Similarities and Differences among Protein Dynamics Studied by Variable Temperature Nuclear Magnetic Resonance Relaxation

Understanding and describing the dynamics of proteins is one of the major challenges in biology. Here, we use multifield variable-temperature NMR longitudinal relaxation (R-1) measurements to determine the hierarchical activation energies of motions of four different proteins: two small globular proteins (GB1 and the SH3 domain of alpha-spectrin), an intrinsically disordered protein (the C-terminus of the nucleoprotein of the Sendai virus, Sendai Ntail), and an outer membrane protein (OmpG). The activation energies map the motions occurring in the side chains, in the backbone, and in the hydration shells of the proteins. We were able to identify similarities and differences in the average motions of the proteins. We find that the NMR relaxation properties of the four proteins do share similar features. The data characterizing average backbone motions are found to be very similar, the same for methyl group rotations, and similar activation energies are measured. The main observed difference occurs for the intrinsically disordered Sendai Ntail, where we observe much lower energy of activation for motions of protons associated with the protein-solvent interface as compared to the others. We also observe variability between the proteins regarding side chain N-15 relaxation of lysine residues, with a higher activation energy observed in OmpG. This hints at strong interactions with negatively charged lipids in the bilayer and provides a possible mechanistic clue for the "positive-inside" rule for helical membrane proteins. Overall, these observations refine the understanding of the similarities and differences between hierarchical dynamics in proteins

Solid-state NMR at natural isotopic abundance for the determination of conformational polymorphism - the case of designed beta-turn peptides containing di-prolines

The proton double quantum-carbon single quantum correlation experiment has been applied to designed peptides in the solid state in natural isotopic abundance. Analogous to nOe studies in solution, through-space double-quantum connectivities have been exploited to obtain the cis-trans conformational polymorphism of diproline residues occurring at beta-turns in the peptides

Proton-detected 3D N-15/H-1/H-1 isotropic/anisotropic/isotropic chemical shift correlation solid-state NMR at 70 kHz MAS

Chemical shift anisotropy (CSA) tensors offer a wealth of information for structural and dynamics studies of a variety of chemical and biological systems. In particular, CSA of amide protons can provide piercing insights into hydrogen-bonding interactions that vary with the backbone conformation of a protein and dynamics. However, the narrow span of amide proton resonances makes it very difficult to measure H-1 CSAs of proteins even by using the recently proposed 2D H-1/H-1 anisotropic/isotropic chemical shift (CSA/CS) correlation technique. Such difficulties due to overlapping proton resonances can in general be overcome by utilizing the broad span of isotropic chemical shifts of low-gamma nuclei like N-15. In this context, we demonstrate a proton-detected 3D N-15/H-1/H-1 CS/CSA/CS correlation experiment at fast MAS frequency (70 kHz) to measure 1H CSA values of unresolved amide protons of N-acetyl-N-15-L-valyl-N-15-L-leucine (NAVL). (C) 2016 Elsevier Inc. All rights reserved

Use of reverse cross-polarization for editing solid state proton NMR spectra

We present here the use of the cross-polarization- reverse cross-polarization (CP-RCP) method for editing proton spectra by identifying protons attached exclusively to a nucleus such as carbon or nitrogen. Experiments have been done at moderate spinning speeds utilizing the Combined Rotation and Multi-Pulse Scheme (CRAMPS) technique which provided resolution of the spectral lines that is sufficient for several small molecular systems. This approach, in addition to being an editing tool, helps to increase resolution further and also leads to identifying protons such as the OH and the SH protons. Here we present results of the application of the CP-RCP scheme to systems at natural abundance of the nuclei C-13 and N-15. The utility of the method has been illustrated for the case several amino acids, a tri-peptide and a synthesized cocrystal. (C) 2018 Elsevier B.V. All rights reserved

Solvent suppression in DNP enhanced solid state NMR

We show how DNP enhanced solid-state NMR spectra can be dramatically simplified by suppression of solvent signals. This is achieved by (i) exploiting the paramagnetic relaxation enhancement of solvent signals relative to materials substrates, or (ii) by using short cross-polarization contact times to transfer hyperpolarization to only directly bonded carbon-13 nuclei in frozen solutions. The methods are evaluated for organic microcrystals, surfaces and frozen solutions. We show how this allows for the acquisition of high-resolution DNP enhanced proton-proton correlation experiments to measure inter-nuclear proximities in an organic solid.This article is published as Yarava, Jayasubba Reddy, Sachin Rama Chaudhari, Aaron J. Rossini, Anne Lesage, and Lyndon Emsley. "Solvent suppression in DNP enhanced solid state NMR." Journal of Magnetic Resonance 277 (2017): 149-153. doi: 10.1016/j.jmr.2017.02.016. Posted with permission.</p

