CPMG Experiments Research Articles

NMR measurement of the off rate from the first calcium-binding site of the synaptotagmin I C 2A domain

The off rate from the first calcium-binding site of the C 2A domain of synaptotagmin I, a putative calcium receptor in neurotransmitter release, has been determined by 15N-nuclear magnetic resonance relaxation dispersion measurements. The exchange rate was obtained by fitting the dependence of the transverse relaxation rates on the interval between 180° pulses in relaxation-compensated CPMG experiments at 3.2 μM calcium concentration. The measured k ex is 2.0×10 3 s −1. The calcium on rate of 3.5±1×10 7 s −1, determined from the measured off rate and the dissociation constant (5.3×10 −5 M), is close to the diffusion limit. These results are consistent with the proposed role of synaptotagmin I as a calcium sensor in release, but suggest that additional factors may help to accelerate the diffusion of Ca 2+ to the sensor.

Slow dynamics in folded and unfolded states of an SH3 domain.

(15)N relaxation dispersion experiments were applied to the isolated N-terminal SH3 domain of the Drosophila protein drk (drkN SH3) to study microsecond to second time scale exchange processes. The drkN SH3 domain exists in equilibrium between folded (F(exch)) and unfolded (U(exch)) states under nondenaturing conditions in a ratio of 2:1 at 20 degrees C, with an average exchange rate constant, k(ex), of 2.2 s(-1) (slow exchange on the NMR chemical shift time scale). Consequently a discrete set of resonances is observed for each state in NMR spectra. Within the U(exch) ensemble there is a contiguous stretch of residues undergoing conformational exchange on a micros/ms time scale, likely due to local, non-native hydrophobic collapse. For these residues both the F(exch) <--> U(exch) conformational exchange process and the micros/ms exchange event within the U(exch) state contribute to the (15)N line width and can be analyzed using CPMG-based (15)N relaxation dispersion measurements. The contribution of both processes to the apparent relaxation rate can be deconvoluted numerically by combining the experimental (15)N relaxation dispersion data with results from an (15)N longitudinal relaxation experiment that accurately quantifies exchange rates in slow exchanging systems (Farrow, N. A.; Zhang, O.; Forman-Kay, J. D.; Kay, L. E. J. Biomol. NMR 1994, 4, 727-734). A simple, generally applicable analytical expression for the dependence of the effective transverse relaxation rate constant on the pulse spacing in CPMG experiments has been derived for a two-state exchange process in the slow exchange limit, which can be used to fit the experimental data on the global folding/unfolding transition. The results illustrate that relaxation dispersion experiments provide an extremely sensitive tool to probe conformational exchange processes in unfolded states and to obtain information on the free energy landscape of such systems.

Off-resonance effects in 15N T2 CPMG measurements.

The systematic difference between T2 values obtained from CPMG and T1p experiments was observed for backbone 15N nuclei of bacterial ribonuclease barnase. Theoretical consideration suggests that the observed difference is caused by off-resonance effects of 180 degree pulses of the CPMG pulse train. Namely, at off-resonance conditions T1-dependent secondary echo coherence pathways considerably contribute to the signal decay in the CPMG experiment and result in systematic (up to 10%) offset-dependent overestimation of 15N T2 measured by the CPMG technique. Under certain circumstances off-resonance effects result in dependence of 15N T2 on CPMG frequency, which might be erroneously interpreted as conformational exchange on the millisecond time-scale. A procedure for numerical correction of 15N T2 (CPMG) data is proposed.

Estimating the time scale of chemical exchange of proteins from measurements of transverse relaxation rates in solution.

Chemical (conformational) exchange on the ms--microsecond time scale is reliably identified by the observation of transverse relaxation rates, Rex, that depend upon the strength of the effective field (omega 1eff = gamma B1eff) used in spin lock or CPMG experiments. In order to determine if the exchange correlation time, tau ex, is the fast or slow limit, measurements of (i) signal line shape and (ii) temperature dependence of Rex have been commonly used in studies of stable, small molecules. However, these approaches are often not applicable to proteins, because sample stability and solubility, respectively, limit the temperature range and signal sensitivity of experiments. Herein we use a complex, but general, two-site exchange equation to show when the simple fast exchange equations for Rex are good approximations, in the case of proteins. We then present a simple empirical equation that approximately predicts Rex in all exchange regimes, and explains these results in a clear, straightforward manner. Finally we show how one can reliably determine whether tau ex is in the fast or slow exchange limit.

