ratio of 1.11. This magnetic field dependence of the T₁ and T₂ relaxation times suggests that the spectral density contribution from fast internal motions is not negligible, and that the chemical shift anisotropy of peptide backbone amides, on average, is larger than the 160 ppm value commonly used in ¹⁵N relaxation studies of proteins.     

Comparison of 15N- and 13C-determined parameters of mobility in melittin

Backbone and tryptophan side-chain mobilities in the 26-residue, cytolytic peptide melittin (MLT) were investigated by ¹⁵N and ¹³C NMR. Specifically, inverse-detected ¹⁵N T₁ and steady-state NOE measurements were made at 30 and 51 MHz on MLT at 22 °C enriched with ¹⁵N at six amide positions and in the Trp¹⁹ side chain. Both the disordered MLT monomer (1.2 mM peptide at pH 3.6 in neat water) and -helical MLT tetramer (4.0 mM peptide at pH 5.2 in 150 mM phosphate buffer) were examined. The relaxation data were analyzed in terms of the Lipari and Szabo model-free formalism with three parameters: _m, the correlation time for the overall rotation; S², a site-specific order parameter which is a measure of the amplitude of the internal motion; and _e, a local, effective correlation time of the internal motion. A comparison was made of motional parameters from the ¹⁵N measurements and from ¹³C measurements on MLT, the latter having been made here and previously [Kemple et al. (1997) Biochemistry, 36, 1678–1688]. _m and _e values were consistent from data on the two nuclei. In the MLT monomer, S² values for the backbone N-H and C-H vectors in the same residue were similar in value but in the tetramer the N-H order parameters were about 0.2 units larger than the C-H order parameters. The Trp side-chain N-H and C-H order parameters, and _e values were generally similar in both the monomer and tetramer. Implications of these results regarding the dynamics of MLT are examined.     

NMR study of 17O from H217O in human erythrocytes

Human erythrocytes were incubated in a Ringer's solution enriched with 10–18% H₂¹⁷O. The longitudinal relaxation time (T₁) of the ¹⁷O was determined separately in samples of red cell suspesions, packed cells, and supernatant. The longitudinal relaxation of ¹⁷O in erythrocyte suspensions was non-exponential, reflecting water exchange across the cell membranes as well as relaxation processes inside and outside the cell.The T₁ of intracellular ¹⁷O is 4–5 times shorter than in the supernatant, similar to the enhancement of proton relaxation by hemoglobin in erythrocytes and free solution at the frequency applied (8.13 MHz). This datum is consistent with the thesis that hemoglobin modifies the NMR relaxation behavior of water inside cells and in free solution in the same way.The rate constant

for water exchange was calculated to be 60 and 107 s⁻¹ at 25 and at 37° C, respectively. The apparent activation energy for

over the temperature range 23–37° C was 8.7±1.0 kcal/mole.     

Protein dynamics studied by rotating frame 15N spin relaxation times

Summary Conformational rate processes in aqueous solutions of uniformly ¹⁵N-labeled pancreatic trypsin inhibitor (BPTI) at 36°C were investigated by measuring the rotating frame relaxation times of the backbone ¹⁵N spins as a function of the spin-lock power. Two different intramolecular exchange processes were identified. A first local rate process involved the residues Cys³⁸ and Arg³⁹, had a correlation time of about 1.3 ms, and was related to isomerization of the chirality of the disulfide bond Cys¹⁴-Cys³⁸. A second, faster motional mode was superimposed on the disulfide bond isomerization and was tentatively attributed to local segmental motions in the polypeptide sequence-Cys¹⁴-Ala¹⁵-Lys¹⁶-. The correlation time for the overall rotational tumbling of the protein was found to be 2 ns, using the assumption that relaxation is dominated by dipolar coupling and chemical shift anistropy modulated by isotropic molecular reorientation.Abbreviations BPTI basic pancreatic trypsin inhibitor - 2D two-dimensional - COSY 2D correlation spectroscopy - TOCSY 2D total correlation spectroscopy - RF radio frequency - CW continuous wave - TPPI time-proportional phase incrementation - CSA chemical shift anisotropy - T₁ longitudinal relaxation time - T₂ transverse relaxation time - T₁ relaxation time in the rotating frame , correlation time for overall rotational reorientation of the protein - _ex ^s , _ex ^f , correlation times for two conformational exchange processes (slow and fast).     

