Open AccessMeasuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes
Author(s) -
Yong-Guang Gao,
Le Thi My Le,
Xiuhong Zhai,
Иван Болдырев,
Shrawan K. Mishra,
Alexander Tischer,
Toshihiko Murayama,
Atsushi Nishida,
Julian G. Molotkovsky,
Amer Alam,
Rhoderick E. Brown
Publication year - 2020
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.9b05523
Subject(s) - chemistry , vesicle , membrane , förster resonance energy transfer , lipid bilayer , fluorophore , chromatography , bilayer , biophysics , plant lipid transfer proteins , dilution , biochemistry , fluorescence , physics , quantum mechanics , gene , biology , thermodynamics
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.