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Biochem Biophys Rep. 2017 May 4;10:318-324. doi: 10.1016/j.bbrep.2017.05.001. eCollection 2017 Jul.

Protocols and pitfalls in obtaining fatty acid-binding proteins for biophysical studies of ligand-protein and protein-protein interactions.

Wang Q1,2,3, Rizk S1,2,3, Bernard C1,3, Lai MP1,2,3, Kam D1,3, Storch J4, Stark RE1,2,3,5,6,7.

Author information

1
Department of Chemistry and Biochemistry, The City College of New York, New York, NY 10031, USA.
2
Ph.D. Program in Biochemistry, The Graduate Center of the City University of New York, New York, NY 10016, USA.
3
CUNY Institute for Macromolecular Assemblies, New York, NY 10031, USA.
4
Department of Nutritional Sciences, School of Environmental and Biological Sciences, Rutgers University, New Brunswick, NJ 08901, USA.
5
Ph.D. Program in Chemistry, The Graduate Center of the City University of New York, New York, NY 10016, USA.
6
Ph.D. Program in Physics, The Graduate Center of the City University of New York, New York, NY 10016, USA.
7
Ph.D. Program in Biology, The Graduate Center of the City University of New York, New York, NY 10016, USA.

Abstract

Adipocyte fatty acid-binding protein (AFABP: FABP4) is a member of the intracellular lipid-binding protein family that is thought to target long-chain fatty acids to nuclear receptors such as peroxisome proliferator-activated receptor gamma (PPARγ), which in turn plays roles in insulin resistance and obesity. A molecular understanding of AFABP function requires robust isolation of the protein in liganded and free forms as well as characterization of its oligomerization state(s) under physiological conditions. We report development of a protocol to optimize the production of members of this protein family in pure form, including removal of their bound lipids by mixing with hydrophobically functionalized hydroxypropyl dextran beads and validation by two-dimensional NMR spectroscopy. The formation of self-associated or covalently bonded protein dimers was evaluated critically using gel filtration chromatography, revealing conditions that promote or prevent formation of disulfide-linked homodimers. The resulting scheme provides a solid foundation for future investigations of AFABP interactions with key ligand and protein partners involved in lipid metabolism.

KEYWORDS:

AFABP, adipose fatty acid-binding protein; Delipidation; Disulfide bond; ESI-MS, Electrospray Ionization Mass Spectrometry; FABP, fatty acid-binding protein; Fatty acid-binding protein; GF, Gel filtration chromatography; HSQC, [1H–15N] heteronuclear single quantum correlation spectroscopy; Homodimer; LCFA, Long-chain fatty acid; Ligand; MALDI-TOF, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry; NMR, Nuclear Magnetic Resonance; NOESY, 2D nuclear Overhauser spectroscopy; PPAR, peroxisome proliferator-activated receptor; Protein; TCEP, tris(2-carboxyethyl)phosphine; TEV, Tobacco Etch Virus; TOCSY, 2D Total correlation spectroscopy

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