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 Tm 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 (Tm: 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.
All Science Journal Classification (ASJC) codes
- Analytical Chemistry
- Applied Microbiology and Biotechnology
- Molecular Biology
- Organic Chemistry