Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes

Abstract

In vitro assessment of lipid intermembrane transfer activity by cellular proteins typically involves measurement of either radiolabeled or fluorescently labeled lipid trafficking between vesicle model membranes. Use of bilayer vesicles in lipid transfer assays usually comes with inherent challenges because of complexities associated with the preparation of vesicles and their rather short “shelf life”. Such issues necessitate the laborious task of fresh vesicle preparation to achieve lipid transfer assays of high quality, precision, and reproducibility. To overcome these limitations, we have assessed model membrane generation by bicelle dilution for monitoring the transfer rates and specificity of various BODIPY-labeled sphingolipids by different glycolipid transfer protein (GLTP) superfamily members using a sensitive fluorescence resonance energy transfer approach. Robust, protein-selective sphingolipid transfer is observed using donor and acceptor model membranes generated by dilution of 0.5 q-value mixtures. The sphingolipid transfer rates are comparable to those observed between small bilayer vesicles produced by sonication or ethanol injection. Among the notable advantages of using bicelle-generated model membranes are (i) easy and straightforward preparation by means that avoid lipid fluorophore degradation and (ii) long “shelf life” after production (≥6 days) and resilience to freeze–thaw storage. The bicelle-dilution-based assay is sufficiently robust, sensitive, and stable for application, not only to purified LTPs but also for LTP activity detection in crude cytosolic fractions of cell homogenates.

Boron-containing compounds as labels, drugs, and theranostic agents for diabetes and its complications

“…Reactions of BODIPY with some membranes as well as intracellular and plasma lipids have been reported as biomarkers of use in diabetes[ 28 - 31 ]. Furthermore, a mix of BODIPYs could be used to determine more than one metabolite linked to diabetes evolution.…”

Correction: Structural basis of phosphatidylcholine recognition by the C2-Domain of cytosolic phospholipase A2α

“…At the time of original publication, the model membranes used as energy acceptors in the FRET experiments were described as dansyl-PE/POPC/DHPC (5:45:50) bicelles. Based on more recent experiments performed for a 2020 Analytical Chemistry publication ( Gao et al, 2020 ) and further elaborated in a recent (2021) Bio-protocol article ( Gao et al, 2021 ), we now know that describing the energy acceptor model membranes as dansyl-PE/POPC/DHPC bicelles is incorrect. In the Analytical Chemistry paper, we showed that dilution of POPC-DHPC (50:50) ‘bicelle’ lipid mixes to the low micromolar range, as similarly employed and described in the Methods for the FRET experiments involving the C2-domain and mutants, results in nearly immediate transformation into small POPC unilamellar bilayer vesicles containing little or no DHPC.…”

“…Bicelle -dilution vesicle s were formed by mixing the POPC, dansyl-PE and DHPC in chloroform, drying under a stream of nitrogen and placing under vacuum for ~2 h, before resuspending in buffer (20 mM Tris, pH 7.5, 150 mM NaCl and 50 µM CaCl 2 ). Unilamellar POPC vesicle preparation by POPC/DHPC bicelle mix dilution is detailed in Gao et al, 2020 . Binding reactions included C2–domain (0.5 µM) and various amounts of bicelle -dilution vesicle s (PC conc.…”

“…The new references ( Gao et al, 2020 and Gao et al, 2021 ) that document the formation of small homogeneous unilamellar POPC bilayer vesicles by dilution of POPC/DHPC bicelle mixtures (0.5 q-value) have now been added to the REFERENCES section. They are:…”