Characterisation of Soybean and Wheat Seeds by Nuclear Magnetic Resonance Spectroscopy

The effects of equilibration under different air relative humidities (RH, 1 – 90 %) and temperatures (35 and 45 °C) on soybean (Glycine max) and wheat (Triticum aestivum) seeds were studied using different techniques. Seed moisture content, electrical conductivity (EC) of seed leachate and per cent seed germination were measured following standard procedures, and compared with nuclear magnetic resonance spin-spin relaxation time (T₂) measurements. Moisture contents of soybean and wheat seeds, following the reverse sigmoidal trend, were greater at 35 than at 45 °C at any particular RH. Changes in T₂ were related to the changes in germination percentage and leachate EC of both soybean and wheat seeds. Equilibrating soybean seeds at RH 11 % decreased germination percentage with corresponding decrease in T₂. On the contrary, EC of seed leachate increased. In wheat seeds equilibrated at 45 °C, T² was maximal at RH 5.5 %. T₂ declined in seeds equilibrated at high RH (> 80 %) together with low germination percentage.     

Conformation and dynamic structure of poly(inosinic acid) in neutral aqueous solution by 1H-, 2H-, 13C-, and 31P-NMR spectroscopy

The conformation and dynamic structure of single-stranded poly(inosinic acid), poly(I), in aqueous solution at neutral pH have been investigated by nmr of four nuclei at different frequencies: ¹H (90 and 250 MHz), ²H (13.8 MHz), ¹³C (75.4 MHz), and ³¹P (36.4 and 111.6 MHz). Measurements of the proton-proton coupling constants and of the ¹H and ¹³C chemical shifts versus temperature show that the ribose is flexible and that base-base stacking is not very significant for concentrations varying from 0.04 to 0.10M in the monomer unit. On the other hand, the proton T₁ ratios between the sugar protons, T₁ (H₁′)/T₁ (H₃′), indicate a predominance of the anti orientation of the base around the glycosidic bond. The local motions of the ribose and the base were studied at different temperatures by measurements of nuclear Overhauser enhancement (NOE) of protonated carbons, the ratio of the proton relaxation times measured at two frequencies (90 and 250 MHz), and the deuterium quadrupolar transverse relaxation time T₂. For a given temperature between 22 and 62°C, the ¹³C-{¹H} NOE value is practically the same for seven protonated carbons (C₂, C₈, C_1′, C_2′, C_3′, C_4′, C_5′). This is also true for the T₁ ratio of the corresponding protons. Thus, the motion of the ribose–base unit can be considered as isotropic and characterized by a single correlation time, τ_c, for all protons and carbons. The τ_c values determined from either the ¹³C-{¹H} NOE or proton T₁ ratios, T₁(90 MHz)/T₁(250 MHz), and/or deuterium transverse relaxation time T₂ agree well. The molecular motion of the sugar-phosphate backbone (O-P-O) and the chemical-shift anisotropy (CSA) were deduced from T₁ (³¹P) and ³¹P-{¹H} NOE measurements at two frequencies. The CSA contribution to the phosphorus relaxation is about 12% at 36.4 MHz and 72% at 111.6 MHz, corresponding to a value of 118 ppm for the CSA (σ = σ∥ ? σ?). Activation energies of 2–6 kcal/mol for the motion of the ribose–base unit and the sugarphosphate backbone were evaluated from the proton and phosphorus relaxation data.     

Measurement of proton relaxation rates in proteins

Summary Five different types of experiment are described which make it possible to measure various relaxation rates of selected protons in crowded spectra of macromolecules such as proteins: longitudinal spin-lattice relaxation rates =1/T₁, transverse relaxation rates =1/T₂ measured under conditions of free precession, transverse relaxation rates ¹ _LOCK=1/T₁ measured under conditions of spin-locking, and transverse relaxation rates ^DQC=1/T₂ ^DQC and ^ZQC=1/T₂ ^ZQC of double- and zero-quantum coherences. The surprisingly large discrepancy between the transverse rates ^t and ^t is discussed in detail. To separate overlapping proton signals, the experimental schemes involve one or several magnetization transfer steps, using a doubly selective homonuclear Hartmann-Hahn method. Numerous variants of the basic ideas can be conceived, depending on the extent of signal overlap and on the topology of the networks of scalar couplings. Applications are shown to H and H of Tyr²³, to H, H and H of Cys³⁰, and to H and H of Ala²⁴ in bovine pancreatic trypsin inhibitor (BPTI).     

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.     

An investigation of the dynamics of spermine bound to duplex and quadruplex DNA by <Superscript>13</Superscript>C NMR spectroscopy

