Regulation of Ras Paralog Thermostability by Networks of Buried Ionizable Groups

Protein folding is governed by a
variety of molecular forces including
hydrophobic and ionic interactions. Less is known about the molecular
determinants of protein stability. Here we used a recently developed
computer algorithm (pHinder) to investigate the relationship between
buried charge and thermostability. Our analysis revealed that charge
networks in the protein core are generally smaller in thermophilic
organisms as compared to mesophilic organisms. To experimentally test
whether core network size influences protein thermostability, we purified
18 paralogous Ras superfamily GTPases from yeast and determined their
melting temperatures (Tm, or temperature
at which 50% of the protein is unfolded). This analysis revealed a
wide range of Tm values (35–63
°C) that correlated significantly (R = 0.87)
with core network size. These results suggest that thermostability
depends in part on the arrangement of ionizable side chains within
a protein core. An improved capacity to predict protein thermostability
may be useful for selecting the best candidates for protein crystallography,
the development of protein-based therapeutics, as well as for industrial
enzyme applications.