Protein Immobilization Capabilities of Sucrose and Trehalose Glasses: The Effect of Protein/Sugar Concentration Unraveled by High-Field EPR

• Published in: The journal of physical chemistry letters Vol. 7; no. 23; pp. 4871 - 4877
• Main Authors: Malferrari, Marco, Savitsky, Anton, Lubitz, Wolfgang, Möbius, Klaus, Venturoli, Giovanni
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 01.12.2016
• ISSN: 1948-7185; 1948-7185
• DOI: 10.1021/acs.jpclett.6b02449

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.