A detailed analysis of the ¹³C relaxation of ¹³C-labelled spermine bound to duplex and quadruplex DNA is presented. T₁, T₂ and heteronuclear NOE data were collected at four ¹³C frequencies (75.4, 125.7, 150.9 and 201.2 MHz). The data were analyzed in terms of a frequency-dependent order parameter, S ²(ω), to estimate the generalized order parameter and the contributions to the relaxation from different motional frequencies in the picosecond–nanosecond timescale and from any exchange processes that may be occurring on the microsecond–millisecond timescale. The relaxation data was surprisingly similar for spermine bound to two different duplexes and a linear parallel quadruplex. Analysis of the relaxation data from these complexes confirmed the conclusions of previous studies that the dominant motion of spermine is independent of the macroscopic tumbling of the DNA and has an effective correlation time of ∼50 ps. In contrast, spermine bound to a folded antiparallel quadruplex had faster relaxation rates, especially R ₂. As with the other complexes, a fast internal motion of the order of 50 ps makes a substantial contribution to the relaxation. The generalized order parameter for spermine bound to duplex DNA and the linear quadruplex is small but is larger for spermine bound to the folded quadruplex. In the latter case, there is evidence for exchange between at least two populations of spermine occurring on the microsecond–millisecond timescale. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium

In this paper it is demonstrated that cross-correlated time modulation of isotropic chemical shifts (`conformational exchange') leads to differential relaxation of double- and zero-quantum coherences, respectively. Quantitative information can be obtained from the time dependence of the interconversion between the two two-spin coherences 2I_xS_x and 2I_yS_y, induced by the differential relaxation. The effect is illustrated with an application to ¹³C,¹⁵N-labeled quail CRP2(LIM2), by studying ¹⁵N-¹H^N multiple-quantum relaxation. Significant cross-correlated fluctuations of isotropic chemical shifts were observed for residues which are part of a disordered loop region connecting two -strands in CRP2(LIM2). Differential ¹H^N and ¹⁵N exchange contributions to multiple-quantum relaxation observed at these sites illustrate the complex interplay between hydrogen bonding events and conformational reorientations in proteins.     

Backbone dynamics of an alamethicin in methanol and aqueous detergent solution determined by heteronuclear 1H−15N NMR spectroscopy

Summary The ¹⁵N relaxation rates of the -aminoisobutyric acid (Aib)-rich peptide alamethicin dissolved in methanol at 27°C and 5°C, and dissolved in aqueous sodium dodecylsulfate (SDS) at 27°C, were measured using inverse-detected one-and two-dimensional ¹H–¹⁵N NMR spectroscopy. Measurements of ¹⁵N longitudinal (R_N(N_z)) and transverse (R_N(N_x,y)) relaxation rates and the {¹H} ¹⁵N nuclear Overhauser enhancement (NOE) at 11.7 Tesla were used to calculate (quasi-) spectral density values at 0, 50, and 450 MHz for the peptide in methanol and in SDS. Spectral density mapping at 0, 50, 450, 500, and 550 MHz was done using additional measurements of the ¹H–¹⁵N lingitudinal two-spin order, R_NH(2H _infZ ^supN N_Z), two-spin antiphase coherence, R_NH(2H _infN ^supZ N_x,y), and the proton longitudinal relaxation rate, R_H(H _infN ^supZ ), for the peptide dissolved in methanol only. The spectral density of motions was also modeled using the three-parameter Lipari-Szabo function. The overall rotational correlation times were determined to be 1.1, 2.5, and 5.7 ns for alamethicin in methanol at 27°C and 5°C, and in SDS at 27°C, respectively. From the rotational correlation time determined in SDS the number of detergent molecules associated with the peptide was estimated to be about 40. The average order parameter was about 0.7 and the internal correlation times were about 70 ps for the majority of backbone amide ¹⁵N sites of alamethicin in methanol and in SDS. The relaxation data, spectral densities, and order parameters suggest that the peptide N-H vectors of alamethicin are not as highly constrained as the core regions of folded globular proteins. However, the peptide backbone is clearly not as mobile as the most unconstrained regions of folded proteins, such as those found in the frayed C-and N-termini of some proteins, or in randomcoil peptides. The data also suggest significant mobility at both ends of the peptide dissolved in methanol. In SDS the mobility in the middle and at the ends of the peptide is reduced. The implications of the results with respect to the sterically hindered Aib residues and the biological activities of the peptide are discussed.To whom correspondence should be addressed.     

Effects of temperature and field strength on the NMR relaxation times of 23Na in frog striated muscle

Pulsed NMR techniques have been applied to the study of the relaxation parameters characterizing ²³Na within frog striated muscle. Experiments were performed at 3°C, 22–24°C and 39°C at a Larmor frequency of 15.7 MHz; at 22–24°C, measurements were obtained both at 15.7 MHz and at 7.85 MHz.As previously reported, only a single spine-lattice relaxation time (T₁) was observed, but both slow (T₂)_I and fast (T₂)_II components of the spin-spin relaxation time were measured. The effect of temperature (θ) upon (1/T₁) was qualitatively similar to that reported for ²³Na in free solution; (θ) did not significantly affect (1/T₂) over the range of temperatures studied. (1/T₂)_I, and to a lesser degreee, (1/T₁) exhibited a modest inverse dependence of doubtful significance on the Larmor frequency.The data are examined within the framework of a simple specific model; a conservative values in assumed for the quadrupolar coupling constant characterizing immobilized intracellular Na⁺. Within this framework, the results suggest that the fraction of bound ions whose molecular tumbling is severely restricted does not exceed some few percent of the total sodium population.     

