Spectroscopic characterization of tick anticoagulant peptide.
Tick anticoagulant peptide (TAP) is a disulfide rich potent inhibitor of factor Xa. Although this peptide is of potential clinical utility, very little is known about its higher order structure. Therefore, the secondary structure of recombinant TAP (rTAP) has been examined by circular dichroism (CD) and Fourier transform infrared (FTIR) spectroscopy. Both techniques suggest that rTAP is rich in beta-sheet structure. Disulfide bonds play a significant role in the folding and structural stability of rTAP. This is apparent from the resistance of rTAP to fluorescence-detected unfolding by guanidinium chloride (Gdn-HCl), unless disulfides are first reduced. The protein's tryptophan and tyrosine residues exhibit greater solvent exposure upon reduction of the cystines as indicated by fluorescence spectra and second derivative UV spectroscopy. A considerable amount of beta-structure appears to be retained after reduction of disulfides, although the CD spectrum manifests an increased amount of disordered structure in the reduced peptide. While rTAP does not bind the hydrophobic fluorescence probe 2-p-toludinylnaphthalene-6-sulfonate (TNS) at neutral or acidic pH, the reduced peptide binds TNS at pH 2.0 but not at pH 7.0. The secondary structure of the reduced peptide at pH 2 is, however, similar to that at pH 7 as judged by CD spectroscopy. The reduced form of rTAP at acidic pH thus resembles a molten globule-like state.
Sanyal
G
G
Department of Pharmaceutical Research and Development, Merck Research Laboratories, West Point, PA 19486, USA.
Marquis-Omer
D
D
Waxman
L
L
Mach
H
H
Ryan
J A
JA
O'Brien Gress
J
J
Middaugh
C R
CR
D060829:Arthropod Proteins; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides / Q000737:chemistry*; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D012997:Solvents; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared
D060829:Arthropod Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; D012997:Solvents; C063967:tick anticoagulant peptide