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Time‐resolved fluorescence study of a calcium‐induced conformational change in prothrombin fragment 1
Author(s) -
Hof Martin,
Fleming Graham R.,
Fidler Vlastimil
Publication year - 1996
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/(sici)1097-0134(199604)24:4<485::aid-prot7>3.0.co;2-d
Subject(s) - fluorescence , quenching (fluorescence) , chemistry , picosecond , excited state , tryptophan , photochemistry , time resolved spectroscopy , analytical chemistry (journal) , crystallography , chromatography , atomic physics , quantum mechanics , laser , biochemistry , physics , amino acid , optics
The wavelength dependent fluorescence decay properties of bovine prothrombin fragment 1 have been investigated employing a picosecond time‐correlated single photon counting technique. All observations are discussed with using the crystal structure (Soriano‐Garcia et al., Biochemistry 31:2554–2566, 1992). Fluorescence lifetimes distribution and conventional multiexponential analysis, as well as acrylamide quenching studies lead to the identification of six distinguishable tryptophan excited‐states. Accessibility to the quencher and the known structure are used to associate a fluorescence decay of the tryptophan present in the Gla domain (Trp42) with two red shifted components (2.3 and 4.9 ns). The two kringle domain tryptophans (Trp90 and Trp126) exhibit four decay times (0.06, 0.24, 0.68, and 2.3 ns), which are blue shifted. The calcium‐induced fluorescence quenching is a result of static quenching: the five decay times remain unchanged, whereas the fluorescence intensity of Trp42 is decreased. The static quenching process is a consequence of a ground state interaction between the Cys18‐Cys23 disulfide bridge and Trp42. The monomolecular equilibrium constant for this disulfide‐π‐electron interaction is found as 4.8.