Off-resonance rotating-frame amide proton spin relaxation experiments measuring microsecond chemical exchange in proteins

NMR spin relaxation in the rotating frame (R_1ρ) is a unique method for atomic-resolution characterization of conformational (chemical) exchange processes occurring on the microsecond time scale. Here, we use amide ¹H off-resonance R_1ρ relaxation experiments to determine exchange parameters for processes that are significantly faster than those that can be probed using ¹⁵N or ¹³C relaxation. The new pulse sequence is validated using the E140Q mutant of the C-terminal domain of calmodulin, which exhibits significant conformational exchange contributions to the transverse relaxation rates. The ¹H off-resonance R_1ρ data sample the entire relaxation dispersion profiles for the large majority of residues in this protein, which exchanges between conformations with a time constant of approximately 20 μs. This is in contrast to the case for ¹⁵N, where additional laboratory-frame relaxation data are required to determine the exchange parameters reliably. Experiments were performed on uniformly ¹⁵N-enriched samples that were either highly enriched in ²H or fully protonated. In the latter case, dipolar cross-relaxation with aliphatic protons were effectively decoupled to first order using a selective inversion pulse. Deuterated and protonated samples gave the same results, within experimental errors. The use of deuterated samples increases the sensitivity towards exchange contributions to the ¹H transverse relaxation rates, since dipolar relaxation is greatly reduced. The exchange correlation times determined from the present ¹H off-resonance R_1ρ experiments are in excellent agreement with those determined previously using a combination of ¹⁵N laboratory-frame and off-resonance R_1ρ relaxation data, with average values of and 21 ± 3 μs, respectively.     

<Superscript>15</Superscript>N transverse relaxation measurements for the characterization of µs–ms dynamics are deteriorated by the deuterium isotope effect on <Superscript>15</Superscript>N resulting from solvent exchange

¹⁵N R₂ relaxation measurements are key for the elucidation of the dynamics of both folded and intrinsically disordered proteins (IDPs). Here we show, on the example of the intrinsically disordered protein α-synuclein and the folded domain PDZ2, that at physiological pH and near physiological temperatures amide—water exchange can severely skew Hahn-echo based ¹⁵N R₂ relaxation measurements as well as low frequency data points in CPMG relaxation dispersion experiments. The nature thereof is the solvent exchange with deuterium in the sample buffer, which modulates the ¹⁵N chemical shift tensor via the deuterium isotope effect, adding to the apparent relaxation decay which leads to systematic errors in the relaxation data. This results in an artificial increase of the measured apparent ¹⁵N R₂ rate constants—which should not be mistaken with protein inherent chemical exchange contributions, R_ex, to ¹⁵N R₂. For measurements of ¹⁵N R₂ rate constants of IDPs and folded proteins at physiological temperatures and pH, we recommend therefore the use of a very low D₂O molar fraction in the sample buffer, as low as 1%, or the use of an external D₂O reference along with a modified ¹⁵N R₂ Hahn-echo based experiment. This combination allows for the measurement of R_ex contributions to ¹⁵N R₂ originating from conformational exchange in a time window from µs to ms.     

A TROSY CPMG sequence for characterizing chemical exchange in large proteins

A new NMR spin relaxation experiment is described for measuring chemical exchange time constants from approximately 0.5 ms to 5 ms in ¹⁵N-labeled macromolecules. The pulse sequence is based on the Carr–Purcell–Meiboom–Gill technique [Carr and Purcell (1954) Phys. Rev., 94, 630–638; Meiboom and Gill (1958) Rev. Sci. Instrum., 29, 688–691; Loria et al. (1999) J. Am. Chem. Soc., 121, 2331–2332], but implements TROSY selection [Pervushin et al. (1997) Proc. Natl. Acad. Sci. USA, 94, 12366–12371] to permit measurement of exchange linebroadening contributions to the narrower component of the ¹H-¹⁵N scalar-coupled doublet. This modification extends the size limitation imposed on relaxation measurements due to the fast decay of transverse magnetization in larger macromolecules. The new TROSY-CPMG experiment is demonstrated on a [U-98% ¹⁵ N] labeled sample of basic pancreatic trypsin inhibitor and a [U-83% ²H, U-98% ¹⁵ N] labeled sample of triosephosphate isomerase, a 54 kDa homodimeric protein.     

