title: Interaction of phospholipase C with liposome: A conformation transition of the enzyme is critical and specific to liposome composition for burst hydrolysis and fusion in concert.
abstract: Phospholipase C (PLC)1is known to help the pathogen B. cereus entry to the host cell and human PLC is over expressed in multiple cancers. Knowledge of dynamic activity of the enzyme PLC while in action on membrane lipids is essential and helpful to drug design and delivery. In view of this, interactions of PLC with liposome of various lipid compositions have been visualized by testing enzyme activity and microenvironments around the intrinsic fluorophores of the enzyme. Overall change of the protein's conformation has been monitored by fluorescence spectroscopy and circular dichroism (CD). Liposome aggregation and fusion were predicted by increase in turbidity and vesicle size. PLC in solution has high fluorescence and exhibit appreciable shift in its emission maxima, upon gradual change in excitation wavelength towards the red edge of the absorption band. REES fluorescence studies indicated that certain Trp fluorophores of inactive PLC are in motionally restricted compact/rigid environments in solution conformation. PLC fluorescence decreased in association with liposome and Trps loosed rigidity where liposome aggregation and fusion occurred. We argue that the structural flexibility is the cause of decrease of fluorescence, mostly to gain optimum conformation for maximum activity of the enzyme PLC. Further studies deciphered that the enzyme PLC undergoes change of conformation when mixed to LUVs prepared with specific lipids. CD data at the far-UV and near-UV regions of PLC in solution are in excellent agreement with the previous reports. CD analyses of PLC with LUVs, showed significant reduction of α-helices, increase of β-sheets; and confirmed dramatic change of orientations of Trps. In case of liposome composed of lipid raft like composition, the enzyme binds very fast, hydrolyze PC with higher rate, exhibit highest structural flexibility and promote vesicle fusion. These data strongly suggest marked differences in conformation transition induced PLC activation and liposome fusion on the lipid composition.
authors: Department of Life Science, National Institute of Technology, Rourkela, Orissa, India. Electronic address: skpatra_99@yahoo.com.
authors: Samir Kumar
authors: SK
authors: Patra
authors: Department of Life Science, National Institute of Technology, Rourkela, Orissa, India.
authors: Dipta
authors: D
authors: Sengupta
authors: Department of Life Science, National Institute of Technology, Rourkela, Orissa, India.
authors: Moonmoon
authors: M
authors: Deb
authors: Department of Life Science, National Institute of Technology, Rourkela, Orissa, India.
authors: Swayamsiddha
authors: S
authors: Kar
authors: Department of Life Science, National Institute of Technology, Rourkela, Orissa, India.
authors: Chahat
authors: C
authors: Kausar
mesh_terms: D002942:Circular Dichroism; D006868:Hydrolysis; D050356:Lipid Metabolism; D008081:Liposomes / Q000737:chemistry* / Q000378:metabolism*; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D012996:Solutions; D013050:Spectrometry, Fluorescence; D010738:Type C Phospholipases / Q000737:chemistry* / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism
chemical_list: D008081:Liposomes; D010713:Phosphatidylcholines; D012996:Solutions; D053835:Unilamellar Liposomes; D010738:Type C Phospholipases
keywords: Fluorescence spectroscopy; Lipid rafts; Liposome; Membrane fusion; Phospholipase C; Protein structure