Effects of site-directed mutations on the folding, assembly, and function of the 33 kDa protein of photosystem II.

The 33-kDa extrinsic protein of photosystem II (PSII), also called the manganese-stabilizing protein (MSP), stabilizes the oxygen-evolving manganese cluster and accelerates oxygen evolution activity. This dissertation reports results from in vitro mutagenesis and reconstitution experiments aimed at relating structural features in spinach MSP to its known functions in spinach PSII. Cross-reconstitution of spinach PSII with recombinant Arabidopsis MSP shows evolutionary conservation of structures required for specific binding interactions between MSP and PSII. Replacement of Cys28 and Cys51 in MSP with Ala demonstrates that, contrary to previous reports, the conserved intramolecular disulfide bridge in MSP is not required for binding or function. In contrast, replacement of only Cys51 with Ala caused a specific binding or functional defect. Substitution of Val235 with Ala caused a cold-sensitive assembly defect and also weakened binding of MSP to one of its two binding sites on PSII. Characterization of this mutant protein thus provides the first evidence that MSP binds to two non-identical sites on PSII. The existence of non-identical MSP binding sites is proposed as a necessary consequence of the pseudo two-fold symmetry of the reaction center core complex. Addition of a Met residue to the N-terminus also caused a cold-sensitive assembly defect; by comparison, the wild-type MSP precursor polypeptide, which has an N-terminal extension of 84 amino acids, assembled efficiently independent of temperature. Neither N-terminal extension affected MSP function in PSII. Finally, deletion of two amino acids from the C-terminus of MSP had no effect on binding or function, but deletion of four amino acids abolished binding. A structural model is proposed for the oxygen-evolving complex of PSII in which symmetrical but non-identical binding sites exist for MSP on CP47 and CP43, two intrinsic PSII proteins. The structure of MSP in solution has been described as "loose and expansive", and it is further proposed here that MSP retains its non-globular structure following assembly.PhDBiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/104913/1/9624570.pdfDescription of 9624570.pdf : Restricted to UM users only