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Chaperones mediated mechanical protein folding

Final Report Summary - FORCECHAPERONES (Chaperones mediated mechanical protein folding)

The main goal of the ForceChap Project was to understand the molecular mechanisms by which proteins equilibrate under the effect of a constant stretching force and the effect of molecular chaperones in the mechanical folding of an individual protein. We have used the newly developed single molecule force-clamp spectroscopy technique to elucidate the conformational dynamics of a single refolding protein during its individual folding trajectory from highly extended states and how different chaperones affect this refolding process. This two-year Project has allowed the fellow to increase her knowledge in the protein folding field using a different and novel technology, namely force-clamp spectroscopy. During this time, she has learnt how to use an Atomic Force Microscope and to understand and analyze the obtained data. Now, she is independent in setting up experiments using Atomic Force Microscopes and in the analysis of results. After this period she has improved her capacity to propose new experiments, resolve possible problems and set the foundations of a completely novel research field
In relation to the ForceChaperones project, Dr. Perales-Calvo has studied the mechanical (un)folding of three different substrates (I27, Ubiquitin and Z1) in the absence and presence of molecular chaperones (DnaJ and DnaK individually, and the DnaK system (DnaK, DnaJ and GrpE)). She has observed that DnaJ works as a holdase, preventing the refolding of the substrates. Given the capability of the force-clamp technique to monitor the complete refolding trajectory of an individual protein under force, it is possible to study the conformational dynamics of the protein substrate followed during the individual unfolding and refolding in all the conformations along its (un)folding pathways. The fellow has demonstrated that DnaJ binds preferentially the extended conformation of Ubiquitin and this binding takes place in a force dependent manner. By contrast, DnaJ binds with more affinity the collapsed state of I27. The different behaviour of the same chaperone on two different substrates highlights the importance of a consensus sequence in the binding of the chaperone to different substrates. In the case of DnaK, Dr. Perales-Calvo has demonstrated that this chaperone recognizes with high affinity the collapsed conformation of both Ubiquitin and I27. This is the first time that a technique allows us to discriminate between unfolded and collapsed states regarding chaperone binding. Finally, she has confirmed, at the single molecule level, that the complex of chaperones composing the DnaK system is able to increase the refolding yield of the three different substrates, regardless of their ability to refold on their own. This work is in preparation to be published.
In addition, during these two years, the fellow has developed a parallel project designed to study the mechanical unfolding of a chaperone. She has described that the Hsp40 DnaJ protein follows a sequential mechanical unfolding pathway that does not follow a typical hierarchical unfolding. Also, the DnaJ structure, which contains two zinc finger motifs, has allowed to study in detail the mechanochemistry of this small structural protein domain. Using single molecule force spectroscopy, Dr. Perales-Calvo has demonstrated the mechanical lability (90 pN) of the individual Zn-S bond and she has designed different DnaJ mutants, which have enabled direct identification of the chemical determinants that regulate the interplay between zinc binding and disulfide formation. Finally, she has showed that binding of hydrophobic peptides to the chaperone drastically increases its mechanical stability. The manuscript associated with this work (“The mechanochemistry of a structural Zinc Finger motif”) was submitted at the end of the first year of this Project and it is now published in "The Journal of Physical Chemistry Letters". The paper was selected as editors choice (Judit Perales-Calvo, Ainhoa Lezamiz and Sergi Garcia-Manyes. “The Mechanochemistry of a structural Zinc Finger” (2015). Journal of Physical Chemistry Letters 6: 3335-3340).

Considering that diseases as Alzheimer´s or Parkinson´s disease have their origin when a single molecule undergoes a conformational change and is not able to fold back to its native structure anymore, it is therefore really challenging to discover how this process at the early stages can be prevented. The results obtained during these two years pave the way to better understanding the effect of molecular chaperones in the refolding of proteins and the conformations that these molecular players are able to recognize.
This work, together with the other projects carried out in the lab, can be found in the group website: