A Combined Genetic, Biochemical, and Biophysical Analysis of the A1 Phylloquinone Binding Site of Photosystem I from Green Algae

This project has resulted in the increase in our understanding of how proteins interact with and influence the properties of bound cofactors. This information is important for several reasons, including providing essential information for the re-engineering of biological molecules, such as proteins, for either improved function or entirely new ones. In particular, we have found that a molecule, such as the phylloquinone used in Photosystem I (PS1), can be made a stronger electron donor by placing it in a hydrophobic environment surrounded by negative charges. In addition, the protein is constrained in its interactions with the phylloqinone, in that it must bind the cofactor tightly, but not in such a way that would stabilize the reduced (negatively-charged) version of the molecule. We have used a combination of molecular genetics, in order to make specific mutations in the region of the phylloquinone, and an advanced form of spectroscopy capable of monitoring the transfer of electrons within PS1 using living cells as the material. This approach turned out to produce a significant savings in time and supplies, as it allowed us to focus quickly on the mutants that produced interesting effects, without having to go through laborious purification of the affected proteins.more » We followed up selected mutants using other spectroscopic techniques in order to gain more specialized information. In addition to the main project funded by this work, this grant supported several related side-projects that also increased our understanding about related issues.« less