Probing Protein-Chaperone Interactions with Single-Molecule Fluorescence Spectroscopy
| Autor: | Michelle Grandin, Daniel Nettels, Marcus Textor, Benjamin Schuler, Sonja Geister, Dominik Hänni, Frank Hillger |
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| Přispěvatelé: | University of Zurich, Hillger, F |
| Rok vydání: | 2008 |
| Předmět: |
Protein Folding
1503 Catalysis 1600 General Chemistry Fluorescence Polarization Catalysis Protein Interaction Mapping 10019 Department of Biochemistry Fluorescence Resonance Energy Transfer biology Chemistry Proteins Chaperonin 60 General Chemistry Single-molecule FRET Single-molecule experiment GroEL Thiosulfate Sulfurtransferase Molecular machine Förster resonance energy transfer Chaperone (protein) biology.protein Biophysics 570 Life sciences Protein folding Two-dimensional nuclear magnetic resonance spectroscopy Molecular Chaperones |
| Zdroj: | Angewandte Chemie International Edition. 47:6184-6188 |
| ISSN: | 1521-3773 1433-7851 |
| DOI: | 10.1002/anie.200800298 |
| Popis: | Molecular chaperones are an essential part of the cellular machinery that aids protein folding and assembly in vivo. Particularly remarkable are the members of the Hsp60 class, which encapsulate the folding protein in a central, closed cavity; the most well-studied example is the bacterial GroEL/ ES system. Work of the past two decades has resolved many aspects of the processes involved. However, remarkably little is known about the influence of the chaperone on the conformational distributions and folding mechanisms of its substrate proteins. Because of the structural heterogeneity of the nonnative substrate bound to a molecular machine in the 10 Da range, its experimental investigation has been difficult with established ensemble methods. Since singlemolecule spectroscopy, in particular in combination with F.rster resonance energy transfer (FRET), can provide distance and orientational information free of ensemble averaging and allows intramolecular distance dynamics to be observed at equilibrium, it is a promising approach to address such questions. Herein, we show how single molecule FRET can be utilized to investigate the nonnative conformation and dynamics of bovine rhodanese, a classic chaperone substrate protein, upon interaction with GroEL. To obtain a transfer efficiency signature suitable for discriminating native and nonnative conformations, two rhodanese variants with complementary donor and acceptor positions (Figure 1) were investigated. Figure 1c–j shows the transfer efficiency histograms determined from photon bursts originating from individual labeled rhodanese molecules freely diffusing through the observation volume of the |
| Databáze: | OpenAIRE |
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