Measuring Lipid Transfer Protein Activity Using Bicelle-Dilution Model Membranes

Yong-Guang Gao, Thi My Le Le, Xiuhong Zhai, Ivan A. Boldyrev, Shrawan K Mishra, Alexander Tischer, Toshihiko Murayama, Atsushi Nishida, Julian G. Molotkovsky, Amer Alam, Rhoderick E Brown

Research output: Contribution to journalArticlepeer-review

1 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)3417-3425
Number of pages9
JournalAnalytical Chemistry
Volume92
Issue number4
DOIs
StatePublished - Feb 18 2020

Bibliographical note

Funding Information:
This work was supported in whole or part by grants from the National Institutes of Health (NHLBI HL125353 and NIGMS GM45928), the Russian Foundation for Basic Research 015-04-07415, and the Hormel Foundation.

Publisher Copyright:
Copyright © 2020 American Chemical Society.

PubMed: MeSH publication types

  • Journal Article
  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

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