Redox sensitive cysteine residues in calbindin D(28)k are structurally and functionally important

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Redox sensitive cysteine residues in calbindin D(28)k are structurally and functionally important. / Cedervall, Tommy; Berggård, Tord; Borek, V; Thulin, Eva; Linse, Sara; Akerfeldt, KS.

In: Biochemistry, Vol. 44, No. 2, 2005, p. 684-693.

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Cedervall, Tommy ; Berggård, Tord ; Borek, V ; Thulin, Eva ; Linse, Sara ; Akerfeldt, KS. / Redox sensitive cysteine residues in calbindin D(28)k are structurally and functionally important. In: Biochemistry. 2005 ; Vol. 44, No. 2. pp. 684-693.

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TY - JOUR

T1 - Redox sensitive cysteine residues in calbindin D(28)k are structurally and functionally important

AU - Cedervall, Tommy

AU - Berggård, Tord

AU - Borek, V

AU - Thulin, Eva

AU - Linse, Sara

AU - Akerfeldt, KS

PY - 2005

Y1 - 2005

N2 - Human calbindin D-28k is a Ca2+ binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cystein e-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D-28k appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D-28k binds Ca2+ with lower affinity than does the reduced protein.

AB - Human calbindin D-28k is a Ca2+ binding protein that has been implicated in the protection of cells against apoptosis. In this study, the structural and functional significance of the five cysteine residues present in this protein have been investigated through a series of cystein e-to-serine mutations. The mutants were studied under relevant physiological redox potentials in which conformational changes were monitored using ANS binding. Urea-induced denaturations, as monitored by intrinsic tryptophan fluorescence, were also carried out to compare their relative stability. It was shown that the two N-terminal cysteine residues undergo a redox-driven structural change consistent with disulfide bond formation. The other cysteine residues are not by themselves sufficient at inducing structural change, but they accentuate the disulfide-dependent conformational change in a redox-dependent manner. Mass spectrometry data show that the three C-terminal cysteine residues can be modified by glutathione. Furthermore, under oxidizing conditions, the data display additional species consistent with the conversion of cysteine thiols to sulfenic acids and disulfides to disulfide-S-monoxides. The biological function of calbindin D-28k appears to be tied to the redox state of the cysteine residues. The two N-terminal cysteine residues are required for activation of myo-inositol monophosphatase, and enzyme activation is enhanced under conditions in which these residues are oxidized. Last, oxidized calbindin D-28k binds Ca2+ with lower affinity than does the reduced protein.

U2 - 10.1021/bi049232r

DO - 10.1021/bi049232r

M3 - Article

VL - 44

SP - 684

EP - 693

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 2

ER -