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Published online before print December 22, 2006, 10.1110/ps.062448907
Protein Science (2007), 16:227-238. Published by Cold Spring Harbor Laboratory Press. Copyright © 2007 The Protein Society
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Thermodynamic stability of a cold-adapted protein, type III antifreeze protein, and energetic contribution of salt bridges

Olga García-Arribas1, Roberto Mateo1, Melanie M. Tomczak2, Peter L. Davies2, and Mauricio G. Mateu1

1 Centro de Biología Molecular "Severo Ochoa" (UAM-CSIC), Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain
2 Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada

(RECEIVED July 17, 2006; FINAL REVISION October 16, 2006; ACCEPTED November 1, 2006)

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°C, ~3 kcal/mol, and presents a remarkably downshifted stability–temperature curve, reaching a maximum of 5 kcal/mol around 0°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 (~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°C. This study suggests that type III AFPs may have evolved for a minimally acceptable stability at the restricted, low temperature range (around 0°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.

Keywords: psychrophilic protein; cold adaptation; antifreeze protein; thermodynamic stability; salt bridge


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