Characterization of parvalbumin and polcalcin divalent ion binding by isothermal titration calorimetry.
The elucidation of structure-affinity relationships in EF-hand proteins requires a reliable assay of divalent ion affinity. In principle, isothermal titration calorimetry (ITC) should be capable of furnishing estimates for Ca2+- and Mg2+-binding constants in these systems. And because the method yields the binding enthalpy directly, ITC can provide a more detailed view of binding energetics than methods that rely on 45Ca2+ or fluorescent indicators. For several reasons, however, it is generally not possible to extract reliable binding parameters from single ITC experiments. Ca2+ affinity is often too high, and Mg2+ affinity is invariably too low. Moreover, least-squares minimization of multisite systems may not afford a unique fit because of strong parameter correlations. This chapter outlines a strategy for analyzing two-site systems that overcomes these obstacles. The method--which involves simultaneous, or global, least-squares analysis of direct and competitive ITC data--yields binding parameters for both Ca2+ and Mg2+. Application of the method is demonstrated for two systems. The S55D/E59D variant of rat alpha-parvalbumin, noteworthy for its elevated metal ion affinity, binds divalent ions noncooperatively and is amenable to analysis using an independent two-site model. On the other hand, Phl p 7, a pollen-specific EF-hand protein from timothy grass, binds Ca2+ with positive cooperativity. Divalent ion-binding data for the protein must be analyzed using a two-site Adair model.
Henzl
Michael T
MT
Department of Biochemistry, University of Missouri, Columbia, Missouri, USA.
D000818:Animals; D002021:Buffers; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D002151:Calorimetry / Q000379:methods*; D002614:Chelating Agents; D007477:Ions / Q000737:chemistry / Q000302:isolation & purification; D010320:Parvalbumins / Q000737:chemistry* / Q000378:metabolism*; D011485:Protein Binding; D017186:Titrimetry
D002021:Buffers; D002135:Calcium-Binding Proteins; D002614:Chelating Agents; D007477:Ions; D010320:Parvalbumins