Phd in BIOCHEMISTRY

Thesis title: Interdomain communication in protein folding and allostery - the case of Grb2 protein

Proteins have evolved by increasing their structural complexity through the combination of multiple domains. Most proteins are multidomain: two-thirds of all prokaryotic proteins and over 80% of eukaryotic proteins contain more than one domain. Although multidomain proteins constitute the majority of the proteome, biophysical studies on these complex systems have only begun recently. This study presents two examples illustrating how the behavior of a single domain changes within its natural multidomain context. The experimental system used was the adaptor protein Grb2, composed of a central SH2 domain flanked by two SH3 domains. In the first part of the study, the influence of the SH2 domain on the binding activity of the C-SH3 domain was explored using an approach that combines mutagenesis and biophysical measurements, revealing allosteric interactions within the multidomain protein. This study shows that the allosteric effects of Grb2 are subtle and detectable only through extensive analysis, identifying an allosteric site at the SH2–C-SH3 interface, which may represent a potential target for modulating the selectivity of the C-SH3 domain. These findings suggest that Grb2 acts as an active signal integrator rather than a passive adaptor, with its function finely tuned by interdomain allosteric modulation. The second part of the thesis focuses on comparing the folding pathways of full-length Grb2 and the SH2 domain expressed in isolation. While the isolated domain generally folds according to a minimally frustrated energy landscape, in the multidomain context, interdomain interactions can modify the frustration pattern, increasing the likelihood of misfolded states. Although modular organization enhances the functional versatility of proteins, it also introduces greater complexity into the folding process, as domains must contend not only with their intrinsic energy landscapes but also with possible interdomain interactions. Kinetic unfolding and refolding experiments, combined with Φ-value analysis and in silico studies, made it possible to identify the patterns responsible for Grb2 misfolding, providing new insights into the mechanisms of communication and coordination between domains in complex multidomain systems.