17 August 1994 Probing the structure of human tissue factor by site-directed mutagenesis of tryptophan residues and in-vivo incorporation of tryptophan analogs
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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.
© (1994) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.
Carol A. Hasselbacher, Carol A. Hasselbacher, Elena Rusinova, Elena Rusinova, Evan Waxman, Evan Waxman, Wan Lam, Wan Lam, Arabinda Guha, Arabinda Guha, Radda Rusinova, Radda Rusinova, Yale Nemerson, Yale Nemerson, J. B. Alexander Ross, J. B. Alexander Ross, W. H. Konigsberg, W. H. Konigsberg, } "Probing the structure of human tissue factor by site-directed mutagenesis of tryptophan residues and in-vivo incorporation of tryptophan analogs", Proc. SPIE 2137, Time-Resolved Laser Spectroscopy in Biochemistry IV, (17 August 1994); doi: 10.1117/12.182739; https://doi.org/10.1117/12.182739
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