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Multi-protein interaction kinetics by single molecule methods

Periodic Reporting for period 4 - PROSINT (Multi-protein interaction kinetics by single molecule methods)

Reporting period: 2020-10-01 to 2021-09-30

Objective of this project was to obtain a real time picture of how components of a multi-protein complex interact to perform complex regulated tasks, in particular to see how a protein system might be more than the sum of its components.

Proteins have many important functions in our body. For example, they regulate the reproduction of cells or the defense against viruses. However, proteins do not operate on their own, but almost always in combination with other proteins. These protein complexes are not static, but very dynamic, often constantly exchanging their interaction partners. In addition, many proteins work out of equilibrium, e.g. by using the energy of ATP hydrolysis.
The overall objective of this proposal was to gain insights into how several proteins dynamically interact. This is the first step to understand how the right proteins find each other at the right time to perform complex cellular tasks. Therefore, we developed methods to follow the real time kinetics of multi-protein interactions in and out of equilibrium with single-molecule methods.

We have developed multi-color single-molecule FRET to monitor the succession of association and dissociation steps as well as simultaneously large conformational changes within the proteins . We have built a combined single-molecule FRET and Magnetic Tweezers setup to observe at the same time the folding state of proteins. We have also developed a combination of microfluidics for fast mixing and single pair FRET to investigate interactions of low affinity. The performance of the setups has been tested on the example of the heat shock protein Hsp90 system, which consists of cochaperones, clients and nucleotides.

This resulted already in deep insights into the Hsp90 system and several publications. We were able to follow multi-protein dynamics in equilibrium and out of equilibrium on timescales from nanoseconds to hours (see Figure). The studies did already have considerable impact on the understanding of the Hsp90 machinery as well as general principles of multi-component protein systems. We will further push these methods to directly observe and understand cellular processes.
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):
- 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).
- We have developed and published a multi-color single-molecule FRET approach to study protein dynamics and interaction simultaneously in and out of equilibrium.

- We have developed and published a diffusion-independent microfluidic mixing device to investigate the kinetics of transient protein complexes.

- We have developed and published a tool to determine dynamic protein structures from single molecule FRET networks.

- We have developed and published an integrated approach to use neutron spin echo data, x-ray crystal data, MD simulations and self-consistent FRET networks to determine dynamic structures of multi-domain proteins from nanoseconds to seconds.

- 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.

- We have extended single-molecule FRET into living cells.
Summary Figure PROSINT