Date of Award

1992

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Terry M. Bricker

Abstract

This work focuses on the production and characterization of crosslinked products obtained from PS II preparations, with the aim of better understanding the structural relationships between the manganese stabilizing protein (MSP) and the interior antenna protein CPa-1. Two oxygen evolving reaction center (OERC) preparations, OERC complex and OERC core, produced various crosslinked products with the hydrophobic crosslinker dithio-bis-(succinimidyl propionate) (DTSP). Crosslinking experiments with OERC complex demonstrated that a close association of MSP to CPa-1 occurs, as both proteins were found to be present simultaneously in various crosslinked products revealed by one- and two-dimensional electrophoretic and immunoblotting techniques. These crosslinked products did not appear in preparations from which MSP had been removed. These results confirm published work indicating an association between MSP and CPa-1. To better understand this association, a 100 kDa crosslinked product (100 XL) was characterized by cleavage with CNBr or formic acid (FA), followed by immunoblotting. Two-dimensional diagonal LMW-LDS-PAGE of CNBr-cleaved 100 XL demonstrated that several crosslinks occurred among fragments of MSP and CPa-1. Of these, the clearest example was a crosslink between the large CNBr fragment of MSP and the large CNBr fragment of CPa-1. Consistent with this, diagonal electrophoresis of FA-cleaved 100 XL established that a crosslink existed between the small FA fragment of MSP and an $\approx$9 kDa fragment of CPa-1. Sequencing of a band containing these two crosslinked FA fragments demonstrated that they corresponded to the 9.6 kDa carboxyl FA fragment of MSP, and to the 8.0 kDa FA fragment of CPa-1. Within these fragments, the lysine-containing segments only extend from K$\sp{159}$ to K$\sp{236}$ for MSP, and from K$\sp{418}$ to K$\sp{438}$ for CPa-1. The procedures established here should permit the characterization of other crosslinks shown to exist in the OERC complex. Thus, this work lays the groundwork for further studies of the physical interactions among the OERC complex components.

Pages

176

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