Abstract
SH2 domains are protein domains that mediate protein-protein interaction through the recognition and binding of specific sequences containing phosphorylated tyrosines. The p85 protein is the regulatory subunit of the heterodimeric enzyme PI3K, an important enzyme involved in several molecular pathways. In this work we characterize the folding kinetics of the NSH2 domain of p85. Our data clearly reveal peculiar folding kinetics, characterized by an apparent mismatch between the observed folding and unfolding kinetics. Taking advantage of double mixing stopped flow experiments and site directed mutagenesis we demonstrate that such behavior is due to the cis/trans isomerization of the peptide bond between D73 and P74, being in a cis conformation in the native protein. Our data are discussed in comparison with previous works on the folding of other SH2 domains.
Highlights
SH2 domains are conserved domains of about 100 amino acids, usually found in large multidomain proteins in modular architecture with other domains like WW, SH3 and PDZ, that have the biological primary role to recognize and bind specific sequences that contain phosphorylated tyrosine residues
PI3Ks are a family of proteins, composed of three general classes, I II and III, that catalyze the phosphorylation of phosphatidylinositols at their 3′ position
Both CSH2 and NSH2 domains of the p85 subunit interact with p110 when the enzyme is unbound to any substrate, with an inhibitory effect on its catalytic activity
Summary
SH2 domains are conserved domains of about 100 amino acids, usually found in large multidomain proteins in modular architecture with other domains like WW, SH3 and PDZ, that have the biological primary role to recognize and bind specific sequences that contain phosphorylated tyrosine residues. In order to characterize the folding mechanism of the NSH2 domain of p85 we resorted to perform equilibrium and kinetic experiments.
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