Quaternary Structure and Lipid Affinity of PLC Figure 5. Interaction and affinity of CrPLC with phospholipids. (A) Schematic representation of PIP strip indicating the phospholipid present in each dot numbered from 1–16. Nomenclature of different phospholipids spotted on membrane are Lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), phosphatidylinositol (PtdIns), PtdIns(3)P, PtdIns(4)P, PtdIns(5)P, phosphatidylethanolamine (PE), 8-phosphatidylcholine (PC), sphingosine 1-phosphate, PtdIns(3,4)P2, PtdIns(3,5)P2, PtdIns(4,5)P2, PtdIns(3,4,5)P3, phosphatidic acid (PA), phosphatidylserine (PS), blank. (B and C) Lipid overlay using affinity purified CrPLC WT and CrPLC C7A mutant respectively. (D) Bar graph representing the densitometric measurement of binding of CrPLC WT and C7A mutant (E and F) Comparison of lipid binding affinity of the SEC purified monomer and dimer fractions, respectively. (G) Bar graph representing the densitometric measurement of binding of monomer and dimer fractions of SEC purified wild type CrPLC. Results represent the mean 6 standard deviation of three independent experiments. doi:10.1371/journal.pone.0039258.g005 Secondary Structure and Conformation Analysis of CrPLC and its Variants Secondary structure of the CrPLC WT (WT monomer, WT dimer and WT monomer and dimer mixture; 1:1) and C7A mutant were experimentally determined by CD spectroscopy. Three independent far-UV CD spectroscopy experiments were performed. CD spectra of variants of CrPLC WT and C7A mutant between 190 and 260 nm showed strong minimum near 222 nm. CD spectra of CrPLC WT monomer, CrPLC WT dimer and CrPLC C7A mutant were compared. All the proteins were well folded and their secondary structure content including percentage alpha-helicity, turns and random coil content re- mained largely unchanged (Figures 7A and B). Quantification of secondary structure content of CrPLC variants were performed using Raussean et al [31] method, which utilizes experimental data points of the CD spectra to estimate secondary structure, such PLoS ONE | www.plosone.org 7 June 2012 | Volume 7 | Issue 6 | e39258