Estimation of parvalbumin Ca(2+)- and Mg(2+)-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.
Henzl
Michael T
MT
Department of Biochemistry, University of Missouri-Columbia, Columbia, MO 65211, USA. henzlm@missouri.edu
Larson
John D
JD
Agah
Sayeh
S
D019943:Amino Acid Substitution; D000818:Animals; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002151:Calorimetry / Q000379:methods; D003198:Computer Simulation; D003956:Dialysis / Q000379:methods; D004492:Edetic Acid / Q000737:chemistry / Q000378:metabolism; D004533:Egtazic Acid / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D016018:Least-Squares Analysis; D008274:Magnesium / Q000737:chemistry* / Q000378:metabolism; D009010:Monte Carlo Method; D010320:Parvalbumins / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D017186:Titrimetry / Q000379:methods
D010320:Parvalbumins; D011994:Recombinant Proteins; D004533:Egtazic Acid; D004492:Edetic Acid; D008274:Magnesium; D002118:Calcium