Sayeh Agah

Estimation of parvalbumin Ca2+- and Mg2+-binding constants by global least-squares analysis of isothermal titration calorimetry data

The use of competitive isothermal titration calorimetry (ITC) to measure high-affinity binding constants has been largely restricted to systems with a single binding site or multiple identical sites. This study demonstrates the extension of this approach to proteins with two nonequivalent EF-hand Ca(2+)-binding sites--rat beta parvalbumin and the S55D/E59D variant of rat alpha parvalbumin. The method involves simultaneous (global) least-squares analysis of titrations with Ca(2+), with Mg(2+), with Ca(2+) in the presence of Mg(2+), and with Ca(2+) or Mg(2+) in the presence of a competitive chelator (EDTA or EGTA). The Ca(2+) and Mg(2+) binding constants obtained for rat beta agree well with estimates obtained by flow dialysis. Although the Ca(2+) affinity of alpha S55D/E59D is too high to measure by flow dialysis, it was amenable to analysis using the ITC-based approach. The combined S55D and E59D mutations increase the Ca(2+) and Mg(2+) affinities of the mutated binding site by factors of 14 and 26, respectively. This behavior is consistent with that seen previously for the rat beta S55D variant.

Crystal structure of rat α-parvalbumin at 1.05 Å resolution

The crystal structure of rat α‐parvalbumin has been determined at 1.05 Å resolution, using synchrotron data collected at Advanced Photon Source beamline 19‐ID. After refinement with SHELX, employing anisotropic displacement parameters and riding hydrogen atoms, R = 0.132 and Rfree = 0.162. The average coordinate estimated standard deviations are 0.021 Å and 0.038 Å for backbone atoms and side‐chain atoms, respectively. Besides providing a more precise view of the α‐isoform than previously available, these data permit comparison with the 0.91 Å structure determined for pike β‐parvalbumin. Visualization of the anisotropic displacement parameters as thermal ellipsoids yields insight into the atomic motion within the Ca2+‐binding sites. The asymmetric unit includes three parvalbumin (PV) molecules. Interestingly, the EF site in one displays uncharacteristic flexibility. The ellipsoids for Asp‐92 are particularly large and non‐spherical, and the shape of the Ca2+ ellipsoid implies significant vibrational motion perpendicular to the plane defined by the four y and z ligands. The relative dearth of crystal‐packing interactions in this site suggests that the heightened flexibility may be the result of diminished intermolecular contacts. The implication is that, by impeding conformational mobility, crystal‐packing forces may cause serious overestimation of EF‐hand rigidity. The high quality of the data permitted 11 residues to be modeled in alternative side‐chain conformations, including the two core residues, Ile‐97 and Leu‐105. The discrete disorder observed for Ile‐97 may have functional ramifications, providing a mechanism for communicating binding status between the CD and EF binding loops and between the PV metal ion‐binding domain and the N‐terminal AB region.

Divalent ion-binding properties of the two avian β-parvalbumins

Birds express three parvalbumins, one α isoform and two β isoforms. The latter are known as avian thymic hormone (ATH) and avian parvalbumin 3. Although both were discovered in thymus tissue, and presumably function in T‐cell maturation, they have been detected in other tissue settings. We have conducted detailed Ca2+‐ and Mg2+‐binding studies on recombinant ATH and the C72S variant of CPV3, employing global analysis of isothermal titration calorimetry data. In Hepes‐buffered saline, ATH binds Ca2+ with apparent microscopic binding constants of 2.4 ± 0.2 × 108 and 1.0 ± 0.1 × 108 M−1. The corresponding values for CPV3‐C72S are substantially lower, 4.5 ± 0.5 × 107 and 2.4 ± 0.2 × 107 M−1, a 1.9‐kcal/mol difference in binding free energy. Thus, the β‐parvalbumin lineage displays a spectrum of Ca2+‐binding affinity, with ATH and the mammalian β isoform at the high‐ and low‐affinity extremes and CPV3 in the middle. Interestingly, despite its decreased Ca2+ affinity, CPV3‐C72S exhibits increased affinity for Mg2+, relative to ATH. Whereas the latter displays Mg2+‐binding constants of 2.2 ± 0.2 × 104 and 1.2 ± 0.1 × 104 M−1, CPV3‐C72S yields values of 5.0 ± 0.8 × 104 and 2.1 ± 0.3 × 104 M−1. Proteins 2006.