Titolo della tesi: Comparison of the folding and the binding features of domains in isolation and in a multidomain construct
Two thirds of eukaryotic proteins have evolved as multidomain constructs, however, to date, most of the experimental biophysical studies are based on domains in isolation. Traditionally domains have been considered structural and functional independent units, thus, the folding and the binding features of a single domain are generally assumed to resemble what should be observed for more complex structural architectures. However, as most proteins consist of multiple domains it is important to understand the principles of domain combinations and interactions.
For this reason, the first part of my work aim to assess the differences in the folding pathways of the PDZ1 domain of whirlin protein in isolation and in presence of its natively bounded PDZ2. Thanks to the combination of systematic mutational analysis and kinetic measurements (Φ-value analysis), we could pinpointed those residues that differ the most in the structure of the two constructs throughout the folding. In particular, in the early stages of the folding, the presence of the second PDZ domain seems to affect the frustration pattern of the PDZ1, opening the structure to the possibility of engaging misfolded states.
In the second section of this work we tackled the differences in the binding affinities of CrkL protein and its constituent domains. In particular, using the proteomic phage display technique the systems were screened against two libraries that display peptides from the intrinsically disordered region of the intracellular human and viral proteome (HD2 and RiboVD). With this method we identified known and unknown binders for all the constructs and the associated binding motif. Moreover, we validated the novel interactions through equilibrium binding experiment addressing the variation in terms of dissociation constant for the single domain and full-length protein.
These results, together with folding analysis attempt to shed light on the effects of tethered domain within a multidomain construct. In fact, a more comprehensive description of proteins may not be achieved without merging the information obtained from the folding and the binding, or rather the frustration pattern and the allosteric network of a biological system.