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- Title
Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation.
- Authors
Goch, Graźyna; Vdovenko, Sergiusz; Koz&lslash;owska, Hanna; Bierzyñski, Andrzej
- Abstract
S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of twoα subunits, each containing two calcium-binding loops (N and C). At physiological pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca2+-titration curves of Trp90 fluorescence for both the native protein and its Glu32 → Gln mutant with an inactive N-loop. Using a chelator method, we also determined the calcium-binding constant for the S100A1 Glu73 → Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants ofK1 =4 ± 2 × 103 m−1 (C-loops) andK2≈ 102 m−1 (N-loops). Only when both loops are saturated with calcium does the protein change its global conformation, exposing to the solvent hydrophobic patches, which can be detected by 2-p-toluidinylnaphthalene-6-sulfonic acid– a fluorescent probe of protein-surface hydrophobicity.S-Glutathionylation of the single cysteine residue (85) of theα subunits leads to a 10-fold increase in the affinity of the protein C-loops for calcium and an enormous– four orders of magnitude– increase in the calcium-binding constants of its N-loops, owing to a cooperativity effect corresponding toΔΔ G = −6 ± 1 kcal·mol−1. A similar effect is observed upon formation of the mixed disulfide with cysteine and 2-mercaptoethanol. The glutathionylated protein binds TRTK-12 peptide in a calcium-dependent manner. S100A1 protein can act, therefore, as a linker between the calcium and redox signalling pathways.
- Subjects
PROTEINS; CALCIUM; GLUTATHIONE; BLOOD coagulation factors; SALT; PEPTIDES
- Publication
FEBS Journal, 2005, Vol 272, Issue 10, p2557
- ISSN
1742-464X
- Publication type
Article
- DOI
10.1111/j.1742-4658.2005.04680.x