Mapping the Free Energy Change of Unfolding Versus Temperature for Two Homologous Cytochromes C Adapted to Differing Environmental Temperatures

Abstract

As an adaption to function at temperatures at or below the freezing temperature of water, proteins from psychrophiles must evolve lower overall stabilities to retain flexibility when less thermal energy is available. Our model system to examine cold-adaptation consists of two putative cytochrome c6 proteins from homologous diatoms, the psychrophile Fragilariopsis cylindrus and the mesophile Thalassiosira pseudonana. Because cytochrome c6 serves to shuttle electrons between cytochrome b6f and photosystem I during photosynthesis, its sequence is highly conserved even between distant homologs, allowing us to focus on key sequence differences. The presence of the heme cofactor provides numerous spectroscopic handles by which to evaluate the folding state of the protein. We previously reported that, when both proteins are in the reduced state, the melting temperature of the psychrophilic cytochrome c6 is 6.4°C lower than the melting temperature of the T. pseudonana cytochrome c6. We predict that the stability of the two proteins at their physiological temperatures should be similar, that the mesophilic protein should retain stability at higher temperatures, and that these differing properties are caused by a difference in the solvent accessibility of the hydrophobic core of the two proteins. In this study, we determined the change in free energy upon unfolding for both proteins at a range of temperatures between 7 and 35°C. This allows us to map the stability curves for psychrophilic versus mesophilic cytochrome c6 proteins, and to extract values for the change in heat capacity upon unfolding for the two homologous proteins. These data provide insight into the roles of entropic and enthalpic contributions in the differing stability of the mesophilic and psychrophilic cytochrome c6 proteins.