Characterization of the backbone dynamics of an anti-digoxin antibody VL domain by inverse detected 1H–15N NMR: Comparisons with X-ray data for the Fab

The dynamic behavior of the polypeptide backbone of a recombinant anti-digoxin antibody V_L domain has been characterized by measurements of ¹⁵N T₁ and T₂ relaxation times, ¹H–¹⁵N NOE values, and ¹H–²H exchange rates. These data were acquired with 2D inverse detected heteronuclear ¹H–¹⁵N NMR methods. The relaxation data are interpreted in terms of model free spectral density functions and exchange contributions to transverse relaxation rates R₂ (= 1/T₂). All characterized residues display low-amplitude picosecond timescale librational motions. Fifteen residues undergo conformational changes on the nanosecond timescale, and 24 residues have significant R₂ exchange contributions, which reflect motions on the microsecond to millisecond timescale. For several residues, microsecond to millisecond motions of nearby aromatic rings are postulated to account for some or all of their observed R₂ exchange contributions. The measured ¹H–²H exchange rates are correlated with hydrogen bonding patterns and distances from the solvent accessible surface. The degree of local flexibility indicated by the NMR measurements is compared to crystallographic B-factors derived from X-ray analyses of the native Fab and the Fab/digoxin complex. In general, both the NMR and X-ray data indicate enhanced flexibility in the turns, hypervariable loops, and portions of β-strands A, B, and G. However, on a residue-specific level, correlations among the various NMR data, and between the NMR and X-ray data, are often absent. This is attributed to the different dynamic processes and environments that influence the various observables. The combined data indicate that certain regions of the V_L domain, including the three hypervariable loops, undergo dynamic changes upon V_L:V_H association and/ or complexation with digoxin. Overall, the 26–10 V_L domain exhibits relatively low flexibility on the ps–ns timescale. The possible functional consequences of this result are considered. © 1993 Wiley-Liss, Inc.     

Evaluation of <Superscript>15</Superscript>N-detected H–N correlation experiments on increasingly large RNAs

Recently, ¹⁵N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a ¹⁵N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three ¹⁵N-detected H–N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established ¹⁵N-detected BEST-TROSY experiment, which proves to be the most sensitive ¹⁵N-detected H–N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the ¹⁵N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the ¹H- than in the ¹⁵N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that ¹⁵N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.     

Paramagnetic relaxation enhancement to improve sensitivity of fast NMR methods: application to intrinsically disordered proteins

We report enhanced sensitivity NMR measurements of intrinsically disordered proteins in the presence of paramagnetic relaxation enhancement (PRE) agents such as Ni²⁺-chelated DO2A. In proton-detected ¹H-¹⁵N SOFAST-HMQC and carbon-detected (H-flip)¹³CO-¹⁵N experiments, faster longitudinal relaxation enables the usage of even shorter interscan delays. This results in higher NMR signal intensities per units of experimental time, without adverse line broadening effects. At 40 mmol·L⁻¹ of the PRE agent, we obtain a 1.7- to 1.9-fold larger signal to noise (S/N) for the respective 2D NMR experiments. High solvent accessibility of intrinsically disordered protein (IDP) residues renders this class of proteins particularly amenable to the outlined approach.     

