Interaction of CPa-1 with the Manganese-Stabilizing Protein of Photosystem II:Identification of Domains Cross-Linked by 1-Ethyl-3-[ 3-(dimethylamino)propyl]carbodiimide
Abstract
The structural organization of photosystem II proteins has been investigated by use of the zero-length protein cross-linking reagent 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide and monoclonal and polyclonal antibody reagents. Photosystem II membranes were treated with 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide which cross-links amino groups to carboxyl groups which are in van der Waals contact. This treatment did not affect the oxygen evolution rates of these membranes and increased the retention of oxygen evolution after CaCl2 washing. Analysis of the proteins cross-linked by this treatment indicated that two cross-linked species with apparent molecular masses of 95 and 110 kDa were formed which cross-reacted with antibodies against both the 33-kDa manganese-stabilizing protein and the chlorophyll protein CPa-1. Cleavage of the 110-kDa cross-linked species with cyanogen bromide followed by N-terminal sequence analysis was used to identify the peptide fragments of CPa-1 and the manganese-stabilizing protein which were cross-linked. Two cyanogen bromide fragments were identified with apparent molecular masses of 50 and 25 kDa. N-Terminal sequence analysis of the 50-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa fragment of CPa-1 and the intact manganese-stabilizing protein. This strongly suggests that the manganese-stabilizing protein is cross-linked to the large extrinsic loop domain of CPa-1. N-Terminal analysis of the 25-kDa cyanogen bromide fragment indicates that this consists of the C-terminal 16.7-kDa peptide of CPa-1 and the N-terminal 8-kDa peptide of the manganese-stabilizing protein. These results indicate that the domain 364E-440D 0f CPa-1 is cross-linked to the domain 1E-76K of the manganese-stabilizing protein. The large extrinsic loop domain of CPa-1 thus appears to anchor the manganese-stabilizing protein to the photosynthetic membrane via a charge-pair interaction. © 1992, American Chemical Society. All rights reserved.