Low-affinity signature of the rat beta-parvalbumin CD site. Evidence for remote determinants.
Although rat beta-parvalbumin and chicken parvalbumin 3 (CPV3) are identical at 74 of 108 residues, rat beta exhibits perceptibly lower Ca2+ and Mg2+ affinities. At 25 degrees C, in Hepes-buffered saline, at pH 7.4, the overall deltadeltaG degrees ' values are 2.0 and 3.9 kcal/mol, respectively. These differences primarily reflect the disparate behavior of the CD sites in the two proteins. Their respective binding constants for Ca2+, for example, are 1.5 x 10(6) and 2.4 x 10(7) M-1. The extent to which this differential behavior is dictated by local and remote sequence differences is unknown. To explore this question, we performed mutagenesis on rat beta, substituting the corresponding CPV3 codon for residues 49, 50, 57, 58, 59, and 60. The resulting CD site is identical to CPV3 at 27 of 30 positions. The mutations were introduced in four stages, replacing residues 49 and 50 (yielding beta 49/50), then 57 and 58 (beta 49/50/57/58), then 59 (beta 49/50/57/58/59), and finally 60 (beta 49/50/57/58/59/60). Apoprotein stability was examined by scanning calorimetry and chemical denaturation and divalent ion affinity by titration calorimetry. All four variants exhibit elevated Tm values and are between 0.13 and 0.39 kcal/mol more stable at 25 degrees C. Although all four proteins display heightened divalent ion affinity, the increases are small. The maximal deltadeltaG degrees ' values, observed for 49/50/57/58/59/60, are just -0.56 and -0.96 kcal/mol for Ca2+ and Mg2+, respectively. Evidently, structural features beyond the metal ion-binding motif contribute to the unusual divalent ion-binding behavior associated with the rat beta CD site.
Henzl
Michael T
MT
Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA. henzlm@missouri.edu
Ndubuka
Kelly
K
D000595:Amino Acid Sequence; D000818:Animals; D001059:Apoproteins / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002152:Calorimetry, Differential Scanning; D002413:Cations, Divalent / Q000378:metabolism; D002645:Chickens; D002942:Circular Dichroism; D020832:EF Hand Motifs; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010320:Parvalbumins / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
D001059:Apoproteins; D002413:Cations, Divalent; D010320:Parvalbumins; D002118:Calcium