Quantifying Lipari–Szabo modelfree parameters from 13CO NMR relaxation experiments

It is proposed to obtain effective Lipari–Szabo order parameters and local correlation times for relaxation vectors of protein ¹³CO nuclei by carrying out a ¹³CO-R₁ auto relaxation experiment, a transverse CSA/dipolar cross correlation and a transverse ¹³CO CSA/¹³CO–¹⁵N CSA/dipolar cross correlation experiment. Given the global rotational correlation time from ¹⁵N relaxation experiments, a new program COMFORD (CO-Modelfree Fitting Of Relaxation Data) is presented to fit the ¹³CO data to an effective order parameter , an effective local correlation time and the orientation of the CSA tensor with respect to the molecular frame. It is shown that the effective is least sensitive to rotational fluctuations about an imaginary axis and most sensitive to rotational fluctuations about an imaginary axis parallel to the NH bond direction. As such, the information is fully complementary to the ¹⁵N relaxation order parameter, which is least sensitive to fluctuations about the NH axis and most sensitive to fluctuations about the axis. The new paradigm is applied on data of Ca²⁺ saturated Calmodulin, and on available literature data for Ubiquitin. Our data indicate that the order parameters rapport on slower, and sometimes different, motions than the ¹⁵N relaxation order parameters. The CO local correlation times correlate well with the calmodulin’s secondary structure. Electronic Supplementary Material Supplementary material is available to authorized users in the online version of this article at .     

High-frequency1H NMR studies of the effects of growth factors and phorbol esters on normal syrian hamster diploid fibroblast cells

The nuclear magnetic resonance (NMR) parameters, spin-lattice (T₁), and spin-spin (T₂) relaxation time, are usually longer for neoplastic cells than for normal cells of the same cell type. This has generally been true at low NMR frequencies (≤100 MHz) when comparisons have been made between normal and neoplastic cells that have both spent a short time in culture. We have previously demonstrated that although the T₁ values of paired normal and neoplastic Syrian hamster (SH) fibroblastic cells in culture are not significantly different when measured at 300 MHz, the 300 MHz T₂ values for the neoplastic cells are smaller than those of the normal cells. (Xin et al. (1986),Cell Biophysics 8, 213.) Since treatment of normal diploid cells with polypeptide growth factors or tumor promoters frequently results in reversible expression of neoplasia-associated phenotypes, T₁ and T₂ were obtained at 300 MHz for treated and untreated SH cells to see if these compounds could also produce smaller 300 MHz T₂ values. Secondary culture SH fetal fibroblast cells were treated with epidermal growth factor (EGF), fibroblast growth factor (FGF), phorbol-12,13-didecanoate (PDD) and 4-α-phorbol-12,13-didecanoate (4αPDD). Treatment with either growth factor resulted in smaller T₂ values, but a statistically significant decrease was not observed for PDD or 4αPDD. The observed reductions in T₂ values were correlated with the morphological and growth-stimulatory effects of these compounds on the cells.     

High resolution <Superscript>13</Superscript>C-detected solid-state NMR spectroscopy of a deuterated protein

High resolution ¹³C-detected solid-state NMR spectra of the deuterated beta-1 immunoglobulin binding domain of the protein G (GB1) have been collected to show that all ¹⁵N, ¹³C′, ¹³Cα and ¹³Cβ sites are resolved in ¹³C–¹³C and ¹⁵N–¹³C spectra, with significant improvement in T ₂ relaxation times and resolution at high magnetic field (750 MHz). The comparison of echo T ₂ values between deuterated and protonated GB1 at various spinning rates and under different decoupling schemes indicates that ¹³Cα T ₂′ times increase by almost a factor of two upon deuteration at all spinning rates and under moderate decoupling strength, and thus the deuteration enables application of scalar-based correlation experiments that are challenging from the standpoint of transverse relaxation, with moderate proton decoupling. Additionally, deuteration in large proteins is a useful strategy to selectively detect polar residues that are often important for protein function and protein–protein interactions.     

Study of protein dynamics in solution by measurement of 13Cα-13CO NOE and 13CO longitudinal relaxation

Summary ¹³C-¹³CO homonuclear NOE and ¹³CO T₁ relaxation were measured for a 20 kDa protein using tripleresonance pulse sequences. The experiments were sufficiently sensitive to obtain statistically significant differences in relaxation parameters over the molecule. The ¹³C-¹³CO cross-relaxation rate, obtained from these data, is directly proportional to an order parameter describing local motion and it is largely independent of the local correlation time. It is therefore a relatively straightforward observable for the identification of local dynamics.     

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.