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)

The Fungal CPCR1 Protein, Which Binds Specifically to β-Lactam Biosynthesis Genes, Is Related to Human Regulatory Factor X Transcription Factors

“…There are two possibilities to inactivate a dimeric or multimeric protein. A dimer can dissociate in two folded monomers, followed by denaturing the monomers (three-state model), or a dimeric protein undergoes a transition to a partially unfolded state before the dissociation of the monomers (two-state model) (32,33). According to the latter model, no folded monomers can be observed.…”

Aromatic and Cystine Side-Chain Circular Dichroism in Proteins

“…The thermodynamics of binding of specific ligands to trpR has been studied by fluorescence methods Eftink, 1993, 1994). Because of its interesting biological function, its small size, its intertwined quaternary structure, and its dimeric state, there has been considerable interest in elucidating the thermodynamics and kinetics of trpR folding and unfolding (Lane and Jardetzky, 1987;Gittelman and Matthews, 1990;Fernando and Royer, 1992;Eftink et al, 1994). Lane and J ardetzky ( 1987) and emphasized equilibrium measurements of partially unfolded forms, whereas Matthews and co-workers (Gittelman and Matthews, 1990; carried out both equilibrium measurements and kinetic studies on the folding of the dimer.…”

Experimental evolution of a dense cluster of residues in tyrosyl-tRNA synthetase: quantitative effects on activity, stability and dimerization 1 1Edited by A. R. Fersht

“…This prerequisite is well established for dimeric proteins that unfold without intermediate between the native and unfolded states under equilibrium conditions (Neet & Timm, 1994). In contrast, only a handful of papers have reported quantitative analyses of the unfolding equilibria for dimeric proteins that unfold through a dimeric or a monomeric intermediate (Clark et al, 1993;Cheng et al, 1993;Eftink et al, 1994;Grimsley et al, 1997;Park & Bedouelle, 1998). To our knowledge, no detailed study has yet been published on the variations of stability induced by mutations in these types of proteins.…”