Amino acids conserved at the C-terminal half of the ribonuclease T2 family contribute to protein stability of the enzymes

The ribonuclease MC1 (RNase MC1) from the seeds of the bitter gourd belongs to the RNase T2 family. We evaluated the contribution of 11 amino acids conserved in the RNase T2 family to protein folding of RNase MC1. Thermal unfolding experiments showed that substitution of Tyr¹⁰¹, Phe ¹⁰², Ala¹⁰⁵, and Phe¹⁹⁰ resulted in a significant decrease in themostability; the T_m values were 47-58°C compared to that for the wild type (64°C). Mutations of Pro ¹²⁵, Gly¹²⁷, Gly¹⁴⁴, and Val¹⁶⁵ caused a moderate decrease in thermostability (T_m: 60-62°C). In contrast, mutations of Asp¹⁰⁷ and Gly¹⁷³ did little effect on thermostability. The contribution of Tyr¹⁰¹, Phe¹⁰², Pro¹²⁵, and Gly¹²⁷ to protein stability was further corroborated by means of Gdn-HCl unfolding and protease digestions. Taken together, it appeared that Tyr¹⁰¹, Phe¹⁰², Ala ¹⁰⁵, Pro¹²⁵, Gly¹²⁷, Gly¹⁴⁴, Leu¹⁶², Val¹⁶⁵, and Phe¹⁹⁰ conserved in the RNase T2 family play an important role in the stability of the proteins.