title: The unfolding of trp aporepressor as a function of pH: evidence for an unfolding intermediate.
abstract: The urea-induced unfolding of trp aporepressor from Escherichia coli has been studied as a function of pH from 2.5 to 12.0 at 25 degrees C. At pH 7 and above, the unfolding transition, as monitored by changes in the fluorescence intensity at 360 nm, shows a single transition. At low pH, the transition again appears to be a single transition. In the range of 3.5-6.0, the transition is biphasic, indicating the existence of a folding intermediate. The transitions have also been studied using circular dichroism and size exclusion chromatography. The data were fitted by a model in which the dimeric protein first unfolds to form structured monomers, followed by the unfolding of the monomers. From fits with this "folded monomers" model, the free energy change for the dimer<-->monomer dissociation becomes less positive as pH is decreased; the free energy change for the unfolding of the monomers is essentially independent of pH. An alternate model is one in which the dimer first undergoes a transition to a partially unfolded dimeric state, with this intermediate then denaturing to unfolded monomers. Both models give adequate fits to the data obtained at a single protein concentration. From a study of the concentration dependence of the urea-induced unfolding at pH 5, the "folded monomers" model is found to be more consistent with the data. Size exclusion chromatography data support the description of the intermediate state, which is the most populated state at low pH in the absence of urea, as being a relatively compact monomer.(ABSTRACT TRUNCATED AT 250 WORDS)
authors.affiliation: Department of Chemistry, University of Mississippi, University 38677.
authors.forename: M R
authors.initials: MR
authors.lastname: Eftink
authors.forename: K J
authors.initials: KJ
authors.lastname: Helton
authors.forename: A
authors.initials: A
authors.lastname: Beavers
authors.forename: G D
authors.initials: GD
authors.lastname: Ramsay
mesh_terms: D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001426:Bacterial Proteins; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008956:Models, Chemical; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D012097:Repressor Proteins / Q000737:chemistry* / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014508:Urea
chemical_list: D001059:Apoproteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; C027190:TRPR protein, E coli; D014508:Urea