Date of Award

1991

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Terry M. Bricker

Abstract

The structural organization of Photosystem II polypeptides has been investigated using the protein crosslinking reagent 1-ethyl-3-(dimethyl-aminopropyl)-carbodiimide (EDC), and a library of mono- and polyclonal antibodies. The first phase of this research was to generate the antibody probes against specific PS II polypeptides. A murine monoclonal antibody, FQC3, was characterized. It recognized the D2 protein of Spinacia oleracea L. The second phase of this research utilized antibodies against D1, D2, CPa-1, CPa-2, Cyt b$\sb{559}$, 33 kDa manganese stabilizing protein (MSP), 24 kDa extrinsic protein, and the 17 kDa extrinsic protein to identify PS II subunits crosslinked by EDC. Several intra-complex crosslinked products were identified. One crosslinked species at 110 kDa (called XL) was identified by two different antibodies and is composed of CPa-1 and the extrinsic 33 kDa MSP. The third phase of this research characterized XL. This was of particular interest considering both CPa-1 and the 33 kDa MSP are required for maximal O$\sb2$-evolving rates. EDC modified PS II membranes evolve normal rates of O$\sb2$. An increase in EDC concentration results in increased retention of the O$\sb2$-evolving rate by CaCl$\sb2$ washed EDC modified PS II membranes. XL (the crosslinked species between CPa-1 and the 33 kDa MSP) was isolated. Chemical and proteolytic cleavage techniques were used to identify peptide fragments of CPa-1 and the 33 kDA MSP involved in crosslinkage. CNBr generated fragments from XL were identified at 50 kDa and 25 kDa. N-terminal sequence analysis of the 50 kDa species indicated that the 15.7 kDa C-terminal CNBr fragment of CPa-1 is unequivocally crosslinked to the 33 kDa MSP. Additional N-terminal sequence analysis of the 25 kDa species strongly suggests that the 15.7 kDa C-terminal CNBr fragment of CPa-1 is crosslinked to the 7 kDa N-terminal CNBr fragment of the 33 kDa MSP. The large extrinsic loop of the apoprotein of CPa-1 thus appears to anchor the extrinsic 33 kDa MSP of the O$\sb2$-evolving complex to the thylakoid membrane through charge pair interactions. By helping elucidate the nature of the non-covalent interactions among PS II proteins, these results provide a more integrated view of the structural and functional organization of PS II polypeptides.

Pages

115

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