The presence of subsaturating levels of a high-affinity ligand has been demonstrated both by experiment and calculation to have far-reaching consequences on thermally induced protein denaturation due to the coupling between the protein denaturation and ligand-binding equilibria. Under such circumstances, a protein may undergo biphasic denaturation even though in the absence of ligand it exhibits a thermogram comprised of a single essentially symmetric endotherm. Up to now, the presence of just 2 maxima in the thermogram has been presented merely as an experimental observation or as the result of equilibrium computations. Here we develop a thermodynamic description of the linkage between these equilibria in which the number of cusps present in the thermogram correlates with the number of resolved steps in the plot of saturation level of remaining native protein vs temperature (i.e., the thermal binding curve). During thermally induced denaturation, the concentration of native protein decreases; thus, the native protein in effect is titrated with ligand at constant total ligand concentration. The free ligand concentration The free ligand concentration increases substantially through the release of bound ligand by unfolding protein thereby increasing the saturation level of the remaining native protein. The form of this thermal binding curve is a function of the number of ligand-binding sites on the protein, the magnitudes of the association constants, and the total ligand and total protein concentrations. As a result, the model predicts multiphasic denaturation of a single cooperative unit when the thermal binding curve consists of discrete multiple steps. The presence of only 2 maxima (i.e., a single cusp) in a thermogram for a protein with multiple sites on the native species derives from the form of the thermal binding curve, which in this case is a single-step sigmoidal plot, and not from the predominant denaturation of unliganded and fully liganded native species. In addition, it is shown that, in general, the contributions from the denaturation of individual native protein species are decidedly non-two-state in character; thus, simple deconvolution should not be carried out. The effects of nonzero values of delta Cp and d delta Cp/dT for denaturation and of changes in enthalpy and in heat capacity for ligand binding, as well as the interaction of ligand with the denatured protein, are explored also.
The thermal stability of 5% previously unheated, undefatted human albumin monomer in 145 mM Na+, pH 7.0 was investigated by differential scanning calorimetry (DSC) as a function of added caprylate and/or N-acetyl-DL-tryptophanate. Caprylate was substantially more effective than N-acetyl-DL-tryptophanate in protecting the protein against thermal denaturation at a given level or at a saturating level of stabilizer. The tracing of the differential heat capacity versus temperature (thermogram) for this undefatted monomer that contained 1.5 mol endogenous, long-chain fatty acid (LCFA)/mol monomer exhibits two denaturation peaks (endotherms) in the absence of stabilizer. The endotherm with the lower denaturation temperature (Td) comprises 70% of the total heat of denaturation and also corresponds to irreversible denaturation and precipitation of 70% of the albumin. This endotherm is associated with more thermally labile protein species containing low levels of LCFA. The endotherm with the higher Td is associated with more stable protein species containing high levels of LCFA. Thus, the two endotherms are not related to the proposed domain structure of the protein but result from an uneven LCFA distribution that is due to preexisting heterogeneity in the albumin and/or heterogeneity that arises during the DSC experiment. Binding data do not support a preexisting uneven distribution of sufficient magnitude to explain the experimental results. A complementary explanation is that an uneven fatty acid distribution arises during the DSC experiment by migration of LCFA from the more labile species to the more stable as the former unfold; such migration would cause further stabilization of the latter.
Glutamine synthetase (GS), Mr 622,000, from Escherichia coli contains 12 active sites formed at heterologous interfaces between subunits [Almassy, R. J., Janson, C. A., Hamlin, R., Xuong, N.-H., & Eisenberg, D. (1986) Nature (London) 323, 304-309]. Temperature-induced changes in UV spectra from 3 to 68 degrees C were reversible with the Mn2+- or Mg2+-enzyme at pH 7.0 (50 degrees C) in 100 mM KCl. No dissociation or aggregation of dodecamer occurred at high temperatures. The thermal transition involves the exposure of approximately 0.7 of the 2 Trp residues/subunit (by UV difference spectroscopy) and 2 of the 17 Tyr residues/subunit (change in exposure from 4.7 to 6.7 Tyr/subunit by second-derivative spectral analysis). Monitoring changes in Trp and Tyr exposure independently gives data that conform to a two-state model for partial unfolding with Tm values (where delta G unfolding = 0) differing by 2-3 degrees C at each level of [Mn2+] studied and with average delta HvH values of 80 and 94 kcal/mol, respectively. These observations suggest that two regions of the oligomeric structure unfold separately as independent transitions (random model). However, the data can be fit equally with a sequential model in which the Trp transition occurs first upon heating. By fitting with either model, Tm values increase from approximately 47 to approximately 54 degrees C with increasing free [Mn2+] from 3.6 to 49 microM but decrease from approximately 54 to approximately 43 degrees C by further increasing free [Mn2+] from 0.05 to 10 mM; such behavior indicates that the high-temperature form of the enzyme binds Mn2+ more weakly but has more binding sites than the native enzyme. The high-temperature Mn-enzyme form is somewhat less unfolded than is the catalytically inactive apoenzyme, which undergoes no further Trp or Tyr exposure on heating and therefore is assumed to be the high-temperature form of divalent cation-free GS. Adding substrates [ADP, L-Met-(SR)-sulfoximine, Gln, Gln + NH2OH, or Gln + ADP] to Mn.GS increased Tm to varying extents by preferential binding to the folded form. Indeed, the transition-state analogue complex GS.(Mn2.ADP.L-Met-(S)-sulfoximine phosphate)12 was stable in the folded form to at least 72 degrees C. Moreover, an Arrhenius plot for gamma-glutamyl transfer activity was linear from 4 to 72 degrees C with Ea = 18.3 kcal/mol.(ABSTRACT TRUNCATED AT 400 WORDS)
Interactions of two substrates (L-glutamine and ADP) and Mn2+ ions with glutamine synthetase from Escherichia coli have been studied by calorimetry and equilibrium dialysis techniques. In addition, the use of calorimetry for establishing separateness of binding sites of different enzyme ligands is considered. The thermodynamic parameters for the sequential and simultaneous binding of L-glutamine and ADP to the unadenylylated Mn-enzyme have been determined. Thermal saturation curves for the binding of L-glutamine to the enzyme in the presence and absence of ADP were obtained at pH 7.1 and 30 °C. With saturating ADP and without ADP present. AG' values for L-glutamine binding are -3.83 and 16, NO.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.