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data. The continuous lines represent the best fitstothe sum oftwo non-two-

statetransitions;the individual profiles have been displaced inthey-axisfor the sake ofclarity. (Reprinted with permission from Thorolfsson et al., 2002,

10 tu M L-I3hc

Fig. 2.9. Heat capacity versus temperature profiles forthethermal denaturation ofwild-type phenylalanine hydroxylase in the absence and presence oflO mM L-phenylalanine (L-Phe). Open circles and triangles, experimental heat capacity data. The continuous lines represent the best fitstothe sum oftwo non-two-

statetransitions;the individual profiles have been displaced inthey-axisfor the sake ofclarity. (Reprinted with permission from Thorolfsson et al., 2002,

10 tu M L-I3hc

Biochemistry 41: 7573-7585, Copyright (2002), American Chemical Society)

Additional experiments on truncated hPAH forms have revealed that the low-temperature transition corresponds to the denaturation of the regulatory domains, while the high-temperature transition is due to the denaturation of the catalytic domains.

tte thermal denaturation of hPAH was always irreversible (in the absence and in the presence of ligand), as no transition could be detected in reheating runs (not even when the first run had been stopped immediately after the end of the second transition). Furthermore, aggregation was evident in the sample at the end of the experiment. As seen in Sect. 2.4, the analysis of DSC thermograms according to the equilibrium thermodynamics is seriously hampered in such cases of irreversible denaturation. Nevertheless, in a few cases, the analysis of the scan-rate effect on the DSC transitions has supported the equilibrium thermodynamics analysis of irreversible DSC thermograms (Vogl et al. 1997; ttorolfsson et al. 2002). In fact, Fig. 2.10 suggests that at the highest scan-rate kinetic distortions become negligible and the equilibrium thermodynamic analysis is feasible in the case of hPAH. In view of this, the ligand effect on the transition temperatures was studied using the aforementioned binding polynomial formalism. Before describing the results obtained, however, we must note an important result demonstrated, for equilibrium transitions, by Sturtevant and coworkers many years ago (Fukada et al. 1983; Manly et al. 1985): for denaturation processes that involve ligand dissociation, the plot of transition temperature versus ligand concentration does not show a plateau, but the

Scan Rate (deg/min)

Fig. 2.10. Scan-rate effect on the transition temperatures for the two transitions observed in the DSC thermograms for the thermal denaturation of wild-type phenylalanine hydroxylase (see Fig. 2.9). Closed symbols, transition temperatures in the absence of L-Phe. Open symbols, transition temperatures in the presence of 22 mM L-Phe. Note that there appears to be little or no scan-rate effect on the transition temperatures within the range 0.6-1.3 K/min, which suggeststhat, forthose scan rates, kinetic distortions become negligible and the equilibrium thermodynamic analysis is feasible. (Reprinted with permission from Thorolfsson et al., 2002, Biochemistry 41: 7573-7585, Copyright (2002),American Chemical Society)

transition temperature keeps increasing with ligand concentration even at concentrations for which binding sites are essentially fully occupied, tte reason behind this has to do with the contribution to the relevant Gibbs energy change that arises from the increase in ligand translational entropy that takes place upon ligand dissociation, a contribution that is proportional to the logarithm of free-ligand concentration. With this in mind, the analysis of ligand effects on DSC transitions (Fig. 2.11) based upon the general binding polynomial formalism yielded the following relevant results: (1) l-Phe dissociation takes place upon catalytic domain denaturation, consistent with the presence of binding sites in the catalytic domains; (2) there are no binding sites in the regulatory domains and, therefore, the observed effect on the transition temperature is due to the interaction of the regulatory domain with the catalytic domain.

Beyond the application to this particular system, the theoretical approach based on the binding polynomial formalism described by ttorolfsson et al. (2002) provides a general procedure to obtain relevant information regarding ligand-binding processes in complex protein systems.

Fig. 2.11. Effect ofL-Phe concentration on thetransition temperatures forthetwotransitions observed in the DSC thermograms for the thermal denaturation of wild-type phenylalanine hydroxylase (see Fig. 2.9). Note that while the ^ value shows saturation (plateau) at high ligand concentrations, the T2 value does not. (Reprinted with permission fromThorolfsson et al., 2002, Biochemistry 41:7573-7585, Copyright (2002), American Chemical Society)

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