Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification

Authors

  • Mark R. Bray,

    1. Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    2. Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    Current affiliation:
    1. The Ontario Cancer Institute, 610 University Ave., Toronto, Ontario M5G 2M9, Canada
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  • Neil R. Gilkes,

    1. Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    2. Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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  • Douglas G. Kilburn,

    1. Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    2. Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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  • R. Antony J. Warren,

    Corresponding author
    1. Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    2. Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    • Department of Microbiology, 6174 University Boulevard, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada
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  • Lawrence P. Mcintosh,

    1. Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    2. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
    3. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
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  • Philip E. Johnson

    1. Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3
    2. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z1
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Abstract

The cellulose-binding domain (CBDCex) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the β-barrel structure and three exposed on the surface (Xu GY et al., 1995, Biochemistry 34:6993–7009). Although all five tryptophans can be oxidized by N-bromosuccinimide (NBS), stopped-flow measurements show that three tryptophans react faster than the other two. NMR analysis during the titration of CBDCex with NBS shows that the tryptophans on the surface of the protein are fully oxidized before there is significant reaction with the two buried tryptophans. Additionally, modification of the exposed tryptophans does not affect the conformation of the backbone of CBDCex, whereas complete oxidation of all five tryptophans denatures the polypeptide. The modification of the equivalent of one and two tryptophans by NBS reduces binding of CBDCex to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBDCex to cellulose. Although adsorption to cellulose does afford some protection against NBS, as evidenced by the increased quantity of NBS required to oxidize all of the tryptophan residues, the polypeptide can still be oxidized completely when adsorbed. This suggests that, whereas the binding appears to be irreversible overall [Ong E et al., 1989, Bio/Technology 7:604–607], each of the exposed tryptophans interacts reversibly with cellulose.

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