title: Fluorescence study of the conformational properties of recombinant tick anticoagulant peptide (Ornithodorus moubata) using multifrequency phase fluorometry.
abstract: Steady-state and multifrequency phase fluorometry were used to characterize the conformational state and conformational dynamics of recombinant tick anticoagulant peptide (Ornithodorus moubata) (TAP). The TAP contains two tryptophan residues at positions 11 and 37. The fluorescence emission varies sigmoidally as a function of pH with a pKa of 6.01 +/- 0.07. This pH dependency suggests that tryptophan fluorescence is quenched by His43 at low pH. This is confirmed by modification of the histidine with diethylpyrocarbonate. At pH 9 the fluorescence decay is well described by a sum of three exponentials (0.52, 1.9 and 5.4 ns), which decrease all three at pH 4 (0.25, 1.61 and 4.4 ns). From the reactivity of the fluorescence lifetimes toward N-bromosuccinimide and from the calculation of the accessibility we can attribute the long lifetime to Trp11, the short one to Trp37 and the middle one to both. The anisotropy decay was resolved into two components of 3.85 ns and 0.27 ns at pH 4 and 4.5 ns and 0.6 ns at pH 9. The long anisotropy decay time corresponds to the rotational correlation time of the protein, the short one to local mobility of the tryptophan residues.
authors.affiliation: Laboratory for Chemical and Biological Dynamics, University of Leuven, Belgium.
authors.forename: A
authors.initials: A
authors.lastname: Sillen
authors.forename: R
authors.initials: R
authors.lastname: Vos
authors.forename: Y
authors.initials: Y
authors.lastname: Engelborghs
mesh_terms: D000818:Animals; D060829:Arthropod Proteins; D005453:Fluorescence; D005470:Fluorometry / Q000379:methods; D036341:Intercellular Signaling Peptides and Proteins; D007700:Kinetics; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000032:analysis / Q000737:chemistry; D013987:Ticks
chemical_list: D060829:Arthropod Proteins; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011994:Recombinant Proteins; C063967:tick anticoagulant peptide