Dynamics of Cphycocyanin in various deuterated tr

Authors: Ingo Köper Sophie Combet Winfried Petry MarieClaire BellissentFunel

Publish Date: 2008/01/08

Volume: 37, Issue: 6, Pages: 739-748

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Abstract

The molecular understanding of protein stabilization by the disaccharide trehalose in extreme temperature or hydration conditions is still debated In the present study we investigated the role of trehalose on the dynamics of the protein Cphycocyanin CPC by neutron scattering To single out the motions of CPC hydrogen H atoms in various trehalose/water environments measurements were performed in deuterated trehalose and heavy water D2O We report that trehalose decreases the internal CPC dynamics as shown by a reduced diffusion coefficient of protein H atoms By fitting the Elastic Incoherent Structure Factor—which gives access to the “geometry” of the internal proton motions—with the model of diffusion inside a sphere we found that the presence of trehalose induces a significantly higher proportion of immobile CPC hydrogens We investigated by elastic neutron scattering the mean square displacements MSDs of deuterated trehalose/D2Oembedded CPC as a function of temperature in the range of 40–318 K Between 40 and ∼225 K harmonic MSDs of CPC are slightly smaller in samples containing trehalose Above a transition temperature of ∼225 K we observed anharmonic motions in all trehalose/watercoated CPC samples In the hydrated samples MSDs are not significantly changed by addition of 15 trehalose but are slightly reduced by 30 trehalose In opposition no dynamical transition was detected in dry trehaloseembedded CPC whose hydrogen motions remain harmonic up to 318 K These results suggest that a role of trehalose would be to stabilize proteins by inhibiting some fluctuations at the origin of protein unfolding and denaturationTrehalose is a nonreducing disaccharide of glucose isolated from algae bacteria fungi insects invertebrates and yeasts as well as from lower vascular plants and a few flowering plants Clegg and Filosa 1961 Fox 1995 This sugar has been found in large amounts in organisms able to survive extreme external stresses such as high or very low temperatures or periods of complete drought Clegg and Filosa 1961 Crowe et al 1996 Fox 1995 In presence of trehalose the structure of the biomaterial is conserved and life and decay processes are suspended or highly slowed down Clegg and Filosa 1961 Fox 1995 These astonishing bioprotecting properties of trehalose have been confirmed by in vitro experiments in lipids proteins viruses and mammalian cells Bieganski et al 1998 Carpenter and Crowe 1989 Crowe et al 1984 Eroglu et al 2000 SolaPenna et al 1998 Furthermore trehalose has been found to be the most effective bioprotectant in cryopreservation and dessication Bieganski et al 1998 Green and Angell 1989 LópezDíez et al 2004 as well as in thermal preservation SolaPenna et al 1998The “high viscosity” hypothesis according to which the denaturation processes are hindered is based on the very high glass transition temperature of trehalose solutions Affouard et al 2005 Branca et al 1999 Cornicchi et al 2005 Giuffrida et al 2006 Green and Angell 1989 Magazù et al 2007 Mazzobre et al 1997 Rector et al 2001 The amorphous phase formed by trehalose may protect biomolecules at low temperature since the formation of ice crystals followed by the destruction of cellular material is hinderedThe “water entrapment” mechanism according to which trehalose concentrates water close to the biomolecule as shown by infrared and Raman spectroscopy for the residual water molecules in the dry state Belton and Gil 1994 and by molecular dynamics simulations for trehalose/watercoated COmyoglobin MbCO Cottone et al 2002The “water replacement” theory according to which trehalose would replace water molecules normally present at the surface of the biomaterial to conserve its structure Several observations suggest direct interactions through hydrogen bonds between the sugar and the polar groups of proteins and phospholipids Belton and Gil 1994 Carpenter and Crowe 1989 Cottone et al 2002 Crowe et al 1984 Giuffrida et al 2003 Librizzi et al 2002 LópezDíez et al 2004 emphasized the relatively higher stability of hydrogen bonds with the protein in the case of trehalose as compared to other bioprotecting agents Moreover SolaPenna et al 1998 suggested that more water molecules can be substituted by trehalose than by other sugars through its higher hydration volumePreviously we performed inelastic and quasielastic neutron scattering experiments to study the influence of trehalose on Cphycocyanin CPC dynamics CPC is a phycobiliprotein present in the antennae of cyanobacteria and is involved in the first steps of photosynthesis Glazer 1989 CPC has been widely used as a model protein to study water motions at its surface by neutron scattering BellissentFunel et al 1996 By comparing the dynamics of hydrated samples of CPC and of trehalosecoated CPC we observed in a time range from picoseconds to several nanoseconds that trehalose induces a slowing down of the motions of the protein up to two orders of magnitude Köper et al 2002 By using the model of a particle diffusing inside a sphere Volino and Dianoux 1980 we showed that trehalose reduces the proportion of mobile hydrogens in the protein and that the confining sphere radius is independent on trehalose content Köper et al 2002The aim of the present study was to further investigate by quasielastic neutron scattering the nature and the geometry of CPC hydrogen motions in various deuterated trehalose/D2O environments with the high resolution of the backscattering neutron spectrometer IN13 at the Institut LaueLangevin ILL Grenoble France Moreover we report by elastic neutron scattering the evolution of CPC proton MSDs as a function of temperature in the range of 40–318 K Previous studies have already shown the evolution as a function of temperature of the MSDs of trehalosecoated MbCO Cordone et al 1999 Librizzi et al 2001 In these studies the samples were either fully protonated—and measured only in the dry state—Cordone et al 1999 or exchanged with D2O before lyophilization—and studied both in the dry and the D2Orehydrated states—Librizzi et al 2001 In the present study we used deuterated trehalose and D2O to reduce their contributions in incoherent neutron scattering measurements in order to single out CPC dynamics by measuring the motions of the nonexchangeable hydrogen atoms of the protein Furthermore we investigated CPC dynamics in various trehalose/water environments in order to understand up to which extent trehalose could inhibit protein motions

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