Polcalcin divalent ion-binding behavior and thermal stability: comparison of Bet v 4, Bra n 1, and Bra n 2 to Phl p 7.
Polcalcins are pollen-specific proteins containing two EF-hands. Atypically, the C-terminal EF-hand binding loop in Phl p 7 (from timothy grass) harbors five, rather than four, anionic side chains, due to replacement of the consensus serine at -x by aspartate. This arrangement has been shown to heighten parvalbumin Ca(2+) affinity. To determine whether Phl p 7 likewise exhibits anomalous divalent ion affinity, we have also characterized Bra n 1 and Bra n 2 (both from rapeseed) and Bet v 4 (from birch tree). Relative to Phl p 7, they exhibit N-terminal extensions of one, five, and seven residues, respectively. Interestingly, the divalent ion affinity of Phl p 7 is unexceptional. For example, at -17.84 +/- 0.13 kcal mol(-1), the overall standard free energy for Ca(2+) binding falls within the range observed for the other three proteins (-17.30 +/- 0.10 to -18.15 +/- 0.10 kcal mol(-1)). In further contrast to parvalbumin, replacement of the -x aspartate, via the D55S mutation, actually increases the overall Ca(2+) affinity of Phl p 7, to -18.17 +/- 0.13 kcal mol(-1). Ca(2+)-free Phl p 7 exhibits uncharacteristic thermal stability. Whereas wild-type Phl p 7 and the D55S variant denature at 77.3 and 78.0 degrees C, respectively, the other three polcalcins unfold between 56.1 and 57.9 degrees C. This stability reflects a low denaturational heat capacity increment. Phl p 7 and Phl p 7 D55S exhibit DeltaC(p) values of 0.34 and 0.32 kcal mol(-1) K(-1), respectively. The corresponding values for the other three polcalcins range from 0.66 to 0.95 kcal mol(-1) K(-1).
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211, USA. henzlm@missouri.edu
Michael T
MT
Henzl
Meredith E
ME
Davis
Anmin
A
Tan
D000485:Allergens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism; D052179:Antigens, Plant / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D029662:Betula; D029688:Brassica napus; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016384:Consensus Sequence; D007477:Ions / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D055550:Protein Stability
C515594:8-anilino-1-naphthalenesulfonic acid; D000485:Allergens; D000817:Anilino Naphthalenesulfonates; D052179:Antigens, Plant; D002135:Calcium-Binding Proteins; D007477:Ions; C468525:Phl p 7 allergen; D002118:Calcium