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Deamidation and disulfide bridge formation in human calbindin D 28k with effects on calcium binding
Author(s) -
Vanbelle Christophe,
Halgand Frédéric,
Cedervall Tommy,
Thulin Eva,
Åkerfeldt Karin S.,
Laprévote Olivier,
Linse Sara
Publication year - 2005
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1110/ps.041157705
Subject(s) - deamidation , chemistry , cooperativity , calbindin , calcium binding protein , cysteine , aspartic acid , iodoacetic acid , biochemistry , calcium , stereochemistry , amino acid , enzyme , organic chemistry
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°C or 50°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°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 β‐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.