Enhanced Detection Of Lipid Transfer Protein Activity By Resonance Energy Transfer With Tetramethyl-Bodipy Labeled Lipids

Real-time monitoring of lipid transfer between membranes can be achieved with fluorescence-based approaches. Ideally, the fluorophore probe should have a high extinction coefficient, quantum yield, and photostability. We previously showed the usefulness of 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene-8-yl (Me4-BODIPY-8) fluorophore as a lipid-probe for overcoming deficiencies of other probes [1,2]. Here, we have expanded our evaluation of Me4-BODIPY-8 sphingolipids and glycerolipids by using rhodamine 101-diglyceride (Rh101-DG), rhodamine B-diglyceride (RhB-DG), and C18-tetramethylindocarbocyanine-5,5’-disulfonic acid (C18-diI) as energy acceptors during resonance energy transfer (RET). Each RET system was assessed for sensitivity and accuracy of time-dependent, glycolipid intervesicular transfer by human glycolipid transfer protein (GLTP). Me4-BODIPY-8-PC served as negative control for GLTP specificity. A long linker chain for attaching Me4-BODIPY-8 was essential for maintaining low spontaneous transfer of Me4-BODIPY-8 lipids. Use of Rh-B-DG, Rh-101-DG and C18-DiI also minimized spontaneous transfer compared to RhB-PE and avoided the self-quenching observed when DPH-PC was used as energy donor in the RET system. Our results show that tetramethyl-BODIPY-based energy transfer approaches provide increased sensitivity over previous lipid transfer protein assay systems ([2] and references therein) and show promise for direct monitoring of lipid protein transfer activities within the cellular milieu in the visible light range. [Support: NIH/NIGMS GM45928, Hormel Foundation; Russian Foundation for Basic Research 09-04-00313] [1] Boldyrev, I.A., X. Zhai, M.M. Momsen, H.L. Brockman, R.E. Brown, and J.G. Molotkovsky. 2007. New BODIPY lipid probes for fluorescence studies of membranes. J. Lipid Res. 48: 1518-1532. [2] Mattjus, P., J. G. Molotkovsky, J.M. Smaby, R.E. Brown. 1999. A fluorescence resonance energy transfer approach for monitoring protein-mediated glycolipid transfer between vesicle membranes. Anal. Biochem. 268: 297-304.