Fabien Ferrage

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.

Multiple-Timescale Dynamics of Side-Chain Carboxyl and Carbonyl Groups in Proteins by C-13 Nuclear Spin Relaxation

Side-chain carboxyl and carbonyl groups play a major role in protein interactions and enzyme catalysis. A series of (13)C relaxation experiments is introduced to study the dynamics of carboxyl and carbonyl groups in protein side chains on both fast (sub-ns) and slower (micros-ms) time scales. This approach is illustrated on the protein calbindin D(9k). Fast dynamics features correlate with hydrogen- and ion-binding patterns. We also identify chemical dynamics on micros time scales in solvent-exposed carboxyl groups, most probably due to exchange between the carboxylate and carboxylic acid forms.

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.

Time Scales of Slow Motions in Ubiquitin Explored by Heteronuclear Double Resonance

Understanding how proteins function at the atomic level relies in part on a detailed characterization of their dynamics. Ubiquitin, a small single-domain protein, displays rich dynamic properties over a wide range of time scales. In particular, several regions of ubiquitin show the signature of chemical exchange, including the hydrophobic patch and the β4-α2 loop, which are both involved in many interactions. Here, we use multiple-quantum relaxation techniques to identify the extent of chemical exchange in ubiquitin. We employ our recently developed heteronuclear double resonance method to determine the time scales of motions that give rise to chemical exchange. Dispersion profiles are obtained for the backbone NH(N) pairs of several residues in the hydrophobic patch and the β4-α2 loop, as well as the C-terminus of helix α1. We show that a single time scale (ca. 50 μs) can be used to fit the data for most residues. Potential mechanisms for the propagation of motions and the possible extent of correlation of these motions are discussed.

Distribution of Pico- and Nanosecond Motions in Disordered Proteins from Nuclear Spin Relaxation

Intrinsically disordered proteins and intrinsically disordered regions (IDRs) are ubiquitous in the eukaryotic proteome. The description and understanding of their conformational properties require the development of new experimental, computational, and theoretical approaches. Here, we use nuclear spin relaxation to investigate the distribution of timescales of motions in an IDR from picoseconds to nanoseconds. Nitrogen-15 relaxation rates have been measured at five magnetic fields, ranging from 9.4 to 23.5 T (400–1000 MHz for protons). This exceptional wealth of data allowed us to map the spectral density function for the motions of backbone NH pairs in the partially disordered transcription factor Engrailed at 11 different frequencies. We introduce an approach called interpretation of motions by a projection onto an array of correlation times (IMPACT), which focuses on an array of six correlation times with intervals that are equidistant on a logarithmic scale between 21 ps and 21 ns. The distribution of motions in Engrailed varies smoothly along the protein sequence and is multimodal for most residues, with a prevalence of motions around 1 ns in the IDR. We show that IMPACT often provides better quantitative agreement with experimental data than conventional model-free or extended model-free analyses with two or three correlation times. We introduce a graphical representation that offers a convenient platform for a qualitative discussion of dynamics. Even when relaxation data are only acquired at three magnetic fields that are readily accessible, the IMPACT analysis gives a satisfactory characterization of spectral density functions, thus opening the way to a broad use of this approach.

Nuclear spin relaxation in isotropic and anisotropic media.

2010 Elsevier B.V. All rights reserved.

Single or triple gradients

Pulsed Field Gradients (PFGs) have become ubiquitous tools not only for Magnetic Resonance Imaging (MRI), but also for NMR experiments designed to study translational diffusion, for spatial encoding in ultra-fast spectroscopy, for the selection of desirable coherence transfer pathways, for the suppression of solvent signals, and for the elimination of zero-quantum coherences. Some of these experiments can only be carried out if three orthogonal gradients are available, while others can also be implemented using a single gradient, albeit at some expense of performance. This paper discusses some of the advantages of triple- with respect to single-gradient probes. By way of examples we discuss (i) the measurement of small diffusion coefficients making use of the long spin-lattice relaxation times of nuclei with low gyromagnetic ratios gamma such as nitrogen-15, and (ii) the elimination of zero-quantum coherences in Exchange or Nuclear Overhauser Spectroscopy (EXSY or NOESY) experiments, as well as in methods relying on long-lived (singlet) states to study very slow exchange or diffusion processes.

