Rat alpha- and beta-parvalbumins: comparison of their pentacarboxylate and site-interconversion variants.
Introduction of a fifth carboxylate into the ligand array of the CD site (via the combined S55D and E59D mutations) or the EF site (G98D) of rat alpha-parvalbumin substantially increases divalent ion affinity. This behavior, in conflict with that seen in model peptide systems, agrees with existing data for rat beta-parvalbumin [Henzl et al. (1996) Biochemistry 35, 5856-5869]. The complete analysis of the S55D/E59D double variant necessitated characterization of alpha E59D. Whereas the D59E mutation has minimal influence on beta CD site affinity, E59D has a major impact on the alpha CD site, lowering the apparent association constant by a factor of 14. The thermodynamic consequences of exchanging the rat alpha CD and EF site ligand arrays, which differ at the +z and -x coordination positions, were also examined. When the alpha CD array is imported into the EF site, it acquires a low-affinity phenotype, in agreement with previous findings for beta [Henzl et al. (1998) Biochemistry 37, 9101-9111]. However, when the EF ligand array is introduced into the alpha CD binding loop, it retains a high-affinity signature. This result, contrary to that observed in beta, suggests that the influence of the parvalbumin CD site environment supersedes the intrinsic behavior of the ligand array, a conclusion further supported by the disparate impact of the beta D59E and alpha E59D mutations.
Henzl
Michael T
MT
Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211, USA. henzlm@missouri.edu
Agah
Sayeh
S
Larson
John D
JD
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002264:Carboxylic Acids / Q000737:chemistry*; D008274:Magnesium / Q000378:metabolism; D008969:Molecular Sequence Data; D010320:Parvalbumins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics
D002264:Carboxylic Acids; D010320:Parvalbumins; D020033:Protein Isoforms; D008274:Magnesium; D002118:Calcium