Target of rapamycin FATC domain as a general membrane anchor: The FKBP-12 like domain of FKBP38 as a case study
| Autor: | de Cicco, M., Milroy, L.G., Dames, S.A. |
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| Rok vydání: | 2017 |
| Předmět: |
Models
Molecular Saccharomyces cerevisiae Proteins Methods and Applications Protein Conformation Circular Dichroism Recombinant Fusion Proteins Cell Membrane Saccharomyces cerevisiae Molecular Dynamics Simulation Tacrolimus Binding Proteins Phosphatidylinositol 3-Kinases Protein Domains Liposomes Mutation Protein Membrane Anchoring Membrane Mimetic Protein-membrane Interactions Protein-lipid Interactions Nmr Spectroscopy Humans Nuclear Magnetic Resonance Biomolecular Micelles |
| Zdroj: | Protein Sci. 27, 546-560 (2018) |
| ISSN: | 1469-896X |
| Popis: | Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water-soluble and binds to different membrane mimetics would find broad application. The 33-residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506-binding region of the protein FKBP38 (FKBP38-BD) and used H-1-N-15 NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C-terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6-8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl2 and CaCl2). The high water-solubility of y1fatc enables its use for titration experiments against a membrane-localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C-terminal 17-11 residues of the 33-residue long domain by 1D H-1 NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to N-15-labeled target protein for NMR studies. |
| Databáze: | OpenAIRE |
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