Broadband Dipolar Recoupling for Magnetization Transfer in Solid-State NMR Correlation Spectroscopy

Recent developments in solid-state nuclear magnetic resonance have opened the way to detailed structural and dynamic analysis of crystalline and non-crystalline biological solids. In isotopically enriched molecules of biological interest, C–C magnetization transfer via spin-exchange processes (also called spin diffusion) allows one both to assign the resonances and to determine internuclear distances. Chemical-shift correlation experiments commonly employed for such purposes exploit different means of compensating for the energy imbalances of C spins with different resonance frequencies and use a variety of recoupling schemes which inhibit the quenching of homoand heteronuclear dipolar interactions by magic angle spinning (MAS). Spin exchange can be promoted by any of three methods: 1) Proton-driven spin diffusion (PDSD) which does not require any radio-frequency (RF) irradiation and relies on line broadening due to carbon–proton dipolar couplings, so that its efficiency strongly depends on the spinning frequency and on local motions. 2) Rotor-driven spin exchange which occurs in rotational resonance (R) methods, and does not use any RF irradiation either. It strongly favors flip-flop processes between pairs of C spins which have a difference in isotropic chemical shifts that matches an integer multiple of the spinning frequency. 3) RF-driven spin exchange which relies on homoand heteronuclear recoupling of the dipolar interactions. A wide range of recoupling sequences has been developed, including a number of broadband homonuclear recoupling schemes. For optimal performance, especially at high MAS frequencies, most known recoupling methods require high RF power which can lead to excessive sample heating at longer transfer times. Some dipolar recoupling methods employ reduced RF power on C or on H, like dipolar assisted rotational resonance (DARR) or RF-assisted diffusion (RAD) experiments with RF amplitudes adjusted to the spinning frequency nr . This enhances C–C spin exchange through recoupling of homoand heteronuclear dipolar interactions. In fact, rotary resonance recoupling (R) using various ratios n= n1H /nr=1/2, 1 and 2 leads to the restoration of different anisotropic spin interactions. However, the efficiency of rotary resonance recoupling, including DARR/RAD-like experiments, is critically dependent on resonance offsets and on the homogeneity of the RF fields, and may lead to non-uniform C–C spin exchange between various sites. Herein, we introduce a broadband variant of rotary resonance recoupling (BR) aimed at promoting improved C–C transfer. In Figure 1, it is shown that the RF irradiation is ap-

Methods to determine slow diffusion coefficients of biomolecules. Applications to Engrailed 2, a partially disordered protein

We present new NMR methods to measure slow translational diffusion coefficients of biomolecules. Like the heteronuclear stimulated echo experiment (XSTE), these new methods rely on the storage of information about spatial localization during the diffusion delay as longitudinal polarization of nuclei with long T1 such as nitrogen-15. The new BEST-XSTE sequence combines features of Band-selective Excitation Short-Transient (BEST) and XSTE methods. By avoiding the saturation of all protons except those of amide groups, one can increase the sensitivity by 45% in small proteins. The new experiment which combines band-Selective Optimized Flip-Angle Short-Transient with XSTE (SOFAST-XSTE) offers an alternative when very short recovery delays are desired. A modification of the HSQC-edited version of the XSTE experiment offers enhanced sensitivity and access to higher resolution in the indirect dimension. These new methods have been applied to detect changes in diffusion coefficients due to dimerization or proteolysis of Engrailed 2, a partially disordered protein.

Preservation of heteronuclear multiple-quantum coherences in NMR by double-resonance irradiation

A heteronuclear double-resonance (HDR) method based on MLEV-32 or WALTZ-32 pulse sequences has been designed for the investigation of relaxation of heteronuclear multiple-quantum (MQ) coherences. The theoretical analysis of this technique uses average Hamiltonian theory (AHT) to treat the effects of coherent evolution associated with scalar couplings, offsets, and inhomogeneous radiofrequency (rf) fields during the pulse sequence. Under most conditions, the dynamics of the MQ coherences during the HDR sequence is not affected by rf inhomogeneities and scalar couplings for offsets as large as the nutation frequency. The predictions drawn from AHT are supported by numerical simulations and experiments.