Leader: Stefano Gianni (SAPIENZA); Other collaborator(s):
Older age is generally characterized by the emergence of several complex health states that compromise cell functions. From a molecular perspective, this is often associated with the improper folding, or ‘misfolding’, of specific proteins that escape cell quality control systems and lead to the accumulation of pathogenic aggregates. By taking advantage of the synergy between molecular biology and biophysics, our laboratory will investigate the molecular details of the mechanisms of protein folding, misfolding and recognition as well as the interaction with the quality control systems, with the specific aim to design small molecules to modulate these processes in pathogenic conditions.
Brief description of the activities and of the intermediate results
We have characterized extensively the folding and misfolding pathways of different protein systems, both in thier full-length constructs and expressed as a combination of isolated domain. Using site-directed mutagenesis, we introduced specific amino acid substitutions into the target proteins to perturb their folding landscape. These mutations were strategically chosen based on computational predictions and structural analyses to modulate the stability and kinetics of the folding process. Stopped-flow spectroscopy was then employed to monitor the rapid kinetics of protein folding and misfolding events under physiological conditions. Preliminary results indicate that site-directed mutations profoundly influence the folding kinetics of the protein. Conversely, other mutations introduce kinetic traps or destabilize native contacts, resulting in slower folding kinetics and increased propensity for misfolding. Stopped-flow analysis allows us to capture transient folding intermediates and characterize their structural properties. We observed distinct kinetic phases corresponding to the formation of intermediate species with partially folded conformations. By probing these intermediates using spectroscopic techniques, we gain insights into the structural rearrangements occurring during the folding process. Furthermore, our experiments reveal the occurrence of misfolding events triggered by specific mutations. These misfolded species exhibit altered structural conformations and increased aggregation propensity compared to the native state. The kinetics of misfolding events were elucidated through kinetic modeling, highlighting the role of destabilizing mutations in promoting non-native interactions and aggregate formation. Our findings underscore the intricate relationship between protein sequence, folding kinetics, and misfolding propensity. By leveraging site-directed mutagenesis and stopped-flow analysis, we unravel the complex folding pathways and identify critical intermediates involved in protein folding and misfolding. Future research will focus on refining our understanding of the molecular determinants governing protein stability and designing strategies to mitigate misfolding-associated diseases.
Main policy, industrial and scientific implications
At the moment we do not envisage policy/Industry/practice implications
This approach led to a multiplicity of studies corresponding to several publications in peer-reviewed scientific journals. As an example, we addressed in one of these studies the common assumption that protein folding and unfolding experiments are interpreted as if they were microscopically reversible. Investigators generally use single‐domain proteins to illustrate the validity of such an assumption, although reversibility does not necessarily hold under the different conditions typically used for folding/unfolding. In fact, larger multi-domain proteins that often exhibit irreversible unfolding, are generally considered not amenable to folding kinetics studies. In this sample study, we used the X11 PDZ1‐PDZ2 tandem repeat to reveal the different folding and unfolding pathways at work under different conditions, thus reconciling the apparent contradiction between theory and experiment.