title: Deamidation and disulfide bridge formation in human calbindin D28k with effects on calcium binding.
abstract: Calbindin D(28k) (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca(2+) homeostasis and enzyme regulation. A combination of biochemical methods and mass spectrometry has been used to identify post-translational modifications of human calbindin. The protein was studied at 37 degrees C or 50 degrees C in the presence or absence of Ca(2+). One deamidation site was identified at position 203 (Asn) under all conditions. Kinetic experiments show that deamidation of Asn 203 occurs at a rate of 0.023 h(-1) at 50 degrees C for Ca(2+)-free calbindin. Deamidation is slower for the Ca(2+)-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca(2+) ions with high affinity and positive cooperativity, i.e., in a very similar manner to non-deamidated protein. The form with beta-aspartic acid (or iso-Asp 203) has reduced affinity for two or three sites leading to sequential Ca(2+) binding, i.e., the Ca(2+)-binding properties are significantly perturbed. The status of the cysteine residues was also assessed. Under nonreducing conditions, cysteines 94 and 100 were found both in reduced and oxidized form, in the latter case in an intramolecular disulfide bond. In contrast, cysteines 187, 219, and 257 were not involved in any disulfide bonds. Both the reduced and oxidized forms of the protein bind four Ca(2+) ions with high affinity in a parallel manner and with positive cooperativity.
authors.affiliation: Department of Biophysical Chemistry, Lund University, Chemical Centre, P.O. Box 124, S-221 00 Lund, Sweden.
authors.forename: Christophe
authors.initials: C
authors.lastname: Vanbelle
authors.forename: Frédéric
authors.initials: F
authors.lastname: Halgand
authors.forename: Tommy
authors.initials: T
authors.lastname: Cedervall
authors.forename: Eva
authors.initials: E
authors.lastname: Thulin
authors.forename: Karin S
authors.initials: KS
authors.lastname: Akerfeldt
authors.forename: Olivier
authors.initials: O
authors.lastname: Laprévote
authors.forename: Sara
authors.initials: S
authors.lastname: Linse
mesh_terms: D000478:Alkylation; D000595:Amino Acid Sequence; D001216:Asparagine / Q000737:chemistry; D001224:Aspartic Acid / Q000737:chemistry; D064092:Calbindin 1; D064026:Calbindins; D002118:Calcium / Q000378:metabolism*; D003545:Cysteine / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D064030:S100 Calcium Binding Protein G / Q000737:chemistry* / Q000378:metabolism*
chemical_list: C576498:CALB1 protein, human; D064092:Calbindin 1; D064026:Calbindins; D064030:S100 Calcium Binding Protein G; D001224:Aspartic Acid; D001216:Asparagine; D003545:Cysteine; D002118:Calcium
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