Solvent suppression in DNP enhanced solid state NMR

We thank Prof. P. Tordo, Dr. O. Ouari and Dr. G. Casano (Aix-Marseille Universite) for supplying TEKPol and AMUPol, and Prof. Christophe Coperet (ETH Zurich) for supplying the silica based materials.International audienceWe show how DNP enhanced solid-state NMR spectra can be dramatically simplified by suppression of solvent signals. This is achieved by (i) exploiting the paramagnetic relaxation enhancement of solvent signals relative to materials substrates, or (ii) by using short cross-polarization contact times to transfer hyperpolarization to only directly bonded carbon-13 nuclei in frozen solutions. The methods are evaluated for organic microcrystals, surfaces and frozen solutions. We show how this allows for the acquisition of high-resolution DNP enhanced proton-proton correlation experiments to measure inter-nuclear proximities in an organic soli

Measurement of Proton Spin Diffusivity in Hydrated Cementitious Solids

The study of hydration and crystallization processes involving inorganic oxides is often complicated by poor long-range order and the formation of heterogeneous domains or surface layers. In solid-state NMR, H-1-H-1 spin diffusion analyses can provide information on spatial composition distributions, domain sizes, or miscibility in both ordered and disordered solids. Such analyses have been implemented in organic solids but crucially rely on separate measurements of the 1 H-1 spin diffusion coefficients in closely related systems. We demonstrate that an experimental NMR method, in which "holes" of well-defined dimensions are created in proton magnetization, can be applied to determine spin diffusion coefficients in cementitious solids hydrated with O-17-enriched water. We determine proton spin diffusion coefficients of 240 +/- 40 nm(2)/s for hydrated tricalcium aluminate and 140 +/- 20 nm(2)/s for hydrated tricalcium silicate under quasistatic conditions

Exploring the salt-cocrystal continuum with solid-state NMR using natural-abundance samples: implications for crystal engineering

There has been significant recent interest in differentiating multicomponent solid forms, such as salts and cocrystals, and, where appropriate, in determining the position of the proton in the X-H center dot center dot center dot A-Y X-center dot center dot center dot H-A(+)-Y continuum in these systems, owing to the direct relationship of this property to the clinical, regulatory and legal requirements for an active pharmaceutical ingredient (API). In the present study, solid forms of simple cocrystals/salts were investigated by high-field (700 MHz) solid-state NMR (ssNMR) using samples with naturally abundant N-15 nuclei. Four model compounds in a series of prototypical salt/cocrystal/continuum systems exhibiting {PyN center dot center dot center dot H-O-}/{PyN+-H center dot center dot center dot O-} hydrogen bonds (Py is pyridine) were selected and prepared. The crystal structures were determined at both low and room temperature using X-ray diffraction. The H-atom positions were determined by measuring the N-15-H-1 distances through N-15-H-1 dipolar interactions using two-dimensional inversely proton-detected cross polarization with variable contact-time (invCP-VC) H-1 -> N-15 -> H-1 experiments at ultrafast (nu(R) >= 60-70 kHz) magic angle spinning (MAS) frequency. It is observed that this method is sensitive enough to determine the proton position even in a continuum where an ambiguity of terminology for the solid form often arises. This work, while carried out on simple systems, has implications in the pharmaceutical industry where the salt/cocrystal/continuum condition of APIs is considered seriously

Cellulose phosphorylation comparison and analysis of phosphorate position on cellulose fibers

International audienc

Probing Protein Dynamics Using Multifield Variable Temperature NMR Relaxation and Molecular Dynamics Simulation

International audienceUnderstanding the interplay between protein function and dynamics is currently one of the fundamental challenges of physical biology. Recently, a method using variable temperature solid-state nuclear magnetic resonance relaxation measurements has been proposed for the simultaneous measurement of 12 different activation energies reporting on distinct dynamic modes in the protein GB1. Here, we extend this approach to measure relaxation at multiple magnetic field strengths, allowing us to better constrain the motional models and to simultaneously evaluate the robustness and physical basis of the method. The data reveal backbone and side-chain motions, exhibiting low- and high-energy modes with temperature coefficients around 5 and 25 kJ·mol-1. The results are compared to variable temperature molecular dynamics simulation of the crystal lattice, providing further support for the interpretation of the experimental data in terms of molecular motion