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Deamidation and disulfide bridge formation in human calbindin D-2gk with effects on calcium binding

Vanbelle, Christophe LU ; Halgand, F; Cedervall, Tommy LU ; Thulin, Eva LU ; Akerfeldt, K S; Laprevote, O and Linse, Sara LU (2005) In Protein Science 14(4). p.968-979
Abstract
Calbindin D-28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ 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 Ca2+. 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 Ca2+-free calbindin. Deamidation is slower for the Ca2+-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp... (More)
Calbindin D-28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ 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 Ca2+. 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 Ca2+-free calbindin. Deamidation is slower for the Ca2+-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca2+ 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 Ca2+ binding, i.e., the Ca2+-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 Ca2+ ions with high affinity in a parallel manner and with positive cooperativity. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Protein Science
volume
14
issue
4
pages
968 - 979
publisher
The Protein Society
external identifiers
  • pmid:15741335
  • wos:000227738900015
  • scopus:15244343344
ISSN
1469-896X
DOI
10.1110/ps.041157705
language
English
LU publication?
yes
id
e4ea9c45-6865-412d-bad5-f3f0dfae5c0b (old id 151535)
date added to LUP
2007-06-29 15:19:04
date last changed
2017-01-01 05:18:53
@article{e4ea9c45-6865-412d-bad5-f3f0dfae5c0b,
  abstract     = {Calbindin D-28k (calbindin) is a cytoplasmic protein expressed in the central nervous system, which is implied in Ca2+ 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 Ca2+. 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 Ca2+-free calbindin. Deamidation is slower for the Ca2+-saturated protein. The deamidation process leads to two Asp iso-forms, regular Asp and iso-Asp. The form with regular Asp 203 binds four Ca2+ 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 Ca2+ binding, i.e., the Ca2+-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 Ca2+ ions with high affinity in a parallel manner and with positive cooperativity.},
  author       = {Vanbelle, Christophe and Halgand, F and Cedervall, Tommy and Thulin, Eva and Akerfeldt, K S and Laprevote, O and Linse, Sara},
  issn         = {1469-896X},
  language     = {eng},
  number       = {4},
  pages        = {968--979},
  publisher    = {The Protein Society},
  series       = {Protein Science},
  title        = {Deamidation and disulfide bridge formation in human calbindin D-2gk with effects on calcium binding},
  url          = {http://dx.doi.org/10.1110/ps.041157705},
  volume       = {14},
  year         = {2005},
}