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Disaccharide glasses are increasingly used to immobilize proteins at room temperature for structural/functional studies and long-term preservation. To unravel the molecular basis of protein immobilization, we studied the effect of sugar/protein concentration ratios in trehalose or sucrose matrixes, in which the bacterial photosynthetic reaction center (RC) was embedded as a model protein. The structural, dynamical, and H-bonding characteristics of the sugar-protein systems were probed by high-field W-band EPR of a matrix-dissolved nitroxide radical. We discovered that RC immobilization and thermal stabilization, being independent of the protein concentration in trehalose, occur in sucrose only at sufficiently low sugar/protein ratios. EPR reveals that only under such conditions does sucrose form a microscopically homogeneous matrix that immobilizes, via H-bonds, the nitroxide probe. We conclude that the protein immobilization capability depends critically on the propensity of the glass-forming sugar to create intermolecular H-bond networks, thus establishing long-range, homogeneous connectivity within the matrix.

Matrix effects on long-range electron transfer were studied in cyanobacterial Photosystem I (PS I) complexes, embedded into trehalose glasses at different hydration levels. W-band EPR studies demonstrated, via nitroxide spin probes, structural homogeneity of the dry PS I-trehalose matrix and no alteration of cofactors' distance and relative orientation under temperature and matrix variation. In dry trehalose glasses at room temperature (RT), PS I was stable for months. Flash-induced charge recombination kinetics were examined by high-field time-resolved EPR and optical spectroscopies. The kinetics in hydrated PS I-trehalose glasses mostly reflected the reduction of the photooxidized primary donor P700•+ by the reduced terminal iron-sulfur clusters. Upon dehydration, the P700•+ decay accelerated and became more distributed. Continuous distributions of lifetimes τ were extracted from the kinetics by two numerical approaches: a maximum entropy method (MemExp program) and a constrained regularization method (CONTIN program). Both analyses revealed that upon dehydration the contribution of the two slowest components (lifetimes τ ~ 300 ms and ~ 60 ms), attributed to P700•+[FA/FB] − recombination, decreased in parallel with the increase of the fastest component (τ ~ 150 μs), and of additional distributed phases with intermediate lifetimes. Dehydration at RT mimicked the effects of freezing water-glycerol PS I systems, suggesting an impairment of PS I protein dynamics in the dry trehalose glass. Similar effects were observed previously in bacterial reaction centers. The work presented for PS I provides new insights into the crucial issue of protein-matrix interactions for protein functionality as controlled by hydrogen-bond networks of the hydration shell.