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Multi-protein interaction kinetics by single molecule methods

Periodic Reporting for period 2 - PROSINT (Multi-protein interaction kinetics by single molecule methods)

Reporting period: 2017-10-01 to 2019-03-31

Objective of this proposal is 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.
In living organisms many proteins work in complexes to form multicomponent protein machines and to regulate cellular processes. The function of such multicomponent machines is usually addressed by dividing them into a collection of two state systems at equilibrium. Many molecular machines work in large complexes with multiple states out of equilibrium by utilizing the energy of ATP hydrolysis. In this proposal the real time kinetics of multi-protein interactions in and out of equilibrium will be investigated using single molecule methods.

We have developed multicolour single-molecule FRET to monitor the succession of association and dissociation steps as well as large conformational changes within the proteins simultaneously. 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 performence of the setups has been tested on the example of the heat shock protein Hsp90 system, which consists of co-chaperones, clients and nucleotides.
This resulted alreday 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 sub milliseconds to hours.
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 understanding cellular processes.
So far we already had big achievements in the first four work packages and significant progress in the last work package.

- 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 co-chaperones Aha1 and p23. We also 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. Experiments with the Hsp90 dimer, Cdc37 and a kinase are on their way.

- We quantified the flux of energy for several nucleotide conditions and the cochaperone Aha1, which stimulates the ATPase of Hsp90.

-We have investigated the effect of force on the folding and assembly kinetics of Hsp90 in collaboration with Matthias Rief (Physics, TU Munich, holder of an ERC synergy grant). 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.

- We have managed to deliver labelled Hsp90 dimers into living HeLa cells. Unfortunately, it turned out that the fluorescence background in the cytosol is high, such that we now target Hsp90 to the nucleus with first promising results. Here we teamed up with Ritwick Sawarkar (Biology, MPI-IE, Freiburg), who also just received an ERC consolidator grant.

- We developed three data analysis packages and workflows, they are freely available on our homepage (https://www.singlemolecule.uni-freiburg.de/software):
i) SMACKS is a maximum likelihood approach to extract kinetic rate models from noisy single molecule data.
ii) MDA (Multi Domain Arrangement) is a software tool for arranging dynamic protein structures by FRET networks.
iii) 3D FRET is a three-color single-molecule FRET approach to studying correlated interactions in proteins.
- 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 an integrated approach to use x-ray crystal data, MD simulations and self-consistent FRET networks to determine dynamic structures of mulit-domain proteins.
- 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 know to address basic questions like ergodicity and memory effects in single molecules. Here we teamed up with Carsten Sönnichsen (Physics, University of Mainz), who also held an ERC consolidator grant.

We expect to further extend these methods towards multi-component systems in order to understand their kinetics of association and dissociation as well as the flux of energy in these systems.
We expect to obtain a dynamic structure of a protein complex.
We expect to further extend the timescale of dynamics, which can be determined with single-molecule methods
We expect to extend some of these methods to be applied inside living cells. This will yield new insights into how fundamental mechanisms in aqueous environment can be translated into the cellular environment, where e.g. molecular crowding becomes relevant.