Quaternary Structure and Lipid Affinity of PLC Figure 6. Cellular localization of CrPLC. (A) CrPLC localizes in the plasma membrane of C. reinhardtii cell as shown by green channel (B) Magnified image of a single cell. (C) Plasma membrane traced by using FM4-64 tracker dye is shown in red. (D) Merged image represent the overlay, where green and red channels are merged together to confirm CrPLC localization in the plasma membrane. (E) Cells visualized in DIC mode of light microscopy. (F and G) Immunolocalization with pre-immune serum and auto-fluorescence served as negative control. doi:10.1371/journal.pone.0039258.g006 analysis showed comparable percentage of secondary structure content with that of predicted alpha helical component from the calculated tertiary structure model of CrPLC. Moreover, results obtained suggested that there is no significant change in the alpha helical content of the WT monomer (28.2%), WT dimer (26.9%) and monomer of C7A mutant (28.5%). There was a significant change in the content of beta sheet of the C7A mutant (19.7%) compared to that of WT monomer (22.2%). This difference might account for the minor conformational changes that may impact binding affinity of CrPLC to the set of PIPs. Conclusions Disulphide bridges are known to play important roles in maintaining the structure of proteins and might regulate function of the proteins. This study provides strong evidences for the existence of a novel phospholipase from C. reinhardtii (CrPLC) as both monomer and dimer forms under physiological and in vitro conditions. In both the conditions, the disulphide linkages were found to promote dimerization. Our findings showed the importance of disulphide bridges in CrPLC dimerization. Three single mutants (C7A, C325A and C428A) and one double mutant C68/73A were generated for studying the role of cysteine in oligomerization and lipid interactions. Mutation C7A was mainly found to disrupt the dimerization in CrPLC whereas other mutants like C325A, C428A or C68/73A were not found to affect the CrPLC dimer but leading to the formation of higher oligomers of CrPLC protein. C7A mutation disrupted ,95% of dimerization of CrPLC leading to the increase of monomer fraction of CrPLC by ,2-fold and formation of small amount of higher oligomers. Thus, mutational analysis suggested the pivotal role of Cys7 in CrPLC dimer formation and stabilization. Moreover, substitution of Cys7 to alanine also altered the affinity of CrPLC for a set of phospholipids (substrates). Cys7 mutant potentiates the interaction of CrPLC towards a set of PIPs. However, monomer fraction of CrPLC wild type protein bound weakly and dimer fraction showed the poorest lipid interaction to the same set of PIPs. These experiments left no doubt that Cys7 mutation in CrPLC can potentiate the phospholipid interaction of CrPLC It would be interesting to carry out further studies to delineate the functional importance of the cellular localization of Cr.PLC in Chlamydomonas. Materials and Methods Bioinformatic Analysis Putative CrPLC sequence of C. reinhardtii was fetched from its genome database (http://www.genome.jgi-psf.org/chlamy). Con- served domain architecture of CrPLC was analyzed by CDART program (http://www.ncbi.nlm.nih.gov/Structure/lexington/ lexington.cgi) [32]. PLC sequences from different organisms were retrieved from NCBI and aligned using CLUSTALW multiple alignment tool (http://www.ebi.ac.uk/Tools/clustalw2/index. html ) for homology analysis [33]. Tertiary structure of CrPLC was predicted using homology modeling program (http:// swissmodel.expasy.org/) and pdb files were viewed on PyMol. The predicted structure was verified using SAVES tool (http:// nihserver.mbi.ucla.edu/SAVES/) and submitted to protein model database (PM0077883) [34]. Amino acid residues were located and highlighted in the tertiary structure model using PyMol program. Phylogenetic analysis was performed using MEGA version 5 [35]. Bonded Cysteines were predicted using CysPred server [36] and subcellular localization of CrPLC protein was predicted by Plant-mPLoc web-server [37]. Heterologous Expression and Site Directed Mutagenesis of CrPLC EST encoding CrPLC (accession number BP094257) was procured from Kajusa DNA research institute, Japan. A polynu- cleotide sequence that corresponds to CrPLC was PCR amplified with forward and reverse 59-ATTGAATTCATGGG- CAACGTGTTCAGCT-39 and 59-TTACTC- GAGCTTGGGCCCGGCGTACAT-39 primers respectively us- ing EST as a template. Amplicon was cloned into pET21a (+) vector (Novagen, USA) that possess C-terminus hexa-histidine tag. Point mutations were created using the QuickChange mutagenesis kit (Stratagene, USA) and verified by automated DNA sequencing. CrPLC-pET21a was transformed into BL21 (DE3l) E. coli cells. Cells were grown in terrific broth medium at 37uC and induced PLoS ONE | www.plosone.org 8 June 2012 | Volume 7 | Issue 6 | e39258