Philippe Pelupessy

High-Resolution NMR in Magnetic Fields with Unknown Spatiotemporal Variations

NMR Under Stormy Conditions Nuclear magnetic resonance spectroscopy is immensely useful for molecular characterization. However, the resolution necessary for making fine structural distinctions relies on sample isolation in a surrounding magnetic field that exhibits minimal variation in space and time over the course of the measurement; this requirement limits the range of materials that can be easily probed. Pelupessy et al. (p. 1693) present an approach that circumvents the need for spatiotemporal field homogeneity by detecting coherence transfer among coupled spins. Whereas prior methods have compensated for heterogeneity by applying corrective magnetic fields, this technique relies on the inherent insensitivity of the measured coherences to changes in the local magnetic environment and thus can be applied without prior knowledge of the field variation profile. A coherence transfer method overcomes disruptions to nuclear magnetic resonance spectra by magnetic field fluctuations. Nuclear magnetic resonance (NMR) experiments are usually carried out in homogeneous magnetic fields. In many cases, however, high-resolution spectra are virtually impossible to obtain because of the inherent heterogeneity of the samples or living organisms under investigation, as well as the poor homogeneity of the magnets (particularly when bulky samples must be placed outside their bores). Unstable power supplies and vibrations arising from cooling can lead to field fluctuations in time as well as space. We show how high-resolution NMR spectra can be obtained in inhomogeneous fields with unknown spatiotemporal variations. Our method, based on coherence transfer between spins, can accommodate spatial inhomogeneities of at least 11 gauss per centimeter and temporal fluctuations slower than 2 hertz.

Correlated motions of successive amide N-H bonds in proteins

New nuclear magnetic resonance (NMR) methods are described for the measurement of cross-correlation rates of zero- and double-quantum coherences involving two nitrogen nuclei belonging to successive amino acids in proteins and peptides. Rates due to the concerted fluctuations of two NHN dipole-dipole interactions and to the correlated modulations of two nitrogen chemical shift anisotropies have been obtained in a sample of doubly labeled Ubiquitin. Ambiguities in the determination of dihedral angles can be lifted by comparison of different rates. By defining a heuristic order parameter, experimental rates can be compared with those expected for a rigid molecule. The cross-correlation order parameter that can be derived from a model-free approach can be separated into structural and dynamic contributions.

Selective cross-polarization in solution state NMR

In two influential paper, Ernst and coworkers argued that cross-polarization, originally proposed by Hartmann and Hahn, can be very useful in isotropic liqs. to transfer coherence between scalar-coupled nuclei such as protons and carbon-13 (Maudsley, A. A., Muller, L., and Ernst, R. R., 1977; J. Magn. Reson., 28, 463; Muller, L., and Ernst, R. R., 1979, Molec. Phys., 38, 963). Unfortunately, the efficiency of cross-polarization in liqs. tends to be strongly attenuated if the radiofrequency fields are not perfectly homogeneous. In this paper, it is demonstrated by expts. and simulations that imperfections in RF fields have little effect on the efficiency of magnetization transfer, provided that the RF amplitudes are comparable with the magnitudes of the heteronuclear scalar coupling consts. A comparison between selective cross-polarization and selective INEPT shows clearly that cross-polarization is more efficient. Selective cross-polarization does not require any careful calibration and is insensitive to exptl. instabilities. [on SciFinder (R)]

Nanosecond Time Scale Motions in Proteins Revealed by High-Resolution NMR Relaxometry

Understanding the molecular determinants underlying protein function requires the characterization of both structure and dynamics at atomic resolution. Nuclear relaxation rates allow a precise characterization of protein dynamics at the Larmor frequencies of spins. This usually limits the sampling of motions to a narrow range of frequencies corresponding to high magnetic fields. At lower fields one cannot achieve sufficient sensitivity and resolution in NMR. Here, we use a fast shuttle device where the polarization builds up and the signals are detected at high field, while longitudinal relaxation takes place at low fields 0.5 < B0 < 14.1 T. The sample is propelled over a distance up to 50 cm by a blowgun-like system in about 50 ms. The analysis of nitrogen-15 relaxation in the protein ubiquitin over such a wide range of magnetic fields offers unprecedented insights into molecular dynamics. Some key regions of the protein feature structural fluctuations on nanosecond time scales, which have so far been overlooked in high-field relaxation studies. Nanosecond motions in proteins may have been underestimated by traditional high-field approaches, and slower supra-τc motions that have no effect on relaxation may have been overestimated. High-resolution relaxometry thus opens the way to a quantitative characterization of nanosecond motions in proteins.

