Site directed antisera to the d-2 polypeptide subunit of photosystem ii

Abstract

Three synthetic oligopeptides were used for preparation of antibodies against the D-2 polypeptide of thylakoid membranes. Their sequence was chosen from a model of the folding of the amino acid sequence of the D-2 polypeptide subunit through the membrane that predicted these sequences to be exposed at the membrane surface. For the Merrifield solid-phase method on a fully automated synthesizer the Nα-amino group was protected by a fluorenyl-9-methylcarbonyl group. The oligopeptides were coupled to serum albumin by EDAC for immunizations in rabbits. Antisera with high titer were obtained for the two oligopeptides that contained the amino acid sequence of the D-2 protein from amino acid 230 to 235 and from 235 to 241. The antisera reacted with the D-2 polypeptide, separated on SDS gel and agglutinated the thylakoid membrane. It is known that certain photosystem II functions are impaired by short time trypsin treatment of the membrane. The antisera were used to show that under these conditions the D-2 polypeptide in the membrane is very sensitive. The trypsination yielded two cleavage products detected by the two antisera, a 20 kDa fragment blotted by antiserum against amino acids 230 to 235and a 10 kDa fragment blotted by the antiserum against amino acids 235 to 241. As the polypeptide cleavage occurs between the two epitopes, the trypsin cut is therefore at arginine 234. This supports the prediction that the sequence containing this arginine is the mostexposed part of the D-2 polypeptide on the membrane (matrix) surface. It is proposed that the high sensitivity of the D-2 polypeptide accounts for the known effect of membrane trypsination on QA accessibility in photosystem II. © 1987, Walter de Gruyter. All rights reserved.