The dogma of structure-function relationship in protein biophysics has recently been challenged by the striking discovery of many natively unfolded proteins in the human proteome and the concept of functional disorder is revolutionary for scientific community. The characterization of intrinsically unstructured proteins has been, to date, particulary elusive because of their heterogeneous nature.
The aim of this research project is the study of disorder on some selected model system to exemplify both the local and global disorder.
In this context, the binding induced folding of intrinsically disordered systems will be investigated either in protein-protein or protein-DNA interactions. The systems selected are: i) the PDZ domain family, a class of fully native domains displaying frustrated clusters located in the functional sites; ii) the PH domain, a peculiar class of split proteins, whose folding is triggered by the recognition of two complementary halves; iii) Ets domain of SAP-1, a transcription factor whose disorder and flexibility have been suggested to be critical to scan the DNA superstructure.
These systems will be extensively characterized through protein engineering, kinetic and thermodynamic studies.
This scientific project aims at characterizing the function and dynamics of intrinsically disordered proteins, emphasizing the importance of the disorder-to-order transition in the molecular recognition process. To address this target, a combination of different experimental techniques will be used, in particular engineering, kinetic and thermodynamic studies, in combination with in silico molecular dynamics, in order to obtain both theoretical and experimental parameters associated with the binding induced folding process in different systems. We aim at undertaking a complete phi-value analysis of the binding-induced folding events in the different systems analyzed, in order to obtain a complete description at atomic level, of the binding and folding processes.
THE EXPERIMENTAL METHOD: PHI VALUE ANALYSIS phi-value analysis takes advantage of protein engineering in order to introduce a perturbation in the system: by systematically mutating side chains and assessing their effect on the thermodynamics and kinetics of the system, it is possible to map out interaction patterns in the transition state. Usually conservative deletions are introduced in order to cause a small perturbation and analyzing the effects of each mutation on the kinetics allow to map out detailed interaction patterns in folding intermediates and transition states. phi-value analysis allows to structurally characterize folding transition states and folding intermediates, which may accumulate transiently in time-resolved folding experiments prior to the formation of the native state [1]. Phi-values normally range from 0 to 1: a phi-value close to one indicates that the transition state is perturbed in the same way as the native state upon mutation, that means that the residue that has been mutated is involved in the same interactions of the native state. On the other hand, a phi-value close to zero indicates that the transition state is not energetically perturbed by the mutation, i.e. the residue analysed doesn't form any native contacts. The phi value analysis, originally designed for characterizing transition states and intermediates in protein folding studies is a very powerful method, which can be used also to study protein- ligand interactions. We aim to perform phi-analysis on the selected systems in order to provide atomistic details on the mechanisms of recognition and on the folding upon binding reactions, to try to understand the link between function and disorder in proteins.