Developing a new approach to characterize the self-insertion and - folding mechanisms of single membrane proteins into lipid bilayers
Zusammenfassung der Projektergebnisse
The ambitious research project proceeded much slower and different than anticipated. Because refolding experiments of helical transmembrane proteins did not proceed fast enough we decided to additionally unfold and refold beta-barrel outer membrane proteins OmpA, OmpG and FhuA. Being unfolded the first time by SMFS we could observe a new mechanism of how beta-barrel proteins unfold and refold in the lipid membrane. Transmembrane beta-strands predominantly unfold and refold using beta-hairpins until the entire beta-barrel has been unfolded or folded, respectively. In contrast to the much smaller beta-barrel proteins OmpA and OmpG, the much larger beta-barrel of FhuA could not refold and insert into the lipid membrane. We assume that the size and complexity of FhuA requires co-factors that assist the folding and insertion of the FhuA polypeptide into the lipid membrane. Currently we are testing this and other hypotheses. The unfolding and refolding of antiporters (also transporters) progressed slowly so that in the time frame of the project proposal we could only unfold several transporters and investigate how the functional state (e.g., substrate binding) influenced their folding pathways. Refolding of the transporters is ongoing and now, after many years, starting to provide exciting insights into how lipids and co-factors support protein folding and misfolding. In summary the experimental insights gained in this project contribute to the understanding how membrane proteins can refold in lipid membranes. However, we are at the very beginning of this understanding, which requires more time and effort than originally anticipated.
Projektbezogene Publikationen (Auswahl)
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‘One β-hairpin after the other: Exploring mechanical unfolding pathways of the transmembrane βbarrel protein OmpG’. Angewandte Chemie International Edition (2009) 48, 8306-8308
K.T. Sapra, M. Damaghi, S. Köster, Ö. Yildiz, W. Kühlbrandt & D.J. Müller
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‘High-resolution atomic force microscopy and spectroscopy of native membrane proteins’. Reports on Progress in Physics (2011) 74, 086601
Ch. Bippes & D.J. Muller
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‘Locating an extracellular K+-dependent interaction site that modulates betaine-binding of the Na+ -coupled betaine symporter BetP’. Proc. Natl. Acad. Sci. USA (2011) 108, E890-898
L. Ge, C. Perez, I. Waclavska, C. Ziegler & D.J. Muller
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‘One β-hairpin after the other: Exploring refolding pathways and kinetics of the transmembrane βbarrel protein OmpG’. Angewandte Chemie International Edition (2011) 50, 7422-7424
M. Damaghi, S. Koester, C.A. Bippes, Ö. Yildiz & D.J. Muller
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‘Cholesterol increases kinetic, energetic and mechanical stability of the human β2 adrenergic receptor’. Proc. Natl. Acad. Sci. USA (2012) 109, E3463-3473
M. Zocher, C. Zhang, G.F.S. Rassmussen, B.K. Kobilka & D.J. Muller
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‘KpOmpA a transmembrane protein anchoring the outer membrane of Klebsiella pneumoniae unfolds and refolds in response to tensile load’. Structure (2012) 20, 121-127
P. Bosshart, I. Iordanov, C. Garzon-Coral, P. Demange, A. Engel, A. Milon & D.J. Muller
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‘Ligand-specific interactions modulate kinetic, energetic and mechanical properties of the human β2 adrenergic receptor’. Structure (2012) 20, 1391-1402
M. Zocher, J.J. Fung, B.K. Kobilka & D.J. Muller
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‘Out but not in: β-strands shape the unfolding pathway but not the refolding of the large transmembrane β-barrel protein FhuA’. Structure (2012) 20, 2185-2190
J. Thoma, P. Bosshart, M. Pfreundschuh & D.J. Muller
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‘Peptide transporter DtpA populates two alternate conformations, one of which is promoted by inhibitor binding’. Proc. Natl. Acad. Sci. USA (2013) 110, E3978-3986
C. Bippes, L. Ge, M. Meury, D. Harder, Z. Ucurum, H. Daniel, D. Fotiadis & D.J. Muller
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‘Substrate-induced changes in the structural properties of LacY’. Proc. Natl. Acad. Sci. USA (2014) 111, E1571-E1580
T. Serdiuk, M.G. Madej, J. Sugihara, S. Kawamura, S. Mari, H.R. Kaback & D.J. Muller
