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de Cicco, M.* ; Milroy, L.G.* ; Dames, S.A.

Target of rapamycin FATC domain as a general membrane anchor: The FKBP-12 like domain of FKBP38 as a case study.

Protein Sci. 27, 546-560 (2018)
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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.
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Publikationstyp Artikel: Journalartikel
Dokumenttyp Wissenschaftlicher Artikel
Schlagwörter Protein Membrane Anchoring ; Membrane Mimetic ; Protein-membrane Interactions ; Protein-lipid Interactions ; Nmr Spectroscopy; Pik-related Kinases; Mammalian Target; Binding Domain; Subcellular-localization; Conformational-changes; Phosphatidic-acid; Cd Spectroscopy; Lipid-binding; Amino-acids; Nmr
ISSN (print) / ISBN 0961-8368
e-ISSN 1469-896X
Zeitschrift Protein Science
Quellenangaben Band: 27, Heft: 2, Seiten: 546-560 Artikelnummer: , Supplement: ,
Verlag Wiley
Verlagsort Hoboken
Begutachtungsstatus Peer reviewed