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S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of twoasub-units, each containing two calcium-binding loops (N and C). At physiologi-cal pH (7.2) and NaCl concentration (100 mm), we determined the microscopic binding constants of calcium to S100A1 by analysing the Ca 2+ -titration curves of Trp90 fluorescence for both the native protein and its Glu32fiGln mutant with an inactive N-loop. | ềFEBS Journal Affinity of S100A1 protein for calcium increases dramatically upon glutathionylation Grazyna Goch Sergiusz Vdovenko Hanna Koztowska and Andrzej Bierzynski Institute of Biochemistry and Biophysics Polish Academy of Sciences Poland Keywords calcium binding EF-hand proteins glutathionylation S100A1 Correspondence A. Bierzynski Institute of Biochemistry and Biophysics Polish Academy of Sciences ul. Pawinskiego 5 A 02-106 Warsaw Poland Fax 48 22 823 71 94 Tel 48 22 592 23 71 E-mail ajb@ibb.waw.pl Received 14 January 2005 revised 14 March 2005 accepted 22 March 2005 doi 10.1111 j.1742-4658.2005.04680.x S100A1 is a typical representative of a group of EF-hand calcium-binding proteins known as the S100 family. The protein is composed of two a 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 fi Gln mutant with an inactive N-loop. Using a chelator method we also determined the calcium-binding constant for the S100A1 Glu73 fi Gln mutant with an inactive C-loop. The protein binds four calcium ions in a noncooperative way with binding constants of K1 4 2 X 103 m-1 C-loops and K2 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 the a 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 to AAG -6 1 kcal-mol-1. A similar effect is observed upon formation of the mixed .
