Functional role of polyhydroxy compounds on protein structure and thermal stability studied by circular dichroism spectroscopy.

Polyhydroxy compounds such as cyclitols, acyclic polyols and sugars are produced by a wide variety of organisms under stressful conditions in order to protect macromolecular structure. Plants undergoing abiotic stresses like heat and dehydration accumulate enormous amounts of polyhydroxy compounds (up to 400 mM) in their cellular tissues. Not only do they serve as osmoprotectants ("compatible solutes"), they also protect membrane structure and preserve enzymatic activity. To gain further insight into the mechanism of protein protection by polyhydroxy compounds, we examined the structural and thermal stability of six model proteins (bovine serum albumin, glutamine synthetase of Escherichia coli, malate dehydrogenase of pig heart, SH2 domain of phospholipaseCgamma1, SH2_Myc and GST_MycMax fusion proteins) upon the addition of various polyhydroxy compounds by circular dichroism spectroscopy. Our results show that D-pinitol (1D-3-O-methyl-chiro-inositol), L-quebrachitol (1L-2-O-methyl-chiro-inositol), myo-inositol, D-chiro-inositol, mannitol, glucose and trehalose promoted improved structural and thermal stability for each protein, whereas conduritol (1,4/2,3-cyclohexanetetrol) and glycerol were not effective. An increase in the midpoint denaturation temperature (T(m)) of 3.3 degrees C to 4.7 degrees C was observed for each protein upon the addition of 400 mM myo-inositol. Although the apparent T(m) of each protein was shifted by the addition of polyhydroxy compounds, the influence seems to be dependent on attributes like the protein surface topology, the hydration shell and on the nature of the protective solute, as well as on its concentration. The O-methylated cyclitols D-pinitol and L-quebrachitol were more effective preservatives than the less hydrophobic non-methylated myo-inositol and D-chiro-inositol. Amongst various polyhydroxy compounds, hydrophobic cyclitols were the most effective stabilizers.