HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) All Versions of this Article: ps.041157705v1 14/4/968    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Vanbelle, C. Articles by Linse, S. Search for Related Content PubMed PubMed Citation Articles by Vanbelle, C. Articles by Linse, S. Social Bookmarking                   What's this?

Deamidation and disulfide bridge formation in human calbindin D_28k with effects on calcium binding

¹ Department of Biophysical Chemistry, Lund University, Chemical Centre, Lund, Sweden
² Institut de Chimie des Substances Naturelles, CNRS, Gif-sur-Yvette, France
³ Department of Chemistry, Haverford College, Haverford, Pennsylvania 19041, USA

(RECEIVED October 4, 2004; FINAL REVISION December 8, 2004; ACCEPTED December 13, 2004)

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°C or 50°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°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 -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.

Keywords: calbindin D_28k; deamidation; disulfide bond; calcium binding; mass spectrometry; post-translational modifications; ICAT

Abbreviations: calbindin, calbindin D_28k • quin 2, 2-[[2-[bis(carboxymethyl)amino]-5-methylphenoxy]methyl]-6-methoxy-8-[bis(carboxymethyl)amino]-quinoline • MS, mass spectrometry • EDTA, Ethylenediaminetetraacetic acid • F12, a fragment comprising residues 1–86 of human calbindin • F34, a fragment comprising residues 87–175 of human calbindin • F56, a fragment comprising residues 176–261 of human calbindin • F123, a fragment comprising residues 1–132 of human calbindin • F456, a fragment comprising residues 133–261 of human calbindin

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041157705.

Reprint requests to: Sara Linse, Department of Biophysical Chemistry, Lund University, Chemical Centre, P.O. Box 124, S-221 00 Lund, Sweden; e-mail: sara.linse{at}bpc.lu.se; fax: +46 46 222 45 43.

⁴ These authors contributed equally to this study.

⁵ Present address: INSERM, IFR62/ANIMET, Lyon, F-69372, France

CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. C. Bauer, H. Nilsson, E. Thulin, B. Frohm, J. Malm, and S. Linse Zn2+ binding to human calbindin D28k and the role of histidine residues Protein Sci., April 1, 2008; 17(4): 760 - 767. [Abstract] [Full Text] [PDF]