Effects on the conformation of FVIIa by sTF and Ca(2+) binding : Studies of fluorescence resonance energy transfer and quenching

• The apparent length of FVIIa in buffer solution was estimated by a FRET analysis. Two fluorescent probes, fluorescein linked to an inhibitor (FPR-chloromethyl ketone) and a rhodamine derivative (tetramethylrhodamine-5-maleimide), were covalently attached to FVIIa. The binding site of fluorescein was in the PD whereas rhodamine was positioned in the Gla domain, thus allowing a length measure over approximately the whole extension of the protein. From the FRET measurements the distances between the two probes were determined to 61.4 for free FVIIa and 65.5 Å for FVIIa bound to the soluble TF (sTF). 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, by considering how protein dynamics, based on recently published molecular dynamics simulations of FVIIa and sTF:FVIIa (Ohkubo et al., 2010 J. Thromb. Haemost. 8, 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. Moreover, it is known that Ca2+ binding leads to activation of FVIIa, and we have for the first time demonstrated conformational changes in the environment of the active site upon Ca2+ binding by direct measurements, previously suggested based on indirect measurements (Persson & Petersen, 1995 Eur. J. Biochem. 234, 293-300). Interestingly, this Ca2+-induced conformational change can be noted even in the presence of an inhibitor. By forming the sTF:FVIIa complex the conformational change of the active site is further developed, leading to a more inaccessible active-site located probe.

Keyword

Factor VIIa

Fluorescence quenching

Fluorescence resonance energy transfer

Tissue factor

Fluorescein

Rhodamine

Conformational dynamics

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