Pressure effect on the dynamics of an isolated α-helix studied by 15N-1H NMR relaxation

Dynamics and structure of (1–36)bacteriorhodopsin solubilized in chloroform/methanol mixture (1:1) were investigated by ¹H-¹⁵N NMR spectroscopy under a hydrostatic pressure of 2000 bar. It was shown that the peptide retains its spatial structure at high pressure. ¹⁵N transverse and longitudinal relaxation times, ¹⁵N{¹H} nuclear Overhauser effects, chemical shifts and the translation diffusion rate of the peptide at 2000 bar were compared with the respective data at ambient pressure [Orekhov et al. (1999) J. Biomol. NMR, 14, 345–356]. The model free analysis of the relaxation data for the helical 9–31 fragment revealed that the high pressure decreases the overall rotation and translation diffusion, as well as apparent order parameters of fast picosecond internal motions (S² _f) but has no effect on internal nanosecond motions (S² _s and _s) of the peptide. The decrease of translation and overall rotation diffusion was attributed to the increase in solvent viscosity and the decrease of apparent order parameters S² _f to a compression of hydrogen bonds. It is suggested that this compression causes an elongation of H-N bonds and a decrease of absolute values of chemical shift anisotropy (CSA). In particular, the observed decrease of S² _f at 2000 bar can be explained by 0.001 nm increase of N-H bond lengths and 10 ppm decrease of ¹⁵N CSA values.     

Fast evaluation of protein dynamics from deficient <Superscript>15</Superscript>N relaxation data

Simple and convenient method of protein dynamics evaluation from the insufficient experimental ¹⁵N relaxation data is presented basing on the ratios, products, and differences of longitudinal and transverse ¹⁵N relaxation rates obtained at a single magnetic field. Firstly, the proposed approach allows evaluating overall tumbling correlation time (nanosecond time scale). Next, local parameters of the model-free approach characterizing local mobility of backbone amide N–H vectors on two different time scales, S² and R _ex , can be elucidated. The generalized order parameter, S², describes motions on the time scale faster than the overall tumbling correlation time (pico- to nanoseconds), while the chemical exchange term, R _ex , identifies processes slower than the overall tumbling correlation time (micro- to milliseconds). Advantages and disadvantages of different methods of data handling are thoroughly discussed.     

Sensitivity enhancement in NMR of macromolecules by application of optimal control theory

NMR of macromolecules is limited by large transverse relaxation rates. In practice, this results in low efficiency of coherence transfer steps in multidimensional NMR experiments, leading to poor sensitivity and long acquisition times. The efficiency of coherence transfer can be maximized by design of relaxation optimized pulse sequences using tools from optimal control theory. In this paper, we demonstrate that this approach can be adopted for studies of large biological systems, such as the 800 kDa chaperone GroEL. For this system, the ¹H–¹⁵N coherence transfer module presented here yields an average sensitivity enhancement of 20–25% for cross-correlated relaxation induced polarization transfer (CRIPT) experiments.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s10858-005-3592-0     

Backbone dynamics of the human CC-chemokine eotaxin

Eotaxin is a CC chemokine with potent chemoattractant activity towards eosinophils. ¹⁵N NMR relaxation data have been used to characterize the backbone dynamics of recombinant human eotaxin. ¹⁵N longitudinal (R₁) and transverse (R₂) auto relaxation rates, heteronuclear ¹H-¹⁵N steady-state NOEs, and transverse cross-relaxation rates (_xy) were obtained at 30 °C for all resolved backbone secondary amide groups using ¹ H-detected two-dimensional NMR experiments. Ratios of transverse auto and cross relaxation rates were used to identify NH groups influenced by slow conformational rearrangement. Relaxation data were fit to the extended model free dynamics formalism, yielding parameters describing axially symmetric molecular rotational diffusion and the internal dynamics of each NH group. The molecular rotational correlation time (_m) is 5.09±0.02 ns, indicating that eotaxin exists predominantly as a monomer under the conditions of the NMR study. The ratio of diffusion rates about unique and perpendicular axes (D/D) is 0.81±0.02. Residues with large amplitudes of subnanosecond motion are clustered in the N-terminal region (residues 1–19), the C-terminus (residues 68–73) and the loop connecting the first two -strands (residues 30–37). N-terminal flexibility appears to be conserved throughout the chemokine family and may have implications for the mechanism of chemokine receptor activation. Residues exhibiting significant dynamics on the microsecond–millisecond time scale are located close to the two conserved disulfide bonds, suggesting that these motions may be coupled to disulfide bond isomerization.     

