Site-directed mutagenesis of a lipid binding site on the intramembrane surface of the bacterial reaction centre

Abstract

X-ray crystallography has shown that the Rhodobacter sphaeroides reaction centre engages in a specific interaction with a molecule of the anionic phospholipid cardiolipin (diphosphatidyl glycerol). The polar head-group of the cardiolipin participates in a number of binding interactions with the protein surface, including direct contacts with the side chains of residues Arg M267 and His M145. To investigate the consequences of disrupting this specific protein-lipid interaction for the structure and functional properties of the reaction centre, residue Arg M267 was mutated to Leu (RM267L), and residue His M145 was mutated to Phe (HM145F). The mutations do not affect the expression level or absorbance properties of the reaction centre, or its ability to support photosynthetic growth. The RM267L mutation does affect the purification characteristics of the reaction centre, and isothermal scanning calorimetry also shows an effect of this mutation on the thermal stability of the complex. The X-ray crystal structure of the RM267L mutant has been determined, and indicates that cardiolipin is not bound to the mutant complex.