Neutrons

Neutron spectroscopy is a unique tool to characterise the forces in protein structures and the thermal motions associated with the biological activity (Zaccai, 2000 ; Gabel et al., 2002). In the elastic domain it is possible to measure two important parameters: the mean square atomic fluctuation and structural resilience within the macromolecules – the last parameter being a mean force which maintains the protein structure folded and active (Bicout & Zaccai, 2001 ; Gabel, 2005). Furthermore the neutrons are diffused differently according to the isotopes and the replacement of hydrogen atoms by deuterium allows to study the specific components of complex systems. We are using neutron scattering to explore the role of the molecular dynamics for the biological function and their selection by evolution to permit to the protein structures to maintain their stability and activity in different extreme conditions. The considered systems are membranes and extremophile organism proteins, bacteria and archaea cells adapted to extreme temperatures (psychrophiles or thermophiles) and to extreme saline conditions (halophiles) and animal cells in different metabolic or stress states.

Molecular dynamics

Purple membrane

The Halobacterium salinarum purple membrane (PM) consists of proteins, the bacteriorhodopsine (BR), and lipids. The BR binds a retina molecule and acts as a proton pump activated by light. We study in details the dynamics of the PM and of its various components (proteins, lipids, water) by deuterium labelling in situ and its relation to the biological activity (Zaccai, 2000a, 2000b, 2003, 2004; Lehnert et al., 2005, and in preparation). In particular we examine systematically the interne dynamics of different amino acids in BR (Wood et al., in preparation).

Malate-lactate deshydrogenases

We studied molecular dynamics of homologues proteins of the Malate-lactate deshydrogenases group : halophile malate deshydrogenase (Hm MalDH), hyperthermophilic malate dehydrogenase from Methanococcus jannaschii (Mj MalDH) and a mesophilic lactate dehydrogenase from Oryctolagus cunniculus (rabbit) muscle (LDH) (Tehei et al., 2005a). The mean square atomic fluctuations of the three proteins were found to be similar ( 1.5 Å) at their optimal activity temperature, suggesting the existence of an “optimised flexibility” to assure the optimal activity. However the measured hyperthermophilic resilience has a value (1.5 N/m) ten times that of mesophilic MalDH (0.15 N/m). We thus concluded that the thermoadaptation was favoured by the selection of forces increasing the structural rigidity to maintain the protein structure at high temperature and simultaneously to allow the flexibility required to assure the activity. The solvent effect on the stability of the extremophilic proteins was also studied (Tehei et al., 2001).

Extremophilic organisms

The purified protein results are in agreement with these obtained from entire cells of psychrophile, mesophile, thermophile and hyperthermophile bacteria (Tehei et al., 2004, 2005b). The macromolecular population dynamics of each bacterium was quantified in vivo and a mean square amplitude fluctuation of about 1 Å was determined for each bacterium at its physiological temperature. The mean resilience increases with the physiological temperature, by 0.2 N/m for the psychrophiles (growth temperature 4° C) and by 0.6 N/m for the hyperthermophiles (growth temperature 85° C) – this allows a thermoadaptation through the rigidity of the macromolecular structures to assure the flexibility required at low temperature and the stability required at high temperature. It is interesting to notice that these measures, having been achieved in vivo, correspond to conditions of high molecular accumulation (Ebel & Zaccai, 2004).

Extremely slow cell water

Experiences on the instruments IN6 and IN16 (ILL) and on BSS (Jülich) revealed the intracellular water dynamics in Haloarcula marismortui, an extremely halophilic organism originally isolated from the Dead Sea (Tehei et al., 2007a, 2007b). The study was motivated by the fact that K+ was retained within the cell, despite a high membrane permeability. The time scales accessible by the two instruments IN6 and IN16 are of the order of 1 ps and 1 ns, respectively. From IN6 data a translational diffusion constant of 1.3 x 10-5 cm² s-1 was determined at 285 K. This value is close to that found previously for other cells. A very slow water component was discovered from the IN16 data: at 285 K the water-protons of this component displays a residence time of 411 ps (compared with a few ps in bulk water). No such water was found in Escherichia coli measured on BSS. It is hypothesised that the slow mobility of a large part of H. marismortui cell water indicates a specific water structure responsible for the large amounts of K+ bound within these extremophile cells.

