title: Unfolding of a small protein proceeds via dry and wet globules and a solvated transition state.
abstract: Dissecting a protein unfolding process into individual steps can provide valuable information on the forces that maintain the integrity of the folded structure. Solvation of the protein core determines stability, but it is not clear when such solvation occurs during unfolding. In this study, far-UV circular dichroism measurements suggest a simplistic two-state view of the unfolding of barstar, but the use of multiple other probes brings out the complexity of the unfolding reaction. Near-UV circular dichroism measurements show that unfolding commences with the loosening of tertiary interactions in a native-like intermediate, N(∗). Fluorescence resonance energy transfer measurements show that N(∗) then expands rapidly but partially to form an early unfolding intermediate IE. Fluorescence spectral measurements indicate that both N(∗) and IE have retained native-like solvent accessibility of the core, suggesting that they are dry molten globules. Dynamic quenching measurements at the single tryptophan buried in the core suggest that the core becomes solvated only later in a late wet molten globule, IL, which precedes the unfolded form. Fluorescence anisotropy decay measurements show that tight packing around the core tryptophan is lost when IL forms. Of importance, the slowest step is unfolding of the wet molten globule and involves a solvated transition state.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, India.
authors.forename: Saswata Sankar
authors.initials: SS
authors.lastname: Sarkar
authors.forename: Jayant B
authors.initials: JB
authors.lastname: Udgaonkar
authors.forename: Guruswamy
authors.initials: G
authors.lastname: Krishnamoorthy
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D058767:Protein Unfolding; D012997:Solvents / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; D012997:Solvents; C057438:barstar protein, Bacillus amyloliquefaciens