Quaternary Structure and Lipid Affinity of PLC Figure 2. Quaternary structures of recombinant CrPLC. (A) Size exclusion chromatogram of recombinant CrPLC. Straight line depicts the calibration of standard molecular weight marker including, thyroglobulin (670 kDa), c-globulin (158 kDa), ovalbumin (44 kDa), myoglobin (17 kDa) and vitamin B12 (1.35 kDa). (B) Size-exclusion profile of CrPLC of various concentrations. Elution profiles of CrPLC corresponding to various concentrations of recombinant CrPLC (5, 10 and 50 mM) were recorded using analytical grade size exclusion chromatography column and shown in different line patterns as mentioned in the inset of figure. (C and D) Size exclusion chromatogram of reloaded monomer and dimer fractions using preparatory grade chromatography column are shown in black, overlaid with chromatogram of CrPLC (dotted line). doi:10.1371/journal.pone.0039258.g002 crosslinking experiments. Chlamydomonas total cell lysate (Cr TCL) was cross-linked and CrPLC was probed using specific antibody. Interestingly, by glutaraldehyde crosslinking of Cr TCL, we were able to detect both CrPLC dimer and monomer forms. The formation of higher oligomer was also observed in in vivo conditions, which could be due to some degree of nonspecific crosslinking (Figure 3B). Thus, our findings suggested that CrPLC exist as dimer in both in vitro and in vivo conditions. Various non- covalent forces and covalent interactions like disulphide bridges are known to play a role in the formation and stabilization of the quaternary structure of the proteins. To explore the primary driving force involved in the stabilization of quaternary structure of CrPLC, effect of reducing and non-reducing conditions were compared simultaneously. In SDS-PAGE, CrPLC migrated predominantly as a dimer of molecular weight 110 kDa under non-reducing conditions. This higher molecular weight band totally disappeared under reducing conditions and CrPLC was detected as a monomer of 55 kDa only (data not shown). Immunoblotting of SEC purified monomer and dimer peak fractions (elution volume 70 and 82 ml respectively) under reducing and non-reducing conditions also showed the presence of dimer under non reducing conditions (Figure 3C). Small amount of higher oligomers were also detected by immunoblotting under non reducing condition (Figure 3C). Moreover, size exclusion chromatography with 5 mM DTT in the elution buffer resulted in elimination of the dimer peak (Figure 3D). To find out the involvement of disulphide bond(s) in native CrPLC, CrPLC was probed in the Chlamydomonas total cell protein by immuno- blotting under both reducing and non-reducing conditions. Similar to that of recombinant CrPLC, under non-reducing conditions, both monomer and dimer were detected, whereas in the presence of DTT, dimer completely disappeared (Figure 3E). Thus, disulphide bonds were found to contribute significantly in CrPLC dimerization in the native protein as well. Cys 7 Plays Key Role in CrPLC Dimerization Disappearance of CrPLC dimer under reducing conditions, both in vitro and in vivo and the presence of eight cysteine residues in CrPLC protein sequence provided strong basis for inferring the involvement of disulphide bridge(s) in CrPLC dimerization. Moreover, bioinformatics analysis also suggested the existence of Cys7 residue in a bonded state. Hence, to investigate cysteine residues involved in the formation of disulphide bond in CrPLC, the most exposed cysteine residues, present at the N-terminal sequence (C7), catalytic domains (C68, C73) as well as C2 domain (C325 and C428) were mutated to alanine. The chromatographic data representing the elution profile of all CrPLC mutants were overlaid with the wild type (Figure 4A). As presented earlier, wild type CrPLC eluted as two major peaks of approximately equal ratio. In case of C7A mutant, the peak of dimer was found to be dramatically reduced and CrPLC C7A eluted as a monomer. SEC profile of C325A and C428A CrPLC were found to be similar to that of wild type protein. C68A and C73A double mutant formed higher oligomers with relatively less dimeric form. Immunoblot- ting of the corresponding elution fractions of CrPLC C7A mutant when compared with that of wild type and other mutants, showed PLoS ONE | www.plosone.org 4 June 2012 | Volume 7 | Issue 6 | e39258