Src Homology 2 (SH2) domains are a class of protein domains that present a conserved three-dimensional structure and possess a crucial role in mediating protein-protein interactions. Despite their importance and abundance in the proteome, knowledge about the folding properties of SH2 domain is limited. Here we present an extensive mutational analysis (Φ value analysis) of the folding pathway of the N-SH2 domain of the Src homology region 2 domain-containing phosphatase-2 (SHP2) protein, a 104 residues domain that presents the classical SH2 domain fold (two α-helices flanking a central β-sheet composed of 3-5 antiparallel β-strands), with a fundamental role in mediating the interaction of SHP2 with its substrates and triggering key metabolic pathways in the cell. By analysing folding kinetic data we demonstrated that the folding pathway of N-SH2 presents an obligatory on-pathway intermediate that accumulates during the folding reaction. The production of 24 conservative site-directed variants allowed us to perform a Φ value analysis, by which we could fully characterize the intermediate and the transition state native-like interactions, providing a detailed quantitative analysis of the folding pathway of N-SH2. Results highlight the presence of a hydrophobic nucleus that stabilizes the intermediate, leading to a higher degree of native-like interactions in the transition state. Data are discussed and compared with previous works on SH2 domains.
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