Explicit models of motions to analyze NMR relaxation data in proteins

Nuclear Magnetic Resonance (NMR) is a tool of choice to characterize molecular motions. In biological macromolecules, pico- to nano-second motions, in particular, can be probed by nuclear spin relaxation rates which depend on the time fluctuations of the orientations of spin interaction frames. For the past 40 years, relaxation rates have been successfully analyzed using the Model Free (MF) approach which makes no assumption on the nature of motions and reports on the effective amplitude and time-scale of the motions. However, obtaining a mechanistic picture of motions from this type of analysis is difficult at best, unless complemented with molecular dynamics (MD) simulations. In spite of their limited accuracy, such simulations can be used to obtain the information necessary to build explicit models of motions designed to analyze NMR relaxation data. Here, we present how to build such models, suited in particular to describe motions of methyl-bearing protein side-chains and compare them with the MF approach. We show on synthetic data that explicit models of motions are more robust in the presence of rotamer jumps which dominate the relaxation in methyl groups of protein side-chains. We expect this work to motivate the use of explicit models of motion to analyze MD and NMR data

Simple method for the generation of multiple homogeneous field volumes inside the bore of superconducting magnets

International audienceStandard Magnetic Resonance magnets produce a single homogeneous field volume, where the analysis is performed. Nonetheless, several modern applications could benefit from the generation of multiple homogeneous field volumes along the axis and inside the bore of the magnet. In this communication, we propose a straightforward method using a combination of ring structures of permanent magnets in order to cancel the gradient of the stray field in a series of distinct volumes. These concepts were demonstrated numerically on an experimentally measured magnetic field profile. We discuss advantages and limitations of our method and present the key steps required for an experimental validation

The spalling decay of building bioclastic limestones of Provence (South East of France): From clay minerals swelling to hydric dilation

International audienceMedieval historical monuments of the Provence region (South East of France) were erected with bioclastic limestones and display different sensitivities to spalling decay. The present study aims at understanding the physical processes at play as well as the internal properties governing its intensity. Limestones of contrasting sensitivity to spalling were compared to a reference type, unaffected by this decay, by means of petrography, petrophysic, mineralogy, and hydromechanics. The obtained results highlighted that the various sensitivities can be explained by the deformation recorded during water content variation (hydric dilation). A clay fraction was systematically detected within the mineralogical composition except for the reference material, and some swelling layers were identified in ontmorillonite/glauconite mixed layer minerals. A specific quantification procedure based on the combination of transmission electron microscopy coupled to an energy-dispersive X-ray spectrometer (TEM–EDX) and profile modeling of X-ray diffraction patterns was applied. A strong relationship between swelling layer content and hydric dilation of limestones was evidenced and corroborated the spalling sensitivity. Further interpretation of results showed that swelling layers localization within the texture significantly influence hydric dilation kinetics. Eventually, a mechanical softening was measured after water saturation. This behavior seems unrelated to the clay mineral content and its relative influence on spalling should be examined

Understanding the Methyl-TROSY effect over a wide range of magnetic fields

The use of relaxation interference in the methyl Transverse Relaxation-Optimized SpectroscopY (TROSY) experiment has opened new avenues for the study of large proteins and protein assemblies in nuclear magnetic resonance. So far, the theoretical description of the methyl-TROSY experiment has been limited to the slow-tumbling approximation, which is correct for large proteins on high field spectrometers. In a recent paper, favorable relaxation interference was observed in the methyl groups of a small protein at a magnetic field as low as 0.33 T, well outside the slow-tumbling regime. Here, we present a model to describe relaxation interference in methyl groups over a broad range of magnetic fields, not limited to the slow-tumbling regime. We predict that the type of multiple-quantum transitions that show favorable relaxation properties change with the magnetic field. Under the condition of fast methyl-group rotation, methyl-TROSY experiments can be recorded over the entire range of magnetic fields from a fraction of 1 T up to 100 T

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 T-1 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

Cross-correlated relaxation measurements under adiabatic sweeps: determination of local order in proteins

Adiabatically swept pulses were originally designed for the purpose of broadband spin inversion. Later, unexpected advantages of their utilization were also found in other applications, such as refocusing to excite spin echoes, studies of chemical exchange or fragment-based drug design. Here, we present new experiments to characterize fast (ps-ns) protein dynamics, which benefit from little-known properties of adiabatic pulses. We developed a strategy for measuring cross-correlated cross-relaxation (CCCR) rates during adiabatic pulses. This experiment provides a linear combination of longitudinal and transverse CCCR rates, which is offset-independent across a typical amide spectrum. The pulse sequence can be recast to provide accurate transverse CCCR rates weighted by the populations of exchanging states. Sensitivity can be improved in systems in slow exchange. Finally, the experiments can be easily modified to yield residue-specific correlation times. The average correlation time of motions can be determined with a single experiment while at least two different experiments had to be recorded until now

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

High-Resolution Two-Field Nuclear Magnetic Resonance Spectroscopy

International audienceNuclear Magnetic Resonance (NMR) is a ubiquitous branch of spectroscopy that can explore matter on the scale of the atom. Significant improvements in sensitivity and resolution have been driven by a steady increase of static magnetic field strengths. However, some properties of nuclei may be more favourable at low magnetic fields. For example, line-broadening due to chemical shift anisotropy increases sharply at higher magnetic fields. Here, we present a two-field NMR spectrometer that permits the application of rf-pulses and acquisition of NMR signals in two magnetic centres. Our prototype operates at 14.1 T and 0.33 T. The main features of this system are demonstrated by novel NMR experiments that correlate zero-quantum coherences at low magnetic field with single quantum coherences at high magnetic field, so that high resolution can be achieved in both dimensions, despite a ca. 10 ppm inhomogeneity of the low field centre. Two-field NMR spectroscopy offers the possibility to circumvent the limits of high magnetic fields, while benefiting from their exceptional sensitivity and resolution. This approach opens new avenues for NMR above 1 GHz