C-phycocyanin

A methodological approach was established for the interpretation of incoherent elastic neutron scattering on different instruments (IN13 and IN16 at ILL) and for C-phycocyanin (CPC) hydration water dynamics in the presence of trehalose (Gabel & Bellissent-Funel, 2007). Applying the same phenomenological model scattering law, convoluted with the respective instrumental energy resolutions, we were able to extract effective diffusion coefficients, relaxation times and linear dimensions describing the dynamic behaviour of CPC hydration water over a very large temperature range (235 to 320 K). Our methodological approach has several important advantages with respect to conventional approaches such as the Gaussian approximation: No principal restraints on the choice of the instrumental energy resolution and Q-range, a single scattering law can be used to describe the dynamic behaviour of hydration water on several instruments and the signal-to-noise of the data obtained is better than in comparable quasielastic scattering experiments.

Structural studies

The dynamical studies are completed by structural studies to explore the different interactions concerned by the stability mechanism under extreme conditions.

Acetylcholinesterase

Acetylcholinesterase is an enzyme of the neural system representing a high biomedical interest, especially in the framework of senile dementia diseases.

To understand the reasons for the extremely high catalytic turnover number (104 per second) of this enzyme, neutron scattering measurements were carried out to study the effects of the environment and of the inhibitor binding on the molecular dynamics (Gabel et al., 2004, 2005).

Macromolecular assemblies

A high interest of the laboratory is devoted to the mapping of macromolecular dynamics in cellulo . Using the deuteration labelling (in collaboration with the Laboratoire de Deutération de l’ILL, we investigate the dynamics of the ARN component, especially in the framework of the ARN-protein interaction required for the post-transcriptional regulation of the genetic information (Jasnin et al., in preparation). Moreover, the neutron staff participates in a huge European collaboration (Macromolecular assembly and dynamics of Cellular Machines- MAD-Cell) aiming for mapping the structures of big macromolecular complexes in cells. For that purpose different complementary methods can be used, among them small angle neutron scattering combined with specific deuterium labelling to mark the components inside the complexes.

References

2000

Zaccai G. (2000a) How soft is a protein? A protein dynamics force constant measured by neutron scattering. Science. 288:1604-7.

Zaccai G. (2000b) Moist and soft, dry and stiff: A review of neutron experiments on hydration-dynamics-activity relations in the purple membrane of Halobacterium salinarum. Biophys Chem. 86:249-57.

2001

Bicout D & Zaccai G. (2001) An analysis of mean square fluctuations in proteins and the dynamical transition in terms of force constants. Biophys J. 80: 1115-23.

Tehei M, Madern D, Pfister C, Zaccai G. (2001) Fast Dynamics of Halophilic Malate Dehydrogenase and Bovine Serum Albumin measured by Neutron Scattering under various Solvent Conditions influencing Protein Stability. Proc Natl Acad Sci (USA). 98:14356-61.

2002

Gabel F, Bicout D, Lehnert U, Tehei M, Weik M, Zaccai G. (2002) Protein dynamics studied by neutron scattering. Q Rev Biophys 35(4):327-67.

2003

Zaccai G. (2003) Proteins as Nano-machines: Dynamics-Function Relations Studied by Neutron Scattering. J Phys: Condens Matter. 15: S1673-82.

2004

Ebel C & Zaccai G. (2004) Crowding in extremophiles: linkage between solvation and weak protein-protein interactions, stability and dynamics, provides insight into molecular adaptation. J Mol Recognit. 17: 382-9.

Gabel F, Weik M, Doctor BP, Saxena A, Fournier D, Brochier L, Renault F, Masson P, Silman I & Zaccai G. (2004) The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron scattering study. Biophys J. 86: 3152-65.

Tehei M, Franzetti B, Madern D, Ginzburg M, Ginzburg BZ, Giudici-Orticoni MT, Bruschi M, Zaccai G. (2004) Adaptation to extreme environments: Macromolecular thermal dynamics in psychrophile, mesophile and thermophile bacteria compared, in-vivo, by neutron scattering. EMBO Rep. 5:66-70.