An empirical relationship between rotational correlation time and solvent accessible surface area

Structure–dynamics interrelationships are important in understanding protein function. We have explored the empirical relationship between rotational correlation times (τ_c and the solvent accessible surface areas (SASA) of 75 proteins with known structures. The theoretical correlation between SASA and τ_c through the equation SASA = K_rτ_c ^(2/3) is also considered. SASA was determined from the structure, τ_c ^calc was determined from diffusion tensor calculations, and τ_c ^expt was determined from NMR backbone¹³ C or ¹⁵N relaxation rate measurements. The theoretical and experimental values of τ_c correlate with SASA with regression analyses values of K_r as 1696 and 1896 m²s^-(2/3), respectively, and with corresponding correlation coefficients of 0.92 and 0.70.     

Fast hydrogen exchange affects 15N relaxation measurements in intrinsically disordered proteins

Unprotected amide protons can undergo fast hydrogen exchange (HX) with protons from the solvent. Generally, NMR experiments using the out-and-back coherence transfer with amide proton detection are affected by fast HX and result in reduced signal intensity. When one of these experiments, ¹H–¹⁵N HSQC, is used to measure the ¹⁵N transverse relaxation rate (R₂), the measured R₂ rate is convoluted with the HX rate (k_HX) and has higher apparent R₂ values. Since the ¹⁵N R₂ measurement is important for analyzing protein backbone dynamics, the HX effect on the R₂ measurement is investigated and described here by multi-exponential signal decay. We demonstrate these effects by performing ¹⁵N R ₂ ^CPMG experiments on α-synuclein, an intrinsically disordered protein, in which the amide protons are exposed to solvent. We show that the HX effect on R ₂ ^CPMG can be extracted by the derived equation. In conclusion, the HX effect may be pulse sequence specific and results from various sources including the J coupling evolution, the change of steady state water proton magnetization, and the D₂O content in the sample. To avoid the HX effect on the analysis of relaxation data of unprotected amides, it is suggested that NMR experimental conditions insensitive to the HX should be considered or that intrinsic R ₂ ^CPMG values be obtained by methods described herein.     

The use of TROSY for detection and suppression of conformational exchange NMR line broadening in biological macromolecules

The interference between conformational exchange-induced time-dependent variations of chemical shifts in a pair of scalar coupled ¹H and ¹⁵N spins is used to construct novel TROSY-type NMR experiments to suppress NMR signal loss in [¹⁵N,¹H]-correlation spectra of a 14-mer DNA duplex free in solution and complexed with the Antp homeodomain. An analysis of double- and zero-quantum relaxation rates of base ¹H–¹⁵N moieties showed that for certain residues the contribution of conformational exchange-induced transverse relaxation might represent a dominant relaxation mechanism, which, in turn, can be effectively suppressed by TROSY. The use of the new TROSY method for exchange-induced transverse relaxation optimization is illustrated with two new experiments, 2D ^h1 J _HN,^h2 J _NN-quantitative [¹⁵N,¹H]-TROSY to measure ^h1 J _HN and ^h2 J _NN scalar coupling constants across hydrogen bonds in nucleic acids, and 2D (^h2 J _NN+^h1 J _NH)-correlation-[¹⁵N,¹H]-TROSY to correlate ¹H^N chemical shifts of bases with the chemical shifts of the tertiary ¹⁵N spins across hydrogen bonds using the sum of the trans-hydrogen bond coupling constants in nucleic acids.     

Backbone dynamics of the Bacillus subtilis glucose permease IIA domain determined from 15N NMR relaxation measurements.

