Effects on the conformation of FVIIa by sTF and Ca(2+) binding: Studies of fluorescence resonance energy transfer and quenching
2011 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 413, no 4, 545-549 p.Article in journal (Refereed) Published
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.
Place, publisher, year, edition, pages
Elsevier , 2011. Vol. 413, no 4, 545-549 p.
Factor VIIa, Fluorescence quenching, Fluorescence resonance energy transfer, Tissue factor, Fluorescein, Rhodamine, Conformational dynamics
IdentifiersURN: urn:nbn:se:liu:diva-63686DOI: 10.1016/j.bbrc.2011.08.135ISI: 000295912800010OAI: oai:DiVA.org:liu-63686DiVA: diva2:382172
Funding agencies|Swedish Research Council||Knut and Alice Wallenbergs Foundation||2010-12-302010-12-302011-11-10Bibliographically approved