A Mutagenesis Study to Investigate the Role of Alanine to Serine Mutations in the Adaptation of a Diatom Cytochrome c6 to Cold Temperatures

Abstract

Proteins in the cytochrome c family share a covalently bond heme cofactor and clear spectroscopic features in the visible wavelengths, making them ideal model systems to investigate protein folding. We have previously identified two homologous cytochrome c6 proteins, one from the psychrophilic diatom Fragilariopsis cylindrus and another from the mesophilic diatom Thalassiosira pseudonana. We found that, while the two proteins only differ by 11 amino acid positions (89% identity), the F. cylindrus cytochrome c6 melts at a temperature 6.4°C lower than the T. pseudonana homolog. We predict that differences in the solvent accessibility of the hydrophobic cores of the two proteins will account for the differences in their stability over temperature. Using an amino acid sequence alignment, we identified three candidate amino acid positions that contain a non-polar alanine residue in the mesophilic protein, but the hydrophilic residue serine in the psychrophilic protein. Therefore, we predicted that, if serine were substituted for alanine in the context of the amino acid sequence of the mesophilic cytochrome c6, the melting temperature of the resulting variant should more closely match the melting temperature of the psychrophilic protein. In this study, we test our hypothesis by evaluating the melting temperatures of single-site alanine to serine amino acid variants of the T. pseudonana protein. We used site-directed mutagenesis to generate A86S, A93S, and A126S variants of T. pseudonana cytochrome c6, and recombinantly expressed and purified the proteins. Comparison of the melting temperatures of the variants with the melting temperatures of wild-type T. pseudonana and F. cylindrus proteins will provide evidence for the role of hydrophilic residues in destabilizing the hydrophobic core to allow for greater flexibility at lower temperatures.