The backbone dynamics of the uniformly 15N-labeled IIA domain of the glucose permease of Bacillus subtilis have been characterized using inverse-detected two-dimensional 1H-15N NMR spectroscopy. Longitudinal (T1) and transverse (T2) 15N relaxation time constants and steady-state (1H)-15N NOEs were measured, at a spectrometer proton frequency of 500 MHz, for 137 (91%) of the 151 protonated backbone nitrogens. These data were analyzed by using a model-free dynamics formalism to determine the generalized order parameter (S2), the effective correlation time for internal motions (tau e), and 15N exchange broadening contributions (Rex) for each residue, as well as the overall molecular rotational correlation time (tau m). The T1 and T2 values for most residues were in the ranges 0.45-0.55 and 0.11-0.15 s, respectively; however, a small number of residues exhibited significantly slower relaxation. Similarly, (1H)-15N NOE values for most residues were in the range 0.72-0.80, but a few residues had much smaller positive NOEs and some exhibited negative NOEs. The molecular rotational correlation time was 6.24 +/- 0.01 ns; most residues had order parameters in the range 0.75-0.90 and tau e values of less than ca. 25 ps. Residues found to be more mobile than the average were concentrated in three areas: the N-terminal residues (1-13), which were observed to be highly disordered; the loop from P25 to D41, the apex of which is situated adjacent to the active site and may have a role in binding to other proteins; and the region from A146 to S149. All mobile residues occurred in regions close to termini, in loops, or in irregular secondary structure.     

NMR experiments for resonance assignments of 13C, 15N doubly-labeled flexible polypeptides: Application to the human prion protein hPrP(23–230)

A combination of three heteronuclear three-dimensional NMR experiments tailored for sequential resonance assignments in uniformly ¹⁵N, ¹³C-labeled flexible polypeptide chains is described. The 3D (H)N(CO-TOCSY)NH, 3D (H)CA(CO-TOCSY)NH and 3D (H)CBCA(CO-TOCSY)NH schemes make use of the favorable ¹⁵N chemical shift dispersion in unfolded polypeptides, exploit the slow transverse ¹⁵N relaxation rates of unfolded polypeptides in high resolution constant-time [¹H, ¹⁵N]-correlation experiments, and use carbonyl carbon homonuclear isotropic mixing to transfer magnetization sequentially along the amino acid sequence. Practical applications are demonstrated with the 100-residue flexible tail of the recombinant human prion protein, making use of spectral resolution up to 0.6 Hz in the ¹⁵N dimension, simultaneous correlation with the two adjacent amino acid residues to overcome problems associated with spectral overlap, and the potential of the presently described experiments to establish nearest-neighbor correlations across proline residues in the amino acid sequence.     

15N spin-lattice relaxation study of linear peptides: Preliminary results on Leu-enkephalin and Tyr-Gly-Gly-Phe

The ability of ¹⁵N relaxation measurements in conformational analysis of linear peptides was studied using Leu-enkephalin: Tyr-Gly-Gly-Phe-Leu and and related tetrapeptide Tyr-Gly-Gly-Phe 95 % ¹⁵N enriched. ¹⁵N spin-lattice relaxation times measured at different temperatures in Me₂SO solution indicate the presence of highly preferential folded structures in both peptides. A marked dependence of T₁ upon the motional effects (segmental rather than anisotropic overall) was observed, while hydrogen bonding affects weakly the relaxation times. From a comparison of ¹⁵N relaxation parameters it appears that the tetrapeptide exhibits a more rigid structure than Leu-enkephalin, in accordance with previous ¹H NMR studies. This paper provides evidence for the usefulness of ¹⁵N T₁ as a mobility probe (independent from ¹³C) in the investigation of the conformational dynamics of peptides.     

