Thermal denaturation of the core protein of lac repressor

Abstract

The thermal denaturation of the core protein of lac repressor was studied alone and in the presence of the inducer isopropyl beta-D-thiogalactoside (IPTG) and the antiinducer o-nitrophenyl beta-D-fucoside (ONPF) by means of high-sensitivity differential scanning calorimetry. The denaturation that takes place at about 65 degrees C is apparently irreversible; i.e., a rescan of a previously scanned sample of protein solution shows no denaturational endotherm. Despite this irreversibility, the denaturation appeared to follow quantitatively the dictates of equilibrium thermodynamics as embodied in the van't Hoff equation. The results obtained indicate clearly that the tetrameric protein dissociates to monomers during denaturation and that the ligands are not dissociated until denaturation takes place. The enthalpy of denaturation of the protein is 4.57 +/- 0.25 cal g-1 and is independent of temperature. The enthalpies of dissociation of IPTG and ONPF at the denaturation temperature are very large, 37 and 42 kcal (mol of ligand)-1, respectively.

Differential Scanning Calorimetry of the Effects of Ca2+on the Thermal Unfolding ofPseudomonas cepaciaLipase

“…Similar eŠects have been observed for seemingly irreversible denaturations, 12,14,15) and the DSC traces were successfully analyzed by the van't HoŠ equation. The unfolding mechanism of PCL would involve Eq.…”

Domain 4 of the anthrax protective antigen maintains structure and binding to the host receptor CMG2 at low pH

“…Nonetheless, there is empirical evidence from systems studied by Sturtevant that proteins which show irreversibility in the second transition accurately follow thermodynamic behavior in the first heating. [21][22][23][24] Therefore, we present (cautiously) the thermodynamic values for the first heating of domain 4 at pH 8 (12.1 lM) and pH 5 (5 lM), and the data are summarized in Table II.…”

Unfolding of Ervatamin C in the Presence of Organic Solvents: Sequential Transitions of the Protein in the O-state

“…As discussed by Edge et al (1985), calculations for a model system in which protein undergoes a reversible denaturation followed by an irreversible step, show that data for such a system can be analyzed according to the vant Hoff equation and yield parameters close to those originally assigned to the reversible system. Thus, despite the irreversible nature of the thermal denaturation of ervatamin C, it is possible to analyze results in terms of equilibrium thermodynamics, as has been found for other proteins exhibiting irreversible denaturation in DSC experiments (Manly et al, 1985;Edge et al, 1985;Hu et al, 1987;Edge et al, 1988;Brandts et al, 1989). Our differential scanning calorimetric results suggest that during thermal denaturation the domains unfold sequentially which results in two cooperative transitions as the thermogram progresses.…”

“…The propriety of applying equilibrium thermodynamics to apparently irreversible processes has been discussed by Manly et al (1985) in the context of the denaturation of core protein of lac repressor, and by Edge et al (1985) for the ATCase sub unit. As discussed by Edge et al (1985), calculations for a model system in which protein undergoes a reversible denaturation followed by an irreversible step, show that data for such a system can be analyzed according to the vant Hoff equation and yield parameters close to those originally assigned to the reversible system.…”