title: A fast and simple method for probing the interaction of peptides and proteins with lipids and membrane-mimetics using GB1 fusion proteins and NMR spectroscopy.
abstract: The expression of peptides and proteins as fusions to the B1 domain of streptococcal protein G (GB1) is very popular since GB1 often improves the solubility of the target protein and because the first purification step using IgG affinity chromatography is simple and efficient. However, the following protease digest is not always complete or can result in a digest of the target protein. In addition, a further purification step such as RP-HPLC has to be used to get rid of the GB1 tag and undigested fusion protein. Because the protease digest and the following purification step are not only time-consuming but generally also expensive, we tested if GB1 fusion proteins can directly be used for NMR interaction studies using lipids or membrane-mimetics. Based on NMR binding studies using only the GB1 part, this fusion tag does not significantly interact with different membrane-mimetics such as micelles, bicelles, or liposomes. Thus spectral changes observed using GB1-fusion proteins indicate lipid- and membrane interactions of the target protein. The method was initially established to probe membrane interactions of a large number of mutants of the FATC domain of the ser/thr kinase TOR. To demonstrate the usefulness of the approach, we show NMR binding data for the wild type protein and a leucine to alanine mutant.
authors: Department of Chemistry, Biomolecular NMR Spectroscopy, Technische Universität München, Munich, Germany.
authors: Lisa A M
authors: LA
authors: Sommer
authors: Melanie A
authors: MA
authors: Meier
authors: Sonja A
authors: SA
authors: Dames
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry* / Q000378:metabolism; D008567:Membranes, Artificial; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; C034284:IgG Fc-binding protein, Streptococcus; D008563:Membrane Lipids; D008565:Membrane Proteins; D008567:Membranes, Artificial; D010455:Peptides; D011993:Recombinant Fusion Proteins