Picosecond tryptophan fluorescence of membrane-bound prothrombin fragment 1.
The wavelength-dependent tryptophan (Trp) fluorescence decays of Ca-prothrombin fragment 1 (Ca-BF1), which contains three tryptophan residues, in the presence of pure phosphatidylcholine (PC) small unilamellar vesicles (SUV) and PC-SUV containing either 25% phosphatidyl-l-serine (PS), and 25% or 40% phosphatidylglycerol (PG) are characterized, using fluorescence lifetime distribution, conventional multiexponential, and global analysis. In analogy to previous investigations on apo- and Ca-BF1 (M. Hof, G.R. Fleming, V. Fidler, Proteins Struct. Func. Genet. 24 (1996) 485-494), the analysis resulted in a five exponential decay model in all investigated systems, where the five fluorescence lifetimes (e.g. 0. 04+/-0.02 ns (component A), 0.24+/-0.02 ns (B), 0.66+/-0.03 ns (C), 2.4+/-0.3 ns (D), and 5.4+/-0.4 ns (E) for Ca-BF1 in the presence of PC-SUV) are wavelength-independent. The fluorescence lifetimes and the corresponding amplitudes of the 'Gla-Trp' (components D and E) and of the two 'kringle-Trp' (components B, C, and D) remain unchanged when bound to the PS-containing vesicles. Saturation binding to PG-containing membranes leads to a prolongation of the Gla component E from 5.3 in solution to 7.5 ns, indicating a change in the Gla-domain conformation. The results represent the first experimental evidence of a lipid-specific conformational change in the N-terminal 'Gla domain' of a vitamin K-dependent protein.
J. Heyrovský Institute of Physical Chemistry, Academy of Sciences of the Czech Republic, Dolejskova 3, 18223 Prague 8, Czech Republic. hof@jh-inst.cas.cz
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D000818:Animals; D002118:Calcium; D002417:Cattle; D008567:Membranes, Artificial; D010446:Peptide Fragments / Q000737:chemistry*; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D011498:Protein Precursors / Q000737:chemistry*; D011516:Prothrombin / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D008567:Membranes, Artificial; D010446:Peptide Fragments; D010713:Phosphatidylcholines; D010715:Phosphatidylglycerols; D010718:Phosphatidylserines; D011498:Protein Precursors; C029733:prothrombin fragment 1; D014364:Tryptophan; D011516:Prothrombin; D002118:Calcium