Institut de Chimie Moléculaire et des Matériaux d'Orsay

Laboratoire de RMN en milieu orienté - LRMN



 This work has been started during my PhD at Université Claude Bernard Lyon 1. If you are interested in Solid-State NMR of proteins, you can also discover some of the latest developments in this area at CRMN in Lyon. You can also download my thesis manuscript here.

Toward a Site-Specific Analysis of Motions in Crystalline Proteins

 The determination of molecular dynamics in proteins is one of the key challenges in understanding their structure-function relationships, since internal motion has always been suggested as a fundamental modulator of biological function.(1) Solution state NMR, as well as Molecular Dynamics simulations, supply a rich insight into fast motions of the protein backbone. However, relaxation studies in solution are complicated by the superimposition of internal motions and overall tumbling, while MD trajectories of thousands of atoms in a realistic environment are limited to short timescales, and can thus only give a qualitative idea of rates and motional restrictions.

The dimeric form of the protein Crh

  In this context, the link between nuclear relaxation times in solids and motion has been established for decades, and several deuterium NMR lineshape and relaxation studies on solid proteins, as well as nitrogen-15 relaxation measurements(2) have been performed in the past. In these studies, however, it was not possible to obtain widespread site-specific information about the variation of mobility from one part of the molecule to another. The introduction of micro-crystalline protein samples, as well as substantial improvements in the sensitivity and resolution of multidimensional solid-state NMR experiments, has lead to rapid recent progress being made in this field, resulting in the first complete assignments of proteins in the solid-state(3-6) and the first three-dimensional structure of model systems.(7)


 We have shown that it is possible to record longitudinal relaxation experiments, and obtain widespread, site-specific information about the variations in nitrogen-15 spin-lattice relaxation rates.(8)
  We have measured nitrogen-15 nuclear longitudinal relaxation rates (R1) in a micro crystalline, uniformly labeled [15N, 13C] sample of the protein Crh in its domain swapped dimeric form (2 x 10.4 kDa),(3) and provide a qualitative description of the site specific backbone dynamics in the solid-state. We observe substantial differences (up to a factor 7) in R1 along the backbone. It is apparent that there is a strong correlation between measured relaxation rates and a simple but coherent preliminary picture of internal mobility, with increased mobility providing faster R1. In particular we find increased mobility for residues that are not in regular secondary structures, whereas the rates measured for residues in α helices or β sheets show less mobility.(8)


 Since Nitrogen-15 spins can be considered as local probes for the study of internal mobility along protein backbone in solid proteins(2), we also present further development of a theoretical model initially proposed by Torchia and Szabo to quantitatively determine fluctuations in local dynamics, and distinguish between timescale and motional amplitude effects.

 We use Padé approximants to describe autocorrelation functions for nitrogen-15 dipolar relaxation within the model of diffusion in a cone. We examine different averaging schemes in order to propose an analysis of relaxation curves that takes into account the specificity of MAS experiments. In particular, we show that Magic Angle Spinning averages the relaxation rate experienced by a single spin over one rotor period, resulting in individual relaxation curves that are still modulated by the orientation of their corresponding carrousel with respect to the rotor axis. Powder averaging thus leads to a non exponential behaviour in the observed decay curves. To extract dynamic informations from experimental curves, we propose a procedure using a simple motional model. Finally, we apply this study to the analysis of spin-lattice relaxation rates of the microcrystalline protein Crh at two different fields.(9)

 Furthermore, the observation of proton to nitrogen-15 heteronuclear Overhauser effects in the microcrystalline protein Crh is used to confirm that the principal mechanism of relaxation of amide nitrogens is due to the fluctuation of the N-H dipolar couplings caused by N-H bond dynamics.


 Our observations reveal the central role of water as the main source of proton magnetization, and we provide an analysis of the different pathways that could lead to the observed results.(10)

 The effect of nitrogen-15 proton driven spin diffusion on quantitative 15N T1 measurements in solid proteins has also been investigated, and the impact on the measurement of dynamic parameters has been assessed. A simple model of exchange between neighboring nitrogens is used to reproduce the evolution of 15N spin systems whose longitudinal relaxation rates, and exchange rates are compatible with experimental measurements.


 We show that the induced error in the measured T1 and its effect on the determination of dynamics parameters is likely to be less than the current experimental error. The use of deuterated protein samples is shown to have a small but sometimes visible effect, and may also considerably slow down or even suppress the exchange of magnetization due to spin diffusion.(11)

Solid-State NMR Spectroscopy of Paramagnetic Proteins

 Finally, we have shown that the paramagnetic form of a Cu(II) protein, the oxidized form of human, dimeric copper zinc superoxide dismutase (SOD) is accessible to high-resolution solid-state NMR studies when it is in microcrystalline form.


 Due to the long electronic T1, relaxation times of the so-called type II copper center, oxidized SOD is virtually inaccessible to solution NMR studies, with 1H NMR resonances of protons closer than 11 Å from the metal ion broadened beyond detection. We present a series of homonuclear heteronuclear correlation spectra obtained on a microcrystalline sample under MAS (10-20 kHz). Compared to liquid-state experiments that rely on the excitation and detection of proton coherences, solid-state experiments offer better performances for correlating backbone resonances and providing assignments close to the metal.(12)


 These recent progress in methods for obtaining NMR spectra for paramagnetic molecular solids, have yielded the complete assignment of this 32 kDa dimer.(9)


(1) Rasmussen, B. F.; Stock, A. M.; Ringe, D.; Petsko, G. A., Nature. 357, (6377), 423-424 (1992)
H. B. R. Cole and D. Torchia, Chem. Phys. 158, 271-281 (1991)
(3) A. Böckmann, A. Lange, A. Galinier, S. Luca, N. Giraud, M. Juy, H. Heise, R. Montserret, F. Penin, and M. Baldus, J. Biomol. NMR 27, 323-339 (2003)
(4) T. I. Igumenova, A. J. Wand, and A. E. McDermott, J. Am. Chem. Soc. 126, 5323-5331 (2004)
(5) J. Pauli, M. Baldus, B.-J. van Rossum, H. DE Groot, and H. Oschkinat, ChemBioChem 2, 272-281 (2001)
(6) A. Lange, S. Becker, K. Seidel, K. Giller, O. Pongs and M. Baldus, Angew. Chem. Int. Ed. Engl., 44, 2089-2092 (2005)
(7) F. Castellani,B. van Rossum,A. Diehl,M. Schubert,K. Rehbein and H. Oschkinat, Nature 420, 98-102 (2002)
(8) N. Giraud, A. Böckmann,A. Lesage,F. Penin,M. Blackledge and L. Emsley, J. Am. Chem. Soc. 126, (37), 11422-11423 (2004)
(9) Giraud, N., Blackledge, M., Goldman, M., Böckmann, A., Lesage, A, Penin, F. and Emsley, L., J. Am. Chem. Soc., 127(51) pp 18190 - 18201 (2005)
(10) Giraud, N., Sein, J., Lesage, A., Böckmann, A., Blackledge, M. & Emsley, L., J. Am. Chem. Soc., 128 (38): 12398-12399 (2006)
(11) Pintacuda, G., Giraud, N., Pieratelli, R., Böckmann, A., Bertini, I. and Emsley, L., Ang. Chem. Int. Ed. 46 (7): 1079-1082 (2007)
(12) Giraud, N., Blackledge, M., Böckmann, A. & Emsley, L., J. Magn. Res., 184 (1): 51-61 (2006)

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