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Proceedings Paper

Probing the structure of human tissue factor by site-directed mutagenesis of tryptophan residues and in-vivo incorporation of tryptophan analogs
Author(s): Carol A. Hasselbacher; Elena Rusinova; Evan Waxman; Wan Lam; Arabinda Guha; Radda Rusinova; Yale Nemerson; J. B. Alexander Ross; W. H. Konigsberg
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Paper Abstract

Complexation of the extracellular domain of tissue factor to the serine protease factor VIIa is a critical step in the process of blood coagulation following tissue damage. To study the structure and function of the extracellular domain of tissue factor (soluble tissue factor, sTF), we have used site-directed mutagenesis to replace each of sTF's four tryptophans (Trp) with phenylalanine (Phe) or tyrosine (Tyr). Replacement of any one of the four Trps reduced the protein stability against denaturation by guanidinium chloride in a similar manner, indicating that each residue has important structural interactions within the protein. Replacement of Trps 25, 45, and 158 resulted in reduced cofactor activities, indicating that these residues are located in regions important for biological activity. The activities of mutants with Trp 14 or both Trps 14 and 158 replaced were comparable to sTF. From the combination of absorbance and fluorescence spectra of the individual Trps, information is obtained showing that all the Trps are buried in the protein matrix, and Trps 14 and 25 are in highly constrained environments compared to Trps 45 and 158. To directly monitor interactions of sTF with factor VIIa and its substrate factor X, we have undertaken a program to generate spectrally enhanced protein (SEP) analogs of sTF and the sTF Trp mutants by in vivo incorporation of Trp analogs with absorbance and fluorescence distinct from Trp. Attempts to incorporate the Trp analogs 5-hydroxytryptophan (5-OHTrp) and 7-azatryptophan (7- ATrp) into sTF have provided further information on the structural significance of the Trp residues in sTF.

Paper Details

Date Published: 17 August 1994
PDF: 12 pages
Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182739
Show Author Affiliations
Carol A. Hasselbacher, Mount Sinai School of Medicine (United States)
Elena Rusinova, Mount Sinai School of Medicine (United States)
Evan Waxman, Mount Sinai School of Medicine (United States)
Wan Lam, Mount Sinai School of Medicine (United States)
Arabinda Guha, Mount Sinai School of Medicine (United States)
Radda Rusinova, Mount Sinai School of Medicine (United States)
Yale Nemerson, Mount Sinai School of Medicine (United States)
J. B. Alexander Ross, Mount Sinai School of Medicine (United States)
W. H. Konigsberg, Yale Univ. (United States)


Published in SPIE Proceedings Vol. 2137:
Time-Resolved Laser Spectroscopy in Biochemistry IV
Joseph R. Lakowicz, Editor(s)

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