Abstract
Lipoproteins contain fatty acids at their amino termini through which they are anchored in lipid membranes. Here, we describe the use of alkyne fatty acids and click chemistry to label lipoproteins in bacterial cells. Exogenous fatty acids containing an alkyne group are incorporated in phospholipids and lipoproteins during bacterial growth. Protein precipitation followed by click chemistry renders lipoproteins fluorescent that can be visualized by in-gel fluorography. Alkyne phospholipids that are the acyl donor in the maturation process of lipoproteins can also be isolated from bacterial membranes for further enzymatic and kinetic studies of acyltransferase that use phospholipids as substrate.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Legood S, Boneca IG, Buddelmeijer N (2021) Mode of action of lipoprotein modification enzymes-Novel antibacterial targets. Mol Microbiol 115:356–365
Thiele C, Papan C, Hölper D et al (2012) Tracing fatty acid metabolism by click-chemistry. ACS Chem Biol 7:2004–2011
Hannoush RN (2012) Profiling cellular myristoylation and palmitoylation using omega-alkynyl fatty acids. Methods Mol Biol 800:85–94
Rangan KJ, Yang YY, Charron G, Hang HC (2010) Rapid visualization and large-scale profiling of bacterial lipoproteins with chemical reporters. J Am Chem Soc 132:10628–10629
Nozeret K, Boucharlat A, Agou F, Buddelmeijer N (2019) A sensitive fluorescence-based assay to monitor enzymatic activity of the essential integral membrane protein apolipoprotein N-acyltransferase (Lnt). Sci Rep 9:15978
Skipski VP, Peterson RF, Barclay M (1964) Quantitative analysis of phospholipids by thin-layer chromatography. Biochem J 159:e61146
Nozeret K, Pernin A, Buddelmeijer N (2020) Click-chemistry based Fluorometric assay for apolipoprotein N-acyltransferase from enzyme characterization to high-throughput screening. J Vis Exp. https://doi.org/10.3791/61146
Schagger H (2006) Tricine-SDS-PAGE. Nat Protoc 1:16–22
Holzl G, Dormann P (2021) Thin-layer chromatography. Methods Mol Biol 2295:29–41
Hannoush RN, Sun J (2010) The chemical toolbox for monitoring protein fatty acylation and prenylation. Nat Chem Biol 6:498–506
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Nozeret, K., Buddelmeijer, N. (2024). Components Subcellular Localization: Identification of Lipoproteins Using Alkyne Fatty Acids and Click Chemistry. In: Journet, L., Cascales, E. (eds) Bacterial Secretion Systems . Methods in Molecular Biology, vol 2715. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3445-5_5
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3445-5_5
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3444-8
Online ISBN: 978-1-0716-3445-5
eBook Packages: Springer Protocols