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Single Molecule Study of Protease Mechano-Specificity

Periodic Reporting for period 1 - MECHANOPROTEASES (Single Molecule Study of Protease Mechano-Specificity)

Reporting period: 2015-04-01 to 2017-03-31

Fundamental mechanisms in biology rely on the association and recognition among proteins. Especially, enzymes associate specifically to their substrate in order to catalyze precise biochemical reactions in vivo. Proteases form a large family of enzymes found in all life kingdoms and are responsible for the cleavage of peptide bonds. Because a vast number of different proteases are prompt to catalyze various maturation or degradation reactions in the cellular environment, it is therefore important for proteases to have a limited range of potential substrates and avoid promiscuous off-target cleavages. We proposed to uncover at a single molecule level the kinetic and thermodynamic details of the specific recognition and digestion of the endoprotease TEV. The objective of this project is to design a new type of experiments in order to monitor the proteolytic reaction at the single molecule level. In our experiments, the mechanical unfolding of the substrate is accelerated by an Atomic Force Spectrometer in order to activate the reaction and measure the rates of the reaction.
1. Development of the single molecule assay.

The first objective was to set up a reliable Single Molecule assay to study the kinetics of a proteolytic reaction. we had to develop different strategies based on the engineering of protein substrates, that can be stretched with an Atomic Force Microscope. Four different approaches have been developed (Figure1).
Finally, with the combination of an engineered octamer substrate and the design of an adequate force protocol, we successfully obtained experimental recordings that could be used to measure the reaction rates.

2. Extensive measurement of kinetics with different conditions

With this optimized assay (Figure 2), I was able to repeat the experiments in different conditions to measure the effect of Tev concentration and force. Both conditions greatly affect the kinetics of cleavage. A total of 3500 traces could be used across more than 100 experiments. The increase of Tev concentration shows a hyperbolic acceleration of the reaction with a plateau at 0.8 s-1, defining a limiting reaction step that corresponds to the catalytic rate, kcat, measured in bulk.
Alongside, the force dependency displays an unexpected two-regime behavior that could not be explained with polymer physics models.

3. Molecular Modelling of protein stretched by force

Single molecule experiments show that the reaction rate is modulated by force and enzyme concentration. In a simple kinetics model, we propose that the observed rate of cleavage is due to two consecutive reaction steps, the complex formation followed by the catalysis.
We elucidated how forces impact the conformation of the pulled chain and the association rate via molecular simulations.
First, we used steered molecular dynamics (SMD) on two different simplistic chains (10-alanines and 100-alanines). However, these 20 ns long SMD simulations were not sufficient to describe the complete conformational space of the chain. In our experiments, this kinetics occurred in a millisecond-second time scale, a time significantly slower than the ns simulations.
For this reason, we have developed a new method based on free energy calculations and that overcomes the limitations of SMD simulations.
This approach combined simulations with Adaptive Biased Forces and statistical mechanics to describe the elasticity of proteins with a new molecular description. The resulting model successfully provided the new possibility to estimate how the stretching of proteins can modulate the substrate conformation and the association rate. Remarkably, we observed a close correlation between the modeled and experimental force dependencies.

4. Generalization of the approach

We were able to predict the force dependency for different proteases from a simple analysis of the backbone conformation of the substrate bound to its cognate enzyme.
For instance, the holo xray structure of thrombin suggests an optimal cleavage force in the range of the unfolding force (~170pN). A critical strength of the “proof of concept” assay with the Tev was the ability to mechanically unfold the substrate in a range of force that prevents the reaction. Because of this feature, we cannot expand our assay to broadly study other proteases, however, a collaboration in the lab led to a new study that expands the general concept of our approach.
We concluded that mechanical forces can modulate how a protein recognizes a stretched substrate. Experiments on Tev and DnaJ confirmed the accuracy of our model of protein elasticity and the role of conformational dynamics in the kinetics of the formation of a complex.
Our study also presents a novel approach in quantifying the conformational dynamics of intrinsically disordered proteins, an important question in biology that can hardly be addressed with standard biophysical methods.

Moreover, we propose here that the recognition of consecutive residues in a flexible and dynamic peptide is not only sequence specific but can be mechano-specific. This mechanism is particularly relevant in biological systems where proteins under mechanical perturbation are modified by enzymes or associate to a partner. Our findings can also support the engineering of new nanomaterials for therapeutics or industrial needs.