Designed polyanionic coiled-coil proteins: acceleration of heparin cofactor II inhibition of thrombin.
Novel polyanionic proteins were designed to increase the rate of heparin cofactor II (HC) inhibition of alpha-thrombin, an essential protease in the coagulation cascade. Two alpha-helical coiled-coil proteins, a 62-residue dimer containing 8 Glu residues (E8C) and a 104-residue dimer containing 14 Glu residues (E14C), plus two 31-residue control peptides containing 8 Glu residues each (E8A and E8B), were chemically synthesized, structurally characterized and enzymatically assayed. Circular dichroic spectrophotometry indicated that both E8C and E14C formed stable two-chain alpha-helical coiled coils at pH 7 and 25 degrees C. The control peptides were only partially alpha-helical. E14C remained folded at 90 degrees C but E8C was half unfolded at 49 degrees C. Coiled-coil proteins E8C and E14C maximally accelerated by 35- and 33-fold, respectively, the rate of HC inhibition of alpha-thrombin. None of these compounds accelerated antithrombin inhibition of alpha-thrombin, and neither control peptide accelerated HC inhibition of alpha-thrombin. Acceleration of the HC inhibition of alpha-thrombin showed bimodal dependence on the concentration of the polyanionic protein, which is consistent with formation of a HC-coiled-coil-thrombin ternary complex. The results suggest that antithrombotic polyanionic alpha-helical coiled-coil proteins can be designed and synthesized and that the occurrence of secondary structure can be correlated with biological activity.
Melton
L G
LG
Department of Chemistry, University of North Carolina at Chapel Hill, USA.
Church
F C
FC
Erickson
B W
BW
D000595:Amino Acid Sequence; D000838:Anions; D015195:Drug Design; D004355:Drug Stability; D004357:Drug Synergism; D018257:Helix-Loop-Helix Motifs; D015844:Heparin Cofactor II / Q000494:pharmacology*; D006358:Hot Temperature; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid
D000838:Anions; D015844:Heparin Cofactor II