The dependence of chemical exchange on boundary selection in a fibronectin type III domain from human tenascin

The third fibronectin type III domain from human tenascin adopts a compact β-sandwich fold. Its boundaries were originally selected to encode a 90-residue domain (TNfn3 1-90). We conclude that the dynamic properties of TNfn3 are more accurately represented when the C terminus is extended by the two naturally succeeding residues. Longitudinal ( R 1) and transverse ( R 2) 15N relaxation rates, and { 1H- 15N} NOE enhancements at pH 4.9 and 300 K are presented for TNfn3 1-90 and TNfn3 1-92, the extended form, at two field strengths (11.74 and 14.10 T). Nearly identical results confirm their similar motional properties over a broad range of timescales. However, a number of residues near the C terminus in TNfn3 1-90 exhibit elevated transverse relaxation rates and broadened signals in 1H- 15N HSQC spectra. Explicit rates of chemical exchange for five residues in TNfn3 1-90 were determined by measuring transverse relaxation rates in a series of CPMG experiments with spin-echo refocusing delays increasing from 311 to 1436 μs. Calculated exchange rates average 1000(±311) s −1, with individual uncertainties near 20%. Homonuclear TOCSY experiments collected between pH 4 and 7 reveal the coincident titration of two acidic clusters in TNfn3 1-90 at pH 5.64(±0.47). The repulsive electrostatic interaction of the C-terminal carboxylate with one of these clusters may promote chemical exchange in the shorter domain. Additionally, NOE and chemical shift data suggest hydrogen bond formation between the added residues and adjacent loops. The data affirm the importance of judiciously selecting domain boundaries prior to the characterization of molecular properties.

Dynamics of ribonuclease H: temperature dependence of motions on multiple time scales.

The temperature dependence of the backbone motions in Escherichia coli ribonuclease HI was studied on multiple time scales by 15N nuclear magnetic spin relaxation. Laboratory frame relaxation data at 285, 300, and 310 K were analyzed using the model-free and reduced spectral density approaches. The temperature dependence of the order parameters was used to define a characteristic temperature for the motions of the backbone N-H bond vectors on picosecond to nanosecond time scales. The characteristic temperatures for secondary structure elements, loops, and the C-terminus are approximately 1000, approximately 300, and approximately 170 K, respectively. The observed variation in the characteristic temperature indicates that the energy landscape, and thus the configurational heat capacity, is markedly structure dependent in the folded protein. The effective correlation times for internal motions do not show significant temperature dependence. Conformational exchange was observed for a large number of residues forming a contiguous region of the protein that includes the coiled coil formed by helices alpha A and alpha D. Exchange broadening in the CPMG experiments decreased with increased temperature, directly demonstrating that the microscopic exchange rate is faster than the pulse repetition rate of 1.2 ms. The temperature dependence of the exchange contributions to the transverse relaxation rate constant shows approximately Arrhenius behavior over the studied temperature range with apparent activation enthalpies of approximately 20-50 kJ/mol. Numerical calculations suggest that these values underestimate the activation barriers by at most a factor of 2. The present results obtained at 300 K are compared to those reported previously [Mandel, A. M., Akke, M., & Palmer, A. G., III (1995) J. Mol. Biol. 246, 144-163] to establish the reproducibility of the experimental techniques.

Simultaneous Determination of Spin-Lattice and Spin-Spin Relaxation Times in NMR: A Robust and Facile Method for Measuring T2. Optimization and Data Analysis of the Offset-Saturation Experiment

The offset-saturation experiment is shown to be a robust method for the simultaneous measurement of T1 and T2 NMR spin relaxation times. Application of the method to the measurement of T1 and T2 in a sample of distilled water shows that the relaxation times determined by the offset-saturation method are in good agreement with those obtained by conventional inversion-recovery and CPMG experiments for T1 and T2, respectively. Errors in the relaxation times from the nonlinear-least-squares fit to the experimental data are on the order of 5%, provided that the irradiating field γB2 is well calibrated and the sampling points are judiciously chosen. These errors are determined from the intersection of the appropriate parameter axis by the 95% confidence interval tangent. In the limit of small decoupler flip angles, the gated homodecouplers of high-resolution NMR spectrometers can be used as the RF irradiation source. The calibration of γB2 can be performed simply and precisely using the single-spin double-resonance experiment, which is shown to be a generalization of the Bloch-Siegert shift effect. A simple qualitative guideline for optimization of the choice of irradiation resonance offsets and preacquisition delay times is derived from the partial-derivative criteria outlined here.

Diffusion-weighted spatial information from1H relaxation in restricted geometries

NMR relaxation of water1H confined in restricted geometries, whatever is the nature of the system (porous media saturated by water as well as biological tissues), exhibits common characteristics. Artificial microporous media saturated by water have been chosen as model systems to study the longitudinal and transverse relaxation of1H magnetization of water molecules diffusing in restricted geometries. These systems are very stable, easy to prepare, with well-characterized pore size distribution and connections, and with highly homogeneous surface properties. The response was compared with that from more complex natural porous media. Scanning Electron Microscopy techniques demonstrated spatial characteristics and surface properties of the samples. The information content of longitudinal relaxation curves associated with spatial structure and due to restricted diffusion is shown in these samples. The effect on transverse relaxation of self-diffusion in the presence of spatially varying magnetic fields due to susceptibility differences is shown. A simple linear relationship has been found in all samples between the transverse relaxation rate and the interpulse delay in CPMG experiments, in spite of the variety of pore shapes and sizes. In general, one can say that relaxation curves beardiffusion-weighted information on the pore space framework. The role of the investigated relaxation mechanisms is important also in the response of biological tissues, including in the presence of MR Imaging contrast agents inducing microscopic magnetic-field gradients.