WHY DO SOME ORGANISMS USE A UREA-METHYLAMINE MIXTURE AS OSMOLYTE - THERMODYNAMIC COMPENSATION OF UREA AND TRIMETHYLAMINE N-OXIDE INTERACTIONS WITH PROTEIN

Many organisms accumulate low molecular weight substances known as osmolytes when they experience environmental water stress. The main classes of osmolytes are sugars, polyhydric alcohols, amino acids and their derivatives, and methylamines, and all are known to be protein stabilizers. However, marine cartilaginous fishes and the coelacanth use, as osmolytes, a combination of urea and methylamines, i.e., a denaturant and a stabilizer, in a 2:1 molar ratio. Preferential binding and thermal denaturation measurements in the presence of each cosolvent separately and in their mixtures have been carried out using ribonuclease T1 (RNase T1) as the protein. At a 2:1 molar ratio of urea and trimethylamine N-oxide (TMAO), the effects of the two cosolvents on the transition temperature (T-m) were found to be essentially the algebraic sum of their effects when used individually. Preferential interaction measurements of urea, TMAO and urea in its 2:1 molar ratio mixture with TMAO, have shown that the presence of TMAO has no effect on the interaction of urea with the protein in either the native or the unfolded (reduced carboxymethylated RNase T1) state. The preferential interaction of TMAO in the presence of urea could not be measured for technical reasons. Calculations of transfer free energy in the two end states of the denaturation reaction have shown that 2 M urea destabilizes RNase T1 by 3.8 +/- 0.3 kcal/mol whether 1 M TMAO is present or not. The contribution of 1 M TMAO to stabilization is calculated to be 3.1 kcal/mol in the presence of 2 M urea and is measured to be 2.7 kcal/mol in its absence.