Monitoring the Activity of Single Translocons |
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Authors: | Intan Taufik Alexej Kedrov Marten Exterkate Arnold JM Driessen |
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Institution: | Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, and Zernike Institute for Advanced Materials, University of Groningen, Nijenborgh 7, 9747 AG Groningen, The Netherlands |
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Abstract: | Recent studies introduced a novel view that the SecYEG translocon functions as a monomer and interacts with the dimeric SecA ATPase, which fuels the preprotein translocation reaction. Here, we used nanodisc-reconstituted SecYEG to characterize the functional properties of single copies of the translocon. Using a method based on intermolecular Förster resonance energy transfer, we show for the first time that isolated nanodisc-reconstituted SecYEG monomers support preprotein translocation. When several copies of SecYEG were co-reconstituted within a nanodisc, no change in translocation kinetics was observed, suggesting that SecYEG oligomers do not facilitate enhanced translocation. In contrast, nanodisc-reconstituted monomers of the PrlA4 variant of SecYEG showed increased translocation rates. Experiments based on intramolecular Förster resonance energy transfer within the nanodisc-isolated monomeric SecYEG demonstrated a nucleotide-dependent opening of the channel upon interaction with SecA. In conclusion, the nanodisc-reconstituted SecYEG monomers are functional for preprotein translocation and provide a new prospect for single-molecule analysis of dynamic aspects of protein translocation. |
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Keywords: | FRET Fö rster resonance energy transfer FCS fluorescence correlation spectroscopy FCCS fluorescence cross-correlation spectroscopy DDM n-dodecyl β-d-maltoside SEC size-exclusion chromatography |
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