Sequence analysis of photoaffinity-labelled peptides derived by proteolysis of photosystem-2 reaction centres from thylakoid membranes treated with [14C]azidoatrazine.

Abstract

Photosystem-2 reaction centres were prepared from pea thylakoid membranes that had been photoaffinity labelled with [14C]-azidoatrazine (2-azido-4-ethylamino-6-isopropylamino-s-triazine), a derivative of the herbicide atrazine which binds to the secondary plastoquinone electron-acceptor site of photosystem 2. SDS/PAGE of the 14C-labelled reaction centres followed by fluorography revealed photoaffinity-labelled proteins of apparent molecular masses 30 kDa and 55 kDa, which corresponded to the D1 polypeptide and to an SDS-stable heterodimer of the D1 and D2 polypeptides, respectively. To obtain sequence information on the site of photoaffinity labelling, an 8-kDa photoaffinity-labelled peptide, generated by proteolysis of the reaction-centre material with trypsin, was isolated and purified to apparent homogeneity using reverse-phase and size-exclusion HPLC techniques. The amino terminus of the photoaffinity-labelled peptide was determined to be Leu-Gly-Met-Arg-Pro-Xaa-Ile-Ala-Val-Ala-Tyr by Edman sequencing. This corresponds to the amino terminus of a predicted tryptic peptide of D1 and confirms that azidoatrazine photolabels the D1 polypeptide of photosystem 2 in the region Leu137-Arg225. Chymotrypsin/trypsin digestion of photoaffinity-labelled reaction centres followed by reverse-phase HPLC was used to isolate a smaller photoaffinity-labelled peptide. On Edman sequencing, Ser-Ala were identified as the first two residues and 14C was released on the third cycle, after which further degradation was blocked. The two potential peptide fragments with Ser-Ala at the amino terminus in the region Leu137-Arg225 are Ser148-Ala-Pro and Ser212-Ala-Met. Proline is an unlikely target for reaction with the nitrene of the photoactivated azidoatrazine, and the data are thus consistent with Met214 as the site of photoaffinity labelling on D1 when thylakoid membranes are illuminated with ultraviolet irradiation in the presence of [14C]azidoatrazine.