NOVEL METHODOLOGY FOR TOPOLOGICAL STUDIES OF PHOTOSYNTHETIC MEMBRANE-PROTEIN COMPLEXES

Novel techniques have been developed for the investigation of photosyn thetic membrane protein complexes from the thylakoid membrane of spina ch. The protein components of photosystem II were fractionated by a so lid phase extraction procedure using Chromabond cartridges. The cytoch rome b(6)f complex was dissociated with high concentrations of the non ionic detergent n-dodecyl-beta-D-maltoside. Subsequently the subunits of both membrane protein complexes were separated by high resolution r eversed phase HPLC. Nearest neighbour relationships were investigated in the cytochrome b(6)f complex by molecular cross-linking with o-phth aldialdehyde. A methodology has been developed to identify the cross-l inking sites on the molecular level by tryptic digestion of the protei n conjugates and separation of the fragments by high resolution HPLC. Matrix Assisted Laser Desorption Ionization-Mass Spectrometry (MALDI-M S) was used as a sensitive tool to identify the protein components and crosslinked peptide fragments of photosystem II and the cytochrome b( 6)f complex.