Analysis of the heat stability, folding dynamics, and structure of the manganese stabilizing protein of Photosystem II

The Manganese stabilizing protein (MSP), a conserved extrinsic protein found in Photosystem II (PS11) enzyme complexes, is required for the process of water oxidation during photosynthesis. Binding of MSP to PSII is required to maintain the Mn-cluster bound to PSII's active site (the oxygen-evolving complex (OEC)) and for binding of the 23 and 17-kDa extrinsic proteins, whose presence is required to retain the essential cofactors Ca+2 and Cl- in the OEC. Manganese stabilizing protein contains a single, highly conserved disulfide bridge that has no role in MSP's ability to specifically bind to PSII and reconstitute O2 evolution activity in vitro, or its ability to resist heating. The data presented in this thesis show that, without the disulfide bridge, MSP is more disordered and binds non-specifically to PSII. Substitution of the conserved aromatic residues Trp241 and Tyr242 with Phe results in loss of MSP binding to PSII as well as the ability to reconstitute O2 evolution activity; however, both W241F MSP and Y242F MSP possess solution structures estimated to be similar to the wild-type (WT) protein. These are the first MSP mutants to possess intact N-terminal sequences and near-WT solution structures, but which can not bind specifically to PSII and reconstitute O2 evolution activity. Fluorescence excitation and emission spectra of WT and mutant MSPs reveal that Trp emission in WT MSP is quenched by the disulfide bridge and also by tyrosinates in the protein, providing additional support that the disulfide bridge and MSP C-terminus in the WT protein are in close proximity in solution. Manganese stabilizing protein has a highly disordered solution structure and is thermostable, resisting heat-induced aggregation. Data are presented which show that by monitoring changes in MSP solution structure and resistance to heat-induced aggregation as a function of pH, the protein becomes more disordered and also gains resistance to heat as its negative net charge is increased. These properties are proposed to result from the protein's high ratio of charged to hydrophobic residues, which affects its disordered solution structure and ability to dock, fold, and bind simultaneously to multiple partners to form active PSII complexes.