Fluorescence single-molecule study of cobra phospholipase A2 action on a supported gel-phase lipid bilayer.
Previous nanoscale investigations of the gel-state membrane surface structure under the action of phospholipase A(2) (PLA(2)) suggest that single enzymes at work scoot on the membrane surface from the observed defects, which creates nanosized channels oriented along the lipid crystal-packing structure. To date, however, there have been no reports of direct observation of PLA(2) at the single-molecule level focusing on how the enzymes interact with the defects. Herein, we report a single-molecule fluorescence microscopy study on the action of enzymatically active rhodamine B-labeled cobra PLA(2) on a supported lipid membrane with visible packing defects on a glass substrate. Working with a gel-state phospholipid bilayer, the low-activity period (lag phase) of PLA(2) action is followed by the burst binding of PLA(2) molecules from aqueous solution on a few newly created active sites. These active sites are distinguished by a spatial resolution of approximately 40 nm, which is below the diffraction limit. The rate of active-site propagation as reflected by new PLA(2) binding on the membrane surface is estimated to be approximately 5 nm min(-1). This rate is about two orders of magnitude slower than the propagation rate of hydrolyzed channels estimated by AFM studies on bee venom PLA(2) on a similar membrane surface. This direct observation of PLA(2) molecules allows the visualization of different PLA(2) binding modes on the membrane surface and on the membrane boundary.
Chiu
Chang-Ru
CR
Department of Chemistry and Biochemistry, National Chung Cheng University, Chiayi 621, Taiwan.
Huang
Wei-Ning
WN
Wu
Wen-Guey
WG
Yang
Tzyy-Schiuan
TS
D000818:Animals; D004546:Elapid Venoms / Q000737:chemistry*; D017815:Elapidae / Q000378:metabolism*; D005453:Fluorescence; D005782:Gels / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D008954:Models, Biological; D044367:Phase Transition; D054467:Phospholipases A2 / Q000737:chemistry*; D010743:Phospholipids / Q000737:chemistry*
D004546:Elapid Venoms; D005782:Gels; D008051:Lipid Bilayers; D010743:Phospholipids; D054467:Phospholipases A2