During this project we have developed single-molecule FRET based methods from standard two color experiments to up to four color experiments. These allow us to monitor the association and dissociation of multi-protein complexes in real time. In addition, we developed single-molecule FRET to measure the dynamics of proteins in living cells. This is a real breakthrough and opens many new possibilities for investigating dynamic regulation in living cells.
In particular, we have achieved the following:
• We developed a multi-color single-molecule FRET approach to study protein dynamics and interaction simultaneously in and out of equilibrium. This allowed us to determine the association and dissociation kinetics of the Hsp90 dimer with nucleotides and the cochaperones Aha1, p23 and Cdc37. We found that the system is much more dynamic than expected, especially in absence of nucleotides as an energy source.
• We developed a diffusion-independent microfluidic mixing device to investigate the kinetics of transient protein complexes and have already demonstrated its function on the association and dissociation kinetics of the Hsp90 dimer itself. Some three body kinetic networks in the presence and absence of a fourth body have been determined. Our experiments indicate that there is not a strict order on how proteins interact to form a complex. Likely, there are random collisions of the interaction partners until all proteins have the correct conformation to fit together.
• We quantified the flux of energy for several nucleotide conditions and Hsp90 complexes. In general, we find a very weak coupling between the ATPase of Hsp90 and interactions with other proteins.
• We have investigated the effect of force on the folding and assembling of Hsp90 in collaboration with Matthias Rief (TU Munich, holder of an ERC synergy grant) and Katarzyna Tych (University of Groningen). Most interestingly we found that small stretching forces accelerate the folding by preventing the formation of cross-domain misfolding intermediates.
• We developed a new approach based on self-consistent FRET networks to determine the multidomain structure and correlated dynamics of proteins, in particular of Hsp90.
• Together with Carsten Sönnichsen (University of Mainz, ERC consolidator grant), we have developed and published a new method to observe the conformational dynamics of a single protein for 24 hours at video rate. This is an unprecedented bandwidth and allows us to address basic questions like ergodicity and memory effects in single molecules.
• Together with Ritwick Sawarkar (MRC, Cambridge, holder of an ERC consolidator grant) we have managed to inject labelled Hsp90 dimers into living HeLa cells and observe single molecule FRET dynamics. We are now able to track single Hsp90 protein dimers and simultaneously determine the FRET efficiency between two dyes attached to Hsp90 dimers. This breakthrough opens many new possibilities to investigate dynamic regulation in living cells.
• We developed three data analysis packages and workflows, they are freely available on our homepage (
https://www.singlemolecule.uni-freiburg.de/software)(öffnet in neuem Fenster):
- SMACKS is a maximum likelihood approach to extract kinetic rate models from noisy single molecule data.
- MDA (Multi Domain Arrangement) is a software tool for arranging dynamic protein structures by FRET networks.
- 3D FRET is a three-color single-molecule FRET approach to studying correlated interactions in proteins.
• We have disseminated the results in many high impact publications (e.g. PNAS, Nature Methods, Nanoletters, eLife) and several press releases (see:
https://www.singlemolecule.uni-freiburg.de/social-media(öffnet in neuem Fenster)).
