Structural and dynamical characteristics of trehalose and sucrose matrices at different hydration levels as probed by FTIR and high-field EPR.

Some organisms can survive complete dehydration and high temperatures by adopting an anhydrobiotic state in which the intracellular medium contains large amounts of disaccharides, particularly trehalose and sucrose. Trehalose is most effective also in protecting isolated in vitro biostructures. In an attempt to clarify the molecular mechanisms of disaccharide bioprotection, we compared the structure and dynamics of sucrose and trehalose matrices at different hydration levels by means of high-field W-band EPR and FTIR spectroscopy. The hydration state of the samples was characterized by FTIR spectroscopy and the structural organization was probed by EPR using a nitroxide radical dissolved in the respective matrices. Analysis of the EPR spectra showed that the structure and dynamics of the dehydrated matrices as well as their evolution upon re-hydration differ substantially between trehalose and sucrose. The dehydrated trehalose matrix is homogeneous in terms of distribution of the residual water and spin-probe molecules. In contrast, dehydrated sucrose forms a heterogeneous matrix. It is comprised of sucrose polycrystalline clusters and several bulk water domains. The amorphous form was found only in 30% (volume) of the sucrose matrix. Re-hydration leads to a structural homogenization of the sucrose matrix, whilst in the trehalose matrix several domains develop differing in the local water/radical content and radical mobility. The molecular model of the matrices provides an explanation for the different protein-matrix dynamical coupling observed in dried ternary sucrose and trehalose matrices, and accounts for the superior efficacy of trehalose as a bioprotectant. Furthermore, for bacterial photosynthetic reaction centers it is shown that at low water content the protein-matrix coupling is modulated by the sugar/protein molar ratio in sucrose matrices only. This effect is suggested to be related to the preference for sucrose, rather than trehalose, as a bioprotective disaccharide in some anhydrobiotic organisms.

Hydrogen bonding of nitroxide spin labels in membrane proteins.

On the basis of experiments at 275 GHz, we reconsider the dependence of the continuous-wave EPR spectra of nitroxide spin-labeled protein sites in sensory- and bacteriorhodopsin on the micro-environment. The high magnetic field provides the resolution necessary to disentangle the effects of hydrogen bonding and polarity. In the gxx region of the 275 GHz EPR spectrum, bands are resolved that derive from spin-label populations carrying no, one or two hydrogen bonds. The gxx value of each population varies hardly from site to site, significantly less than deduced previously from studies at lower microwave frequencies. The fractions of the populations vary strongly, which provides a consistent description of the variation of the average gxx and the average nitrogen-hyperfine interaction Azz from site to site. These variations reflect the difference in the proticity of the micro-environment, and differences in polarity contribute marginally. Concomitant W-band ELDOR-detected NMR experiments on the corresponding nitroxide in perdeuterated water resolve population-specific nitrogen-hyperfine bands, which underlies the interpretation for the proteins.