Effects on the conformation of FVIIa by sTF and Ca binding: Studies of fluorescence resonance energy transfer and quenching |
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Authors: | Karin Carlsson Egon Persson Mikael Lindgren Uno Carlsson Magdalena Svensson |
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Institution: | aIFM-Department of Chemistry, Linköping University, Linköping, Sweden;bHaemostasis Biochemistry, Novo Nordisk A/S, Måløv, Denmark;cDepartment of Physics, Norwegian University of Science and Technology, Trondheim, Norway |
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Abstract: | The apparent length of FVIIa in solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein (Fl-FPR) and a rhodamine derivative (TMR), were covalently attached to FVIIa. The binding site of Fl-FPR was in the protease domain whereas TMR was positioned in the Gla domain, thus allowing a length measure over virtually the whole extension of the protein. From the FRET measurements, the distances between the two probes were determined to be 61.4 for free FVIIa and 65.5 Å for FVIIa bound to soluble tissue factor (sTF). These seemingly short distances, compared to those anticipated based on the complex crystal structure, require that the probes stretch towards each other. Thus, the apparent distance from the FRET analysis was shown to increase with 4 Å upon formation of a complex with sTF in solution. However, considering how protein dynamics, based on recent molecular dynamics simulations of FVIIa and sTF:FVIIa (Y.Z. Ohkubo, J.H. Morrissey, E. Tajkhorshid, J. Thromb. Haemost. 8 (2010) 1044–1053), can influence the apparent fluorescence signal our calculations indicated that the global average conformation of active-site inhibited FVIIa is nearly unaltered upon ligation to sTF.It is known from amidolytic activity measurements that Ca2+ binding leads to activation of FVIIa, but we have for the first time directly demonstrated conformational changes in the environment of the active site upon Ca2+ binding. Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. Forming a complex with sTF further stabilized this conformational change, leading to a more inaccessible active-site located probe. |
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Keywords: | Abbreviations: EGF epidermal growth factor-like domain FVIIa activated coagulation factor VII Fl fluorescein FPR-chloromethyl ketone Gla γ-carboxyglutamic acid PD protease domain sTF soluble tissue factor (residues 1-219) TF tissue factor TMR tetramethylrhodamine-5-maleimide |
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