title: Critical evaluation of the two-state model describing the equilibrium unfolding of the PI3K SH3 domain by time-resolved fluorescence resonance energy transfer.
abstract: It appears that equilibrium unfolding transitions of many small proteins can be described as two-state transitions, because the probes commonly used to measure such transitions cannot detect the underlying heterogeneity inherent in protein folding and unfolding reactions. Time-resolved fluorescence or Forster resonance energy transfer (TRFRET) measurements have the potential to uncover such heterogeneity and to test the cooperativity of protein folding reactions. Here, TRFRET measurements have been used to study the equilibrium unfolding of the SH3 domain of PI3 kinase. The single tryptophan residue (W53) was used as the FRET donor, and a covalently attached thionitrobenzoate moiety at either of two sites (C17 and C70) was used as the FRET acceptor. The individual lifetime and amplitude components estimated from fitting the fluorescence decay kinetics to the sum of three or four exponentials were determined over a range of denaturant concentrations. The equilibrium unfolding transitions reported by these components were found to be noncoincident, suggesting the presence of multiple conformations in equilibrium during the course of unfolding. Fluorescence lifetime distributions were also generated by the model-free maximum entropy method of analysis. Different segments of the protein were found to show differences in the expansion of the native state at low denaturant concentrations, suggestive of gradual structural transitions. The unfolded protein was found to swell at increasingly high denaturant concentrations. The evolution of the fluorescence lifetime distributions with increasing denaturant concentration was also found to be incompatible with a two-state equilibrium unfolding model.
authors: National Centre for Biological Sciences, Tata Institute of Fundamental Research , Bangalore 560065, India.
authors: Megha
authors: M
authors: Kishore
authors: G
authors: G
authors: Krishnamoorthy
authors: Jayant B
authors: JB
authors: Udgaonkar
mesh_terms: D020816:Amino Acid Motifs; D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D019869:Phosphatidylinositol 3-Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D058767:Protein Unfolding; D018909:src Homology Domains
chemical_list: D019869:Phosphatidylinositol 3-Kinases