Quaternary Structure and Lipid Affinity of PLC Figure 3. CrPLC dimerizes both in vitro and in vivo. (A) Immunoblotting of crosslinked (Glutaraldehyde; GA) dimer and monomer peak fractions. Numbers above each lane are the elution volume in ml with 70 ml (dimer peak fraction) and 82 ml (monomer peak fraction). (B) Cellular detection of CrPLC dimer and monomer species by glutaraldehyde (GA) crosslinking of Chlamydomonas total cell protein (Cr TCL) followed by immunoblotting. (C) Immunoblot analysis of CrPLC dimer and monomer peak fractions of size exclusion chromatography in both presence (reducing) and absence (non- reducing) of dithiothreitol (DTT). (D) Comparative chromatogram of recombinant CrPLC performed under reducing condition using 5 mM DTT (black) and in non-reducing condition (red). (E) Cellular detection of CrPLC in Cr TCL under reducing and non-reducing conditions. Dimer and Monomer in the blots are marked as D and M respectively. doi:10.1371/journal.pone.0039258.g003 a single band corresponding to monomer (Figure 4B; Figures S5A and B). The elution fractions of other CrPLC mutants including C68/73A, C325A and C428A have not shown considerable effect on dimerization (Figures S5C–E). These findings implied the importance of Cys7 mediated disulphide bond in the dimerization of CrPLC. Cys7 Mutation in CrPLC also Affects Lipid Affinity It has been proposed for few PI-PLC, that dimerization on the membrane surface could affect the affinity for its phospholipid substrate [27]. Similarly, the existence of CrPLC in dimerized form in Chlamydomonas could have implications for its interaction with phospholipid substrates. In order to characterize the functional significance of monomer and dimer species of CrPLC, lipid interaction of affinity purified recombinant CrPLC (wild type and mutants) and SEC purified monomer and dimer fractions were performed by lipid overlay experiments [15,28], respectively. CrPLC WT that possess both monomer and dimer form showed association with a set of phospholipids (PIP) such as PI3P, PI4P, PI5P, PI(3,4)P2, PI(3,5) P2, PI94,5)P2, PI(3,4,5)P2 and PA with varying binding affinities (Figure 5B). Interestingly, CrPLC C7A mutant, which is mainly a monomer showed comparatively higher binding affinity with a set of PIPs when compared with the wild type CrPLC (Figures 5B and C). Since, C7A mutant showed significantly increased affinity with PIPs as compared with that of wild type CrPLC (Figure 5D), binding affinity of the dimer and monomer fractions of the wild type CrPLC were also compared (Figures 5E and F). Wild type CrPLC monomer showed binding with the same set of PIPs but with the much lower affinity (Figures 5E and G). The dimer fraction of WT CrPLC showed poorest lipid binding affinity (Figures 5F and G). These results suggested that the C7A mutant monomeric form potentiate the binding affinity of CrPLC to a set of PIPs. These results have left no doubt that the formation of disulphide bond and/or dimerization poses steric hinderance to some residues and prevents them from interacting with the set of PIPs. These results further implied that the process of dimerization might sterically hinder positively charged amino acid residues from interacting with negatively charged phospholipids head groups and hence the dimerization was found to possess lower lipid affinity and mutation in Cys7 residue which disrupted the dimer, had the highest lipid affinity. PLoS ONE | www.plosone.org 5 June 2012 | Volume 7 | Issue 6 | e39258