title: Experimental indication for the existence of multiple Trp rotamers in von Willebrand Factor A3 domain.
abstract: The first step in both normal haemostasis and arterial thrombosis is the interaction between collagen, von Willebrand factor (vWF), and glycoprotein Ib. The A3 domain of vWF forms the principal binding site for collagen type I and type III. Inhibition of the vWF-collagen interaction by an anti-human vWF monoclonal antibody (MoAb) 82D6A3 can be a potential way to prevent arterial thrombosis. Identification of the epitope of MoAb 82D6A3 showed recently that the consensus sequence SPWR obtained by phage display could adopt the conformation of the discontinuous epitope. Modelling showed that Trp982 in the vWF had to obtain a more solvent accessible conformation. We performed a detailed fluorescence study of Trp982 in the vWF A3. Using the method described by Hellings et al. (Biophys J 2003;85:1894-1902), we were able to identify two different low-energy Trp982 rotamers and to link them with their experimentally derived fluorescence lifetimes. Fluorescence anisotropy showed no interconversion in the nanosecond timescale between the two different rotameric states. With these experiments, we gather strong indications for the existence of an exposed rotamer conformation and a rotamer that corresponds to the one observed in the X-ray structure. These results strongly support the modeling work (Vanhoorelbeke et al., J Biol Chem 2003;278:37815-37821).
authors.affiliation: Laboratory of Biomolecular Dynamics, Katholieke Universiteit Leuven, Leuven, Belgium.
authors.forename: Mario
authors.initials: M
authors.lastname: Hellings
authors.forename: Yves
authors.initials: Y
authors.lastname: Engelborghs
authors.forename: Hans
authors.initials: H
authors.lastname: Deckmyn
authors.forename: Karen
authors.initials: K
authors.lastname: Vanhoorelbeke
authors.forename: Marion E
authors.initials: ME
authors.lastname: Schiphorst
authors.forename: Jan Willem N
authors.initials: JW
authors.lastname: Akkerman
authors.forename: Marc
authors.initials: M
authors.lastname: De Maeyer
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray; D005454:Fluorescence Polarization; D006801:Humans; D008958:Models, Molecular; D017434:Protein Structure, Tertiary; D012997:Solvents / Q000378:metabolism; D014364:Tryptophan / Q000378:metabolism*; D014841:von Willebrand Factor / Q000737:chemistry* / Q000378:metabolism
chemical_list: D012997:Solvents; D014841:von Willebrand Factor; D014364:Tryptophan