Characterization ofLigand Binding Effects

Equilibrium thermodynamics analysis of DSC thermograms for protein solutions can, in principle, provide a complete energetic description of the protein de-naturation process. Furthermore, information regarding ligand-binding energetics can also be derived from the analysis of ligand effects on DSC profiles. Significant advances in this regard have been reported recently (Rosgen and Hinz 2001; Rossengarth et al. 2001; Luque et al. 2002; ttorolfsson et al. 2002). In particular, a new theoretical approach, based on the binding partition function (binding polynomial), has been proposed to describe the ligand effects on DSC transition temperatures, ttis is actually a powerful tool as it provides a general, model-independent procedure to analyse data for complex systems (multidomain proteins with multiple and potentially interacting binding sites, for instance). In fact, this approach may allow information regarding localization of binding sites to be derived as well as different binding models to be tested, tte potential of this analysis is clearly shown in the case ofhuman phenylalanine hydroxylase (hPAH) (ttorolfsson et al. 2002).

hPAH is a tetrameric enzyme that catalyses the hydroxylation of l-phenylalanine (l-Phe) to l-tyrosine; a dysfunction of this enzyme causes phenylketonuria. Each subunit in hPAH contains an N-terminal regulatory domain, a catalytic domain and an oligomerization domain. Figure 2.9 shows an illustrative example of DSC thermograms of hPAH in the absence and in the presence of l-Phe. Two partially overlapping transitions affected by addition ofligand are apparent.

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