Zaccai G. (2004) The effect of water on protein dynamics. Phil Trans R Soc Lond. B 359: 1269-75.

2005

Gabel F. (2005a) Protein dynamics in solution and powder measured by incoherent elastic neutron scattering: the influence of Q-range and energy resolution. Eur Biophys J. 34:1-12.

Gabel F (2005b). Influence of solvent on the internal dynamics of butyrlcholinesterase and on the dynamics of water of hydration: a study by incoherent elastic diffusion of neutrons. Journal de Physique IV 130: 133-139.

Gabel F, Weik M, Masson P, Renault F, Fournier D, Brochier L, Doctor BP, Saxena A, Silman I & Zaccai G. (2005) Effects of soman inhibition and of structural differences on cholinesterase molecular dynamics: A neutron scattering study. Biophys J. 89(5): 3303-11.

Kurkal V, Daniel R, Finney J, Tehei M, Dunn RV, Smith JC (2005a). Enzyme activity and Flexibility at very low hydratation activity in enzymes, Biophysical Journal, 89, 1282-1287.

Kurkal V, Daniel R, Finney J, Tehei M, Dunn RV, Smith JC (2005b). Low frequency enzyme dynamics as a function of temperature and hydration: A neutron scattering study, Chem. Phys., 317, 267.

Lehnert U, Reat V, Zaccai G & Oesterhelt D (2005). Proton channel hydration and dynamics of a bacteriorhodopsin triple mutant with an M-state-like conformation. Eur Biophys J. 34(4): 344-52.

Tehei M, Madern D, Franzetti B, Zaccai G (2005a). Neutron scattering reveals the dynamic basis of protein adaptation to extreme temperature. J Biol Chem. 280(49):40974-9.

Tehei M, Smith JC, Monk C, Ollivier J, Oettl M, Kurkal V, Finney J, Daniel R (2005); Dynamics of Immobilized and Native Escherichia coli Dihydrofolate Reductase by Quasielastic Neutron Scattering, Biophys. J., 90, 1090-1097.

Tehei M & Zaccai G. (2005b) Adaptation to extreme environments: macromolecular dynamics in complex systems. Biochim Biophys Acta. 1724(3): 404-10.

Weik M, Lehnert U & Zaccai G. (2005) Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics. Biophys J. 89(5): 3639-46.

2006

Gabel F, Wang D, Madern D, Sadler A, Dayie K, Daryoush MZ, Schwahn D, Zaccai G, Lee X, Williams BR (2006). Dynamic flexibility of double-stranded RNA activated PKR in solution. J. Mol. Biol. 359(3):610-623.

Jasnin M (2006) Bioneutronique : la diffusion de neutrons pour l’étude de la dynamique des protéines. Spectra Analyse. 251:28-32.

Tehei M, Daniel R, Zaccai G (2006); Fundamental and Biotechnological Applications of Neutron Scattering Measurements for Macromolecular Dynamics. Eur. Biophys. J., 35(7):551-8.

2007

Gabel F, Bellissent-Funel M-C (2007). C-phycocyanin hydration water dynamics in the presence of trehalose: an incoherent elastic neutron scattering study at different energy resolutions. Accepted by Biophys. J.

Tehei M, Franzetti B, Wood K, Gabel F, Fabiani E, Jasnin M, Oesterhelt D, Zaccai G, Ginzburg M, Ginzburg BZ (2007a). Halophile water dynamics. Cover Image. Proc Natl Acad Sci USA, 104, n°3.

Tehei M, Franzetti B, Wood K, Gabel F, Fabiani E, Jasnin M, Oesterhelt D, Zaccai G, Ginzburg M, Ginzburg BZ (2007b). Halophile water dynamics. Cover Image. Proc Natl Acad Sci USA, 104, n°3, 766-71.

Natali F, Russo D, Tehei M, Bée M, Deriu A (2007). IN13 Backscattering Spectrometer for the Investigation of the Dynamics of Biological Molecules. Conf: Eighth International Conference on Quasi-Elastic Neutron Scattering, Bloomington, Indiana, USA, from June 14 to June 17, 2006. Accepted on Materials Research Society (2007).

Wood, K (2007). Thèse de doctorat de l’Université Joseph Fourier, Grenoble I, 2007.