Mutational interference with oligomerization properties of OCP-related apo- and holoproteins studied by analytical ultracentrifugation.

In this study, the oligomerization pattern of apo- and holoforms of the Orange Carotenoid Protein (OCP) was examined under different conditions such as photoactivation state, concentration, and carotenoid embedment using analytical ultracentrifugation. Furthermore, studies were conducted on OCP constructs carrying point mutations of amino acid residues affecting OCP oligomerization. Our findings reveal that the concentration-dependent dimerization of dark-adapted OCP holoprotein from Synechocystis sp. PCC 6803 can be effectively prevented by the R27L mutation in the OCP-NTD. By introducing the E258R mutation (also in conjunction with R27L) into the OCP-CTD, monomeric OCP apoprotein can be obtained. Additionally, the holoprotein of the dark-adapted OCP-R27L/E258R variant was monomeric, and, supported by size-exclusion chromatography experiments, the photoactivated form of the OCP-R27L/E258R variant was monomeric as well. This variant, which does not oligomerize in either photocycle state, returns from the photoactivated to the dark-adapted state at a significantly faster rate than the OCP wild-type and the R27L mutant thereof. These observations also highlight the crucial interdependence between OCP dimerization in both photocycle states, the lifetime of the photoactive state of OCP, and the kinetics of the OCP photocycle.