Novel methodology for topological studies of photosynthetic membrane protein complexes

Novel techniques have‐been developed for the investigation of photosynthetic membrane protein complexes from the thylakoid membrane of spinach. The protein components of photosystem II were fractionated by a solid phase extraction procedure using Chromabond cartridges. The cytochrome b 6 f complex was dissociated with high concentrations of the nonionic detergent n‐dodecyl‐β‐D‐maltoside. Subsequently the subunits of both membrane protein complexes were separated by high resolution reversed phase HPLC. Nearest neighbour relationships were investigated in the cytochrome b 6 f complex by molecular cross‐linking with o‐phthaldialdehyde. A methodology has been developed to identify the cross‐linking sites on the molecular level by tryptic digestion of the protein conjugates and separation of the fragments by high resolution HPLC. Matrix Assisted Laser Desorption Ionization‐Mass Spectrometry (MALDI‐MS) 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.