Symmetrical reconversion: measuring cross-correlation rates with enhanced accuracy.

A new strategy has been developed to measure cross-correlation rates with much enhanced accuracy. The method relies on the use of four complementary experiments. Errors due to pulse miscalibration and to uncontrolled attenuation factors associated with relaxation are cancelled out. Problems due to violations of the secular approximation are greatly alleviated. The method has been applied to the measurement of N/NH (CSA/DD) cross-correlated relaxation rates in human ubiquitin.

Frequency-modulated cross-polarization for fast magic angle spinning NMR at high fields: relaxing the Hartmann-Hahn condition

Two novel schemes are described for cross polarization in NMR that make it possible to achieve an efficient transfer of polarization from abundant I spins to dil. S spins under high-speed magic angle spinning spectroscopy, which is useful for systems with large chem. shift anisotropies at high fields. The frequency of one of the RF carriers is modulated sinusoidally during spin-locking while the RF amplitude is kept const. It is shown by expt. that frequency modulation greatly attenuates the crit. character of the Hartmann-Hahn matching condition, and allows one to obtain uniform excitation over a wide range of offsets. Signal intensities of 13C resonances in alanine were obtained as a function of mismatch at spinning speeds of 12 and 15 kHz and a static field strength of 14 T (600 MHz for 1H NMR) to illustrate the advantages of these schemes. [on SciFinder (R)]

Efficient determination of angles subtended by C(alpha)-H(alpha) and N-H(N) vectors in proteins via dipole-dipole cross-correlation

The angle ΘCαHα,NHN subtended by the internuclear vectors 13Cα-Hα and 15N-HN in doubly-labeled proteins can be determined by observing the effect of cross-correlation between the dipolar interactions on zero- and double-quantum coherences involving 13Cα and 15N. Two complementary 2D experiments with the appearance of 15N-HN correlation spectra yield signal intensities that depend on the rate of interconversion through cross-correlated relaxation of in-phase and doubly antiphase zero- and double-quantum coherences. The ratio of the signal intensities in the two experiments bears a simple relationship to the cross-correlation rate, and hence to the angle ΘCαHα,NHN. Assuming planarity of the peptide bond, the dihedral angle Ψ (between Cα and C′) can be determined from the knowledge of ΘCαHα,NHN. The experiments are very time-effective and provide good sensitivity and excellent spectral resolution.

Exchange Rate Constants of Invisible Protons in Proteins Determined by NMR Spectroscopy

Although labile protons that are exchanging rapidly with those of the solvent cannot be observed directly, their exchange rate constants can be determined by indirect detection of scalar‐coupled neighboring nuclei. We have used heteronuclear NMR spectroscopy to measure the exchange rate constants of labile protons in the side chains of lysine and arginine residues in ubiquitin enriched in carbon‐13 and nitrogen‐15 at neutral pH. Exchange rate constants as fast as 40×103 s−1 were thus measured. These results demonstrate that NMR spectroscopy is a powerful tool for the characterization of lysine NH3+ and arginine NH groups in proteins at physiologically relevant pH values.

Speeding up nuclear magnetic resonance spectroscopy by the use of SMAll Recovery Times - SMART NMR

A drastic reduction of the time required for two-dimensional NMR experiments can be achieved by reducing or skipping the recovery delay between successive experiments. Novel SMAll Recovery Times (SMART) methods use orthogonal pulsed field gradients in three spatial directions to select the desired pathways and suppress interference effects. Two-dimensional spectra of dilute amino acids with concentrations as low as 2 mM can be recorded in about 0.1 s per increment in the indirect domain.

Accurate Measurement of Longitudinal Cross-Relaxation Rates in Nuclear Magnetic Resonance

The accuracy of the determination of longitudinal cross-relaxation rates in NMR can be improved by combining symmetrical reconversion with suitable operator swapping methods that lead to the averaging of differences in autorelaxation rates and eliminate the effects of cross relaxation with the environment. The principles are first discussed for an isolated two-spin system comprising a pair of 15N and 1HN nuclei subjected to chemical shift anisotropy and dipole-dipole relaxation, and then extended to include further protons. The gains in accuracy are demonstrated experimentally for the protein ubiquitin.