Impact of proline residues on parvalbumin stability.
Despite its higher net charge and reduced opportunities for favorable tertiary interactions, Ca(2+)-free rat beta-parvalbumin is more stable than rat alpha-parvalbumin. Under conditions wherein alpha denatures at 45.8 degrees C, beta denatures at 53.6 degrees. The homologous chicken beta isoform known as CPV3 also exhibits heightened stability-prompting an inquiry into the stabilizing influence of Pro-21 and Pro-26. Individual P21A and P26A mutations lower the T(m) of rat beta by 3.2 degrees, decreasing conformational stability by 0.74 kcal/mol. Simultaneous replacement of Pro-21 and Pro-26 essentially abolishes the excess stability (DeltaT(m) = -7.6 degrees; DeltaDeltaG(conf) = -1.77 kcal/mol). Significantly, the P21A/P26A variant displays Ca(2+) affinity virtually indistinguishable from wild-type beta, implying that structural alterations in the AB domain do not necessarily influence the divalent ion affinity of the CD-EF domain. The consequences of introducing proline at positions 21 and 26 in rat alpha were also examined. Whereas the H26P mutation raises the T(m) by 5.6 degrees (DeltaDeltaG(conf) = 1.25 kcal/mol), A21P lowers the T(m) by 8.5 degrees (DeltaDeltaG(conf) = -1.9 kcal/mol). Replacement of Ala-21 by proline in an alpha AB/beta CD-EF chimera increases the T(m) by 5.8 degrees (DeltaDeltaG(conf) = 0.95 kcal/mol), implying that the destabilization of alpha by Pro-21 results from steric conflict with a residue in the CD-EF domain. Consistent with that hypothesis, the K80S mutation markedly stabilizes alpha A21P, yielding a protein with a T(m) 2.0 degrees higher than wild-type alpha. The observed differences in stability resulting from proline addition/removal are largely consistent with alterations in main-chain and side-chain conformational entropy.
Agah
Sayeh
S
Department of Biochemistry, University of Missouri-Columbia, 65211, USA.
Larson
John D
JD
Henzl
Michael T
MT
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D002645:Chickens; D004305:Dose-Response Relationship, Drug; D019277:Entropy; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010320:Parvalbumins / Q000737:chemistry*; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D020033:Protein Isoforms; D017434:Protein Structure, Tertiary; D051381:Rats; D013696:Temperature; D013816:Thermodynamics
D010320:Parvalbumins; D020033:Protein Isoforms; D011392:Proline; D002118:Calcium