title: Detection of a hidden folding intermediate of the third domain of PDZ.
abstract: The folding pathway of the third domain of PDZ from the synaptic protein PSD-95 was characterized using kinetic and equilibrium methods by monitoring the fluorescence signal from a Trp residue that is incorporated at a near-surface position. Kinetic folding of this domain showed multiple exponential phases, whereas unfolding showed a single exponential phase. The slow kinetic phases were attributed to isomerization of proline residues, since there are five proline residues in this domain. We found that the logarithms of the rate constants for the fast phase of folding and unfolding are linearly dependent on the concentrations of denaturant. The unfolding free energy derived from these rate constants at zero denaturant was close to the value measured using the equilibrium method, suggesting the absence of detectable sub-millisecond folding intermediates. However, native-state hydrogen exchange experiments detected a partially unfolded intermediate under native conditions. It was further confirmed by a protein engineering study. These data suggest that a hidden intermediate exists after the rate-limiting step in the folding of the third domain of PDZ.
authors.affiliation: Laboratory of Biochemistry, National Cancer Institute, NIH, Building 37, Room 6114E, Bethesda, MD 20892, USA.
authors.forename: Hanqiao
authors.initials: H
authors.lastname: Feng
authors.forename: Ngoc-Diep
authors.initials: ND
authors.lastname: Vu
authors.forename: Yawen
authors.initials: Y
authors.lastname: Bai
mesh_terms: D006859:Hydrogen / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D009419:Nerve Tissue Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010649:Phenylalanine / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D015202:Protein Engineering; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D009419:Nerve Tissue Proteins; C033652:postsynaptic density proteins; D010649:Phenylalanine; D006859:Hydrogen; D014364:Tryptophan