Calcium and phospholipid binding properties of synthetic gamma-carboxyglutamic acid-containing peptides with sequence counterparts in human protein C.
Two peptides with counterpart sequences in the gamma-carboxyglutamic acid (Gla) domain of human protein C (PC) have been synthesized and characterized. One peptide contained 38 amino acids (38-mer) and spanned the region from the amino terminus of the protein to the DNA splice junction between the Gla domain and the following short helical stretch, and the second peptide (48-mer) included a 10 amino acid extension that has been designed to incorporate the exon for the helical segment that is thought to play a role in stabilizing the Ca(2+)-dependent conformation of the Gla domain of proteins of this class. The peptides were synthesized by solid-phase methodology, then oxidized to allow disulfide pairing, and finally purified by FPLC methodology. Chemical characterization showed that each peptide contained its full complement of Gla residues. Two types of Ca(2+)-binding sites were found in these peptides, tighter sights (2-3) with Kd values of 60-370 microM and a weaker set of sites (7-10) with a range of Kd values from 0.8 to 3.1 mM. In general, the 48-mer interacted with Ca2+ more tightly than the 38-mer. As revealed by circular dichroism analysis, and by reactivity with monoclonal antibodies that recognize both the unfolded form of the Gla domain as well as the Ca(2+)-dependent conformation of this same domain, the 38-mer and 48-mer underwent the Ca(2+)-induced conformational changes characteristic of the intact protein. Both peptides displayed Ca(2+)-dependent binding to negatively charged phospholipid vesicles (PL).(ABSTRACT TRUNCATED AT 250 WORDS)
Colpitts
T L
TL
Department of Chemistry and Biochemistry, University of Notre Dame, Indiana 46556.
Castellino
F J
FJ
D015055:1-Carboxyglutamic Acid / Q000737:chemistry*; D000596:Amino Acids; D000911:Antibodies, Monoclonal; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002942:Circular Dichroism; D005449:Fluorenes; D008081:Liposomes / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000378:metabolism*; D011486:Protein C / Q000737:chemistry*; D011487:Protein Conformation / Q000187:drug effects; D017421:Sequence Analysis
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D005449:Fluorenes; D008081:Liposomes; C016456:N(alpha)-fluorenylmethyloxycarbonylamino acids; D010455:Peptides; D010743:Phospholipids; D011486:Protein C; D015055:1-Carboxyglutamic Acid; D002118:Calcium