Alanine Counteracts the Destabilizing Effect that Urea has on RNase-A

Background: It is generally believed that organisms use and accumulate methylamine osmolytes to prevent urea’s damaging effect on protein stability and activity. However, urea-rich cells not only accumulate methylamines but also many other methylated and non-methylated compounds as well. But, so far it is not known whether osmolytes that are not accumulated in urea-rich cells could also confer urea-counteracting properties. Objective: We investigated the behavior of a non-methylamine osmolyte, alanine for its counteracting effect against urea denaturation of a model protein, ribonuclease A (RNase-A). Methods: We have measured structure and thermodynamic parameters (Tm, ΔHm, and ΔGD°) of RNase-A in the presence of alanine, urea and their combination. The results were also compared with the ability of glycine (osmolyte lacking one methyl group when compared with alanine) to counter urea’s effect on protein stability. Results: We observed that alanine but not glycine counteracts urea’s harmful effect on RNase-A stability. Discussion: The results indicated that alanine (in addition to methylamine osmolytes) may serve as an alternate urea-counteractant. Since glycine fails to protect RNase-A from urea’s destabilizing effect, it seems that methylation to glycine might have some evolutionary significance to protect proteins against harmful effects of urea.