PROLINE IN A TRANSMEMBRANE HELIX COMPENSATES FOR CAVITIES IN THE PHOTOSYNTHETIC REACTION-CENTER

A site-specific double mutant, in which the large aromatic residues M2 08Tyr and L181Phe in the interior of the photosynthetic reaction cente r (RC) complex were replaced by smaller threonine residues, showed a d ramatic reduction in the number of assembled complexes and was incapab le of photosynthetic growth. The cavity created by the smaller side-ch ains was thought to interfere with the stability and/or assembly of th e complex. Phenotypic revertants were recovered in which a spontaneous second-site mutation restored photocompetence in the presence of the original site-specific mutations. In these strains, an Ala --> Pro sub stitution in a neighboring transmembrane helix (at M271) resulted in a n increased yield of RC complexes. To test the hypothesis that the ori ginal phenotype was due to a cavity, other mutants were constructed th at created similar-sized voids at other positions in the membrane-span ning interior. These substitutions caused the same phenotype. Coupling of the above proline substitution to these new cavity mutants also re sulted in photocompetent strains that carry increased levels of RC com plexes. Therefore, the proline substitution at M271 serves as a global suppressor of the phenotype caused by these internal cavities. The pr oline substitution slightly increases the thermal stability of the com plex at higher temperatures, but the mutant and suppressor strains hav e about the same stability at the optimal culture temperature, where b oth are less stable than the wild-type strain. Therefore, the proline substitution may suppress the non-photosynthetic phenotype of cavity m utants by facilitating folding of the nascent polypeptides as they ass emble with cofactors to form the transmembranar RC complex. The prolin e replacement occurs at a pre-existing kink in a transmembrane helix w here it can be accommodated without introducing a strain in the struct ure. The function of proline residues in transmembrane helices might b e to promote folding and/or assembly in general. (C) 1995 Academic Pre ss Limited