Binding of calcium to synthetic peptides containing γ‐carboxyglutamic acid

Abstract

The Ca' + binding properties of various y-carboxyglutamic acid (g1a)-containing synthetic peptides with counterpart sequences in human protein C were investigated employing potentiometry with a Ca2 + -selective electrode and titration calorimetric techniques. The shortest peptides, FL(gla)(gla)LR, DF(gla)(gla)AK, and the oxidized form of the cyclic hexapeptide CI(gla)(gla)IC, each of which contains one pair of gla residues, have a weak affinity for Ca2 , with some peptides probably involved in intermolecular bridging of the Ca2 + .The best example of this is the oxidized form of the peptide, CI(gla)(gla)IC, where one g-atom of Ca2 + interacts with 2 mol of peptide ( n = 0.5) with a Kd value of 1.6 mM. A second g-atom of Ca2 + interacts with 2 mol of this same peptide (n = 0.5) and is characterized by a K d of 8.8 mM. A longer peptide containing this same sequence, viz. L(gla)R(gla)CI(gla)(gla)IC, possesses two binding sites (17 = 2.0) for Ca2 + ofKd = 16.1 mM, as well as a tighter site ( n = l), Of Kd = 0.4 mM. An increase in stoichiometry of tight binding sites as the peptide is elongated is observed from binding data obtained on a 38-residue peptide that possesses all nine of the gla-residues of protein C in their proper sequence positions. The strongest Ca2 + binding sites ( n = 3-4) possess an average Kd of 0.3 mM, followed by another class of sites (n = 5-10, average Kd = 1.5-3.0 mM). The affinity and stoichiometry of these stronger sites mimic those observed for binding of Ca2 + to the gla region of prothrombin fragment 1. By selective [ 13C]-labeling of the essential gla 16 residue of the 38-mer peptide, we demonstrate that this particular gla residue participates as a donor for a high-affinity Ca2 + site.These similarities in binding properties between the synthetic peptide containing the entire gla domain and the gla domain as it exists in proteins and protein fragments indicate suitably designed peptides of this type may constitute appropriate models for investigation of the binding of Ca' + to intact gla-containing proteins.Calcium is a required component of the blood coagulation cascade, and functions through its interaction with several proteins, viz. the vitamin K-dependent proteins, that participate in this pathway. Important to Ca2 + binding to these proteins, which include prothrombin, factors VII, IX and X, as well as proteins C and S, is the post-translational modification of specific precursor E residues to gla residues. This latter series of reactions employs vitamin K as a cofactor for the functional y-carboxylase, and results in formation of 9-13 residues of gla, the first 10 of which are present Abbreviations: gla, y-carboxyglutamic acid ; Fmoc, 9-fluorenylmethyloxycarbonyl; HPLC, high-performance liquid chromatography: hPC, human protein C; Ot-Bu, terr-butyl ester; 0-Bzl, benzyl ester; t-Boc, terr-butyloxycarbonyl; Pmc, 2,2,5,7,8-pentamethyIchroman-6-sulfonyl; Bum, terr-butyloxymethyl; Z , benzyloxycarbonyl; FAB, fast atom bombardment.

Hydroxyproline-induced Helical Disruption in Conantokin Rl-B Affects Subunit-selective Antagonistic Activities toward Ion Channels of N-Methyl-d-aspartate Receptors

“…N ␣ -Fmoc-(␥,␥Ј-di-OtBu)-L-Gla was synthesized as described previously (33 , were measured using an AVIV model 62DS spectrometer with a 1-cm path length cell. The relevant peptides were dissolved in a solution of 10 mM HEPES, 10 mM NaCl, pH 7, to a final concentration of 100 M. The ␣-helical contents of these peptides were calculated as a percentage of the molar ellipticity values at 222 nm for Mg 2ϩ /conG, which is known to exist as an end-to-end ␣-helix.…”

Non-strict strand orientation of the Ca2+-induced dimerization of a conantokin peptide variant with sequence-shifted γ-carboxyglutamate residues

“…1, were synthesized, purified, and characterized as previously described [22]. All Fmoc-derivatized amino acids were obtained from either Sigma (St. Louis, MO) or Novabiochem (San Diego, CA) with the exception of Fmoc-γ,γ’-di-o-tBu-L-Gla, which was synthesized as reported earlier [8]. Procedures for obtaining the disulfide-linked peptides used in thiol-disulfide rearrangement assays were similar to those previously described [9].…”