Chapter 8
Testing a Model Domain of Consistent with Infrared
of the Extracellular Human Tissue Fourier Transform Spectroscopy
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Downloaded by MONASH UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: December 14, 1994 | doi: 10.1021/bk-1994-0576.ch008
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J. B. A. Ross , C. A. Hasselbacher , Thomas F. Kumosinski , Gregory King , T. M . Laue , A. Guha , Y. Nemerson , W. H. Konigsberg , E. Rusinova , and E. Waxman 4
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Departments of Biochemistry and Medicine, Mount Sinai School of Medicine, New York, NY 10029 Eastern Regional Research Center, Agricultural Research Service, U.S. Department of Agriculture, 600 East Mermaid Lane, Philadelphia, PA 19118 Department of Biochemistry, University of New Hampshire, Durham, NH 03824 Department of Biochemistry, Yale University, New Haven, CT 06510 3
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Tissue Factor (TF) is a membrane-anchored cell-surface protein that in complex with the serine protease Factor VIIa initiates blood coagulation upon tissue damage. We have cloned and expressed the soluble, cytoplasmic domain of TF (residues 1-218) (sTF) for analysis of structure and function. Global secondary structural elements were determined using FTIR spectroscopy. The amide I band assignments indicated ca. 15% α-helix, 23% extended strands, the remainder being turns, loops, β-sheet, and 'other' structure. Secondary structure prediction algorithms using a knowledge-based approach that was constrained to the FTIR-determined structural elements were used to generate a working model of sTF, which was energy minimized and equilibrated at 300 Κ using a Kollman force field. The predictions of this model were tested by analytical ultracentrifugation, proteolytic cleavage, and absorption and fluorescence spectra of Trp-->Tyr and Trp-->Phe mutants of sTF. Following tissue damage, blood coagulation is initiated by the complexation of the transmembrane protein tissue factor (TF) with the circulating serine protease, factor Vila (Vila). TF is a glycoprotein consisting of an extracellular domain (residues 1219), a single transmembrane domain (residues 220-242), and a cytoplasmic domain (residues 243-263) with a sequence that contains a half-cysteine residue thioesterified to palmitate or stéarate (for review see (i)). After complexation with TF, the proteolytic activity of Vila towards its natural substrate, factor Χ (X), increases by many orders of magnitude (2). 0097-6156/94/0576-0113$08.00/0 © 1994 American Chemical Society In Molecular Modeling; Kumosinski, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1994.
Downloaded by MONASH UNIV on October 26, 2012 | http://pubs.acs.org Publication Date: December 14, 1994 | doi: 10.1021/bk-1994-0576.ch008
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MOLECULAR MODELING
Study of the three-dimensional structure of TF is complicated by the requirement that full-length TF be reconstituted in phospholipid vesicles or solubilized in detergent. To facilitate studies on the three-dimensional structure of TF, we have prepared a soluble TF construct (TF^^; sTF) which lacks the transmembrane and cytoplasmic domains (7). Here we describe a model for the three-dimensional structure of sTF, developed using a knowledge-based approach for protein structure prediction. The prediction algorithms were constrained to the secondary structural elements of sTF, determined separately by FTIR spectrosocopic analysis of the protein amide I absorption bands. The model for the sTF structure was tested by evaluating which of its features are consistent with experimental results obtained from proteolytic cleavage of sTF (3), absorption and fluorescence spectra of sTF and Trp-*Tyr and Trp->Phe mutants (4), and analytical ultracentrirugation (5). Experimental Procedures sTF. Recombinant sTF was cloned, expressed, and purifed as described by Waxman et al. (5). The authenticity of the protein was established by amino acid composition, NH -terminal sequence analysis (10 residues), and carboxyl terminal analysis by carboxypeptidase Ρ digestion. The amino terminal sequence was that expected for human TF. Carboxyl terminal digestion, however, yielded exclusively Arg (residue 218) rather than Glu (residue 219) as predicted by the sequence of the cDNA. We presume that residue 219 was removed by E. coli proteases. 2
Proteolytic Cleavage. Limited proteolytic cleavage was by subtilisin digestion in 20 mM Tris buffer, pH 8, using an sTF:subtilisin ratio of 650:1 (w/w), as described (5). Two fragments recovered after purification by ion-exchange on a mono-Q column followed by HPLC corresponded to residues 1-84 and 87-218 of the 1-218 residue polypeptide chain. sTF tryptophan mutants. sTF has four tryptophan residues. These are located at positions 14, 25, 45, and 158 of the polypeptide chain. Four functionally active mutants were prepared as described (4). In each, a different tryptophan residue was mutated, using site-directed mutagenesis, either to tyrosine or phenylalanine: W14F, W25Y, W45Y, and W158F. Ultraviolet Absorption and Fluorescence Spectroscopy. Concentrations of sTF and sTF mutants were determined by ultraviolet absorption spectroscopy (6). Since the number of tyrosine, phenylalanine, and tryptophan residues varied in the different mutants, it was necessary to determine the relative absorption spectra of the wild-type and mutant proteins. This was accomplished by diluting and denaturing an aliquot of each protein stock in 6 M guanidinium chloride (Gdm»Cl). The concentration of denatured protein was then calculated from knowledge of the protein tryptophan and tyrosine content and the extinction coefficients of tryptophan and tyrosine of a protein denatured in 6 M Gdm«Cl (