1H NMR Relaxometry in Natural Humous Soil Samples: Insights in Microbial Effects on Relaxation Time Distributions

¹H NMR relaxometry is applied for the investigation of pore size distributions in geological substrates. The transfer to humous soil samples requires the knowledge of the interplay between soil organic matter, microorganisms and proton relaxation. The goal of this contribution is to give first insights in microbial effects in the ¹H NMR relaxation time distribution in the course of hydration of humous soil samples. We observed the development of the transverse relaxation time distribution of the water protons after addition of water to air dried soil samples. Selected samples were treated with cellobiose to enhance microbial activity. Besides the relaxation time distribution, the respiratory activity and the total cell counts were determined as function of hydration time. Microbial respiratory activities were 2–15 times higher in the treated samples and total cell counts increased in all samples from 1×10⁹ to 5×10⁹ cells g⁻¹ during hydration. The results of ¹H NMR relaxometry showed tri-, bi- and mono-modal relaxation time distributions and shifts of peak relaxation times towards lower relaxation times of all investigated soil samples during hydration. Furthermore, we found lower relaxation times and merging of peaks in soil samples with higher microbial activity. Dissolution and hydration of cellobiose had no detectable effect on the relaxation time distributions during hydration. We attribute the observed shifts in relaxation time distributions to changes in pore size distribution and changes in spin relaxation mechanisms due to dissolution of organic and inorganic substances (e.g. Fe³⁺, Mn²⁺), swelling of soil organic matter (SOM), production and release of extracellular polymeric substances (EPS) and bacterial association within biofilms.     

A probe to monitor performance of 15N longitudinal relaxation experiments for proteins in solution

The magnitude of the ¹⁵N longitudinal relaxation rate typically decreases as magnetic field strength increases in globular proteins in solution. Thus, it is important to test the performance of ¹⁵N longitudinal relaxation experiments at high field strength. Herein, a tool to investigate systematic errors in ¹⁵N longitudinal relaxation rate, R₁, is introduced. The tool, a difference in R₁ values between the two components of the ¹H-coupled ¹⁵N magnetizations, R ₁ ⁽¹⁾ –R ₁ ⁽²⁾ , conveniently detects inefficiencies in cancellation of cross correlation between ¹H–¹⁵N dipolar coupling and ¹⁵N chemical shift anisotropy. Experiments, in varying conditions, and simulations of a two-spin system indicate that insufficient cancellation of the cross correlation is due to (1) ¹H pulse imperfection and (2) ¹H off-resonance effect, and (3) is further amplified by residual ¹⁵N transverse magnetization that is caused by the ¹⁵N off-resonance effect. Results also show that this problem can be easily and practically remedied by discarding the initial decay points when recording ¹⁵N longitudinal relaxation in proteins.     

A further investigation of the cytochrome<Emphasis Type="Italic"> b</Emphasis><Subscript>5</Subscript>–cytochrome<Emphasis Type="Italic"> c</Emphasis> complex

The interaction of reduced rabbit cytochrome b₅ with reduced yeast iso-1 cytochrome c has been studied through the analysis of ¹H–¹⁵N HSQC spectra, of ¹⁵N longitudinal (R₁) and transverse (R₂) relaxation rates, and of the solvent exchange rates of protein backbone amides. For the first time, the adduct has been investigated also from the cytochrome c side. The analysis of the NMR data was integrated with docking calculations. The result is that cytochrome b₅ has two negative patches capable of interacting with a single positive surface area of cytochrome c. At low protein concentrations and in equimolar mixture, two different 1:1 adducts are formed. At high concentration and/or with excess cytochrome c, a 2:1 adduct is formed. All the species are in fast exchange on the scale of differences in chemical shift. By comparison with literature data, it appears that the structure of one 1:1 adduct changes with the origin or primary sequence of cytochrome b₅.Electronic Supplementary Material Supplementary material is available for this article if you access the article at . A link in the frame on the left on that page takes you directly to the supplementary material.Abbreviations HSQC heteronuclear single quantum correlation spectroscopy - MD molecular dynamics     

Millisecond protein folding studied by NMR spectroscopy

Proteins are involved in virtually every biological process and in order to function, it is necessary for these polypeptide chains to fold into the unique, native conformation. This folding process can take place rapidly. NMR line shape analyses and transverse relaxation measurements allow protein folding studies on a microsecond-to-millisecond time scale. Together with an overview of current achievements within this field, we present millisecond protein folding studies by NMR of the cold shock protein CspB from Bacillus subtilis.