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Effects on the conf...
Effects on the conformation of FVIIa by sTF and Ca(2+) binding : Studies of fluorescence resonance energy transfer and quenching
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- Carlsson, Karin (författare)
- Linköpings universitet,Biokemi,Tekniska högskolan
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- Persson, Egon (författare)
- Haemostasis Biochemistry, Novo Nordisk A/S, DK-2760 Måløv, Denmark
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- Østergaard, Henrik (författare)
- Department of Physics, Norwegian University of Science and Technology, Trondheim, Norway
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- Lindgren, Mikael (författare)
- Linköpings universitet,Biokemi,Tekniska högskolan
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- Carlsson, Uno (författare)
- Linköpings universitet,Biokemi,Tekniska högskolan
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- Svensson, Magdalena (författare)
- Linköpings universitet,Biokemi,Tekniska högskolan
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(creator_code:org_t)
- Elsevier, 2011
- 2011
- Engelska.
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Ingår i: Biochemical and Biophysical Research Communications - BBRC. - : Elsevier. - 0006-291X .- 1090-2104. ; 413:4, s. 545-549
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https://liu.diva-por... (primary) (Raw object)
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http://liu.diva-port...
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https://urn.kb.se/re...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- 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.
Nyckelord
- Factor VIIa
- Fluorescence quenching
- Fluorescence resonance energy transfer
- Tissue factor
- Fluorescein
- Rhodamine
- Conformational dynamics
- NATURAL SCIENCES
- NATURVETENSKAP
Publikations- och innehållstyp
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- art (ämneskategori)
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