Protein-lipid interactions

A Perspective by Anne-Claude Gavin and colleagues, published in Nature Reviews Molecular Cell Biology, discusses recently-developed technologies that enable protein-lipid interactions to be studied on a systems level.

We have highlighted some step-by-step protocols (published by Nature Protocols) that complement the technologies and techniques described in this review. On this page, we give a brief description of the highlighted protocols. More protocols can be found by visiting the Nature Protocols homepage or browsing the protocols categories.

• Mass spectrometry of intact membrane protein complexes. Carol Robinson and colleagues developed a mass spectrometry-based method that can be used to determine the stoichiometry of membrane protein complexes as well to perform protein-lipid binding studies. It was based on the finding of Barrera et al. that detergent micelles protect membrane proteins such that they are stable in the high-vacuum conditions of the mass spectrometer and has been used to investigate two rotary ATPases/synthases, the potassium channel KirBac3.1 and the ATP-binding cassette BtuC2D2.
• Purification of affinity-tagged protein and lipid complexes using TAP labels and size-exclusion chromatography with HPTLC. Aqueous phase protein-lipid interactions have a role in cell physiology, including the transport of newly synthesised lipids to the membranes where they are required. Anne-Claude Gavin and colleagues have developed a protocol for the analysis of in vivo–assembled protein-lipid complexes in S. cerevisiae. This protocol was first applied to the study of lipid transfer proteins and led to the the discovery of phosphatidylserine transport by oxysterol-binding proteins. 
• Measuring genetic interactions using RNAi and imaging. Genetic interaction screens are widely used to identify molecular and functional gene networks. Nature Protocols has published a protocol for using systematic siRNA-mediated pairwise gene knockdown combined with high-content imaging and computational analysis to measure genetic interactions at high throughput in human cells. This method was originally published in Nature Methods and has since been used to study cells in Drosophila.
• Atomic force microscopy of supported lipid bilayers. Atomic force microscopy (AFM) is a promising method to study membrane structure in high-throughput and can be used to monitor dynamic structural changes in lipid membranes on interaction with proteins and other molecules. Mingeot-Leclercq et al. developed a protocol for preparing bilayers that are supported on mica such that AFM methods could be used to analyse their interaction with azithromycin and the simian immunodeficiency virus (SIV) tilted peptide.
• Using patterned supported lipid membranes to investigate the role of receptor organization in intercellular signaling. The interplay between spatial organisation and receptor function can be studied by preparing a lipid membrane that contains the corresponding ligand on a surface containing nanopatterned diffusion barriers. These barriers limit the movement of the receptor proteins on the cell surface and the physical organisation and translocation of the receptor can be monitored by a combination of immunofluorescence and single-cell microscopy. Nair et al. have written a protocol based on the methods developed for the analysis of the EphA2 receptor tyrosine kinase by ephrin-A1 ligands and TCR signaling in immunological synapses.