PDZ domains are protein-protein interaction modules involved in dynamic regulation of signalling pathways and scaffolding. They were first observed in synapses of the mammalian nervous system two decades ago and are now regarded to be one of the most common protein domains in eukaryotes, with some 250 distinct PDZ domains in the human genome. They are usually part of multi-domain proteins and can form supramodules either of multiple PDZ domains or with other domains. The PDZ domains selected are: i) the third PDZ domain from PSD-95 (PDZ3) in presence and absence of its C-terminal ¿-helix extension (¿3); ii) the first PDZ domain (PDZ1) of Na+H+ exchange regulatory factor (NHERF); iii) the first and second PDZ domains (PDZ1 and PDZ2) in tandem in a construct of the N-terminal part from whirlin; iv) the third PDZ domain (PDZ3) in a C-terminal proline-rich construct from whirlin. This project aims at describing the folding and binding mechanisms of PDZ domains both in isolation and in the context of multi-domain constructs. In this framework, we will employ site-directed mutagenesis in synergy with biophysical techniques to unravel the molecular details of folding and binding reaction mechanisms.
This project is focused on the understanding of how associated domains in multi-domain proteins influence the overall folding and function of the protein.
THE EXPERIMENTAL METHODS:
(1) 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.
(2) DOUBLE MUTANT CYCLES is an innovative method using thermodynamic cycles to study folding and binding processes of proteins and address quantitatively the energetic connectivity between remote residues. Fersht and co-workers initially developed the double mutant cycle methodology for probing intramolecular interactions [1-3], but the method can also be used to study intermolecular interactions between a protein and its substrate [4-6]. In a double mutant cycle analysis the kinetics of the wild type protein are compared with those of two single mutants at positions A and B and of the corresponding double mutant (both A and B being mutated). In our work, the residue A will belong to the PDZ domain and the residue B to its corresponding ligand peptide. The binding kinetics for each one of these mutants will be characterized for different peptide ligands. The change in binding free energy of the wild type peptide upon mutation of residue A, B, and AB will be quantified and compared. If the effects on thermodynamics of the single mutations do not sum up to the effect of the double mutant, then we can define a coupling energy which represents the free-energy of the interaction and is defined as the difference of the double mutant binding free energy with the two single mutants binding free energies. In that case, the two mutated residues are hence energetically coupled. On the other hand if the effects on thermodynamics of the single mutations sum up to the effect of the double mutant, then the coupling energy equals to zero and the residues are hence not energetically coupled.
1. Fersht et al (1992) J. Mol. Biol. 224:771
2. Carter, P. J., Winter, G., Wilkinson, A. J., and Fersht, A. R. (1984) Cell 38, ¿835¿ 840 ¿
3. Horovitz, A., and Fersht, A. R. (1992) J. Mol. Biol. 224, 733¿740¿
4. Schreiber, G., and Fersht, A. R. (1995) J. Mol. Biol. 248, 478 ¿ 486¿39
5. Schreiber, G., and Fersht, A. R. (1996) Nat. Struct. Biol. 3, 427¿ 431
6. Gianni, S., Haq, S. R., Montemiglio, L. C., Jürgens, M. C., Engström, Å., Chi, C. N., ... & Jemth, P. (2011). Sequence-specific long range networks in PSD-95/discs large/ZO-1 (PDZ) domains tune their binding selectivity. Journal of Biological Chemistry, 286(31), 27167-27175.