ENVIRONMENTS OF THE 4 TRYPTOPHANS IN THE EXTRACELLULAR DOMAIN OF HUMAN TISSUE FACTOR - COMPARISON OF RESULTS FROM ABSORPTION AND FLUORESCENCE DIFFERENCE SPECTRA OF TRYPTOPHAN REPLACEMENT MUTANTS WITH THE CRYSTAL-STRUCTURE OF THE WILD-TYPE PROTEIN

The local environments of the four tryptophan residues of the extracel lular domain of human tissue factor (sTF) were assessed from differenc e absorption and fluorescence spectra. The difference spectra were der ived by subtracting spectra from single Trp-to-Phe or Trp-to-Tyr repla cement mutants from the corresponding spectrum of the wild-type protei n. Each of the mutants was capable of enhancing the proteolytic activi ty of factor VIIa showing that the mutations did not introduce major s tructural changes, although the mutants were more susceptible to denat uration by guanidinium chloride, The difference spectra indicate that the Trp residues are buried to different extents within the protein ma trix, This evaluation was compared with the x-ray crystal structure of sTF. There is excellent agreement between predictions from the differ ence spectra and the environments of the Trp residues observed in the x-ray crystal structure, demonstrating that difference absorption and particularly fluorescence spectra derived from functional single-Trp r eplacement mutants can be used to obtain information about the local e nvironments of individual Trp residues in multi-tryptophan proteins.