A thermodynamic analysis of a cold-adapted protein, type III anti-freeze protein 
(AFP), was carried out. The results indicate that the folding equilibrium of 
type III AFP is a reversible, unimolecular, two-state process with no populated 
intermediates. Compared to most mesophilic proteins whose folding is two-state, 
the psychrophilic type III AFP has a much lower thermodynamic stability at 25 
degrees C, approximately 3 kcal/mol, and presents a remarkably downshifted 
stability-temperature curve, reaching a maximum of 5 kcal/mol around 0 degrees 
C. Type III AFPs contain few and non-optimally distributed surface charges 
relative to their mesophilic homologs, the C-terminal domains of sialic acid 
synthases. We used thermodynamic double mutant cycles to evaluate the energetic 
role of every surface salt bridge in type III AFP. Two isolated salt bridges 
provided no contribution to stability, while the Asp36-Arg39 salt bridge, 
involved in a salt bridge network with the C-terminal carboxylate, had a 
substantial contribution (approximately 1 kcal/mol). However, this contribution 
was more than counteracted by the destabilizing effect of the Asp36 carboxylate 
itself, whose removal led to a net 30% increase in stability at 25 degrees C. 
This study suggests that type III AFPs may have evolved for a minimally 
acceptable stability at the restricted, low temperature range (around 0 degrees 
C) at which AFPs must function. In addition, it indicates that salt bridge 
networks are used in nature also for the stability of psychrophilic proteins, 
and has led to a type III AFP variant of increased stability that could be used 
for biotechnological purposes.