Protein Science, Vol 5, Issue 11 2311-2318, Copyright © 1996 by Cold Spring Harbor Laboratory Press

ARTICLE

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

M. R. BRAY, P. E. JOHNSON, N. R. GILKES, L. P. MCINTOSH, D. G. KILBURN and RAJ. WARREN
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1Z3 Present address: The Ontario Cancer Institute, 610 University Ave., Toronto, Ontario M5G 2M9, Canada.

The cellulose-binding domain (CBD(Cex)) of the mixed function glucanase-xylanase Cex from Cellulomonas fimi contains five tryptophans, two of which are located within the {beta}-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 CBD(Cex) 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 CBD(Cex), 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 CBD(Cex) to cellulose by 70% and 90%, respectively. This confirms the direct role of the exposed aromatic residues in the binding of CBD(Cex) 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.

This article has been cited by other articles:

				J. Lehtio, J. Sugiyama, M. Gustavsson, L. Fransson, M. Linder, and T. T. Teeri The binding specificity and affinity determinants of family 1 and family 3 cellulose binding modules PNAS, January 21, 2003; 100(2): 484 - 489. [Abstract] [Full Text] [PDF]

				B. W. McLean, A. B. Boraston, D. Brouwer, N. Sanaie, C. A. Fyfe, R. A. J. Warren, D. G. Kilburn, and C. A. Haynes Carbohydrate-binding Modules Recognize Fine Substructures of Cellulose J. Biol. Chem., December 27, 2002; 277(52): 50245 - 50254. [Abstract] [Full Text] [PDF]

				T. Uchiyama, F. Katouno, N. Nikaidou, T. Nonaka, J. Sugiyama, and T. Watanabe Roles of the Exposed Aromatic Residues in Crystalline Chitin Hydrolysis by Chitinase A from Serratia marcescens 2170 J. Biol. Chem., November 2, 2001; 276(44): 41343 - 41349. [Abstract] [Full Text] [PDF]

				Y. Wang, M. B. Slade, A. A. Gooley, B. J. Atwell, and K. L. Williams Cellulose-binding modules from extracellular matrix proteins of Dictyostelium discoideum stalk and sheath Eur. J. Biochem., August 1, 2001; 268(15): 4334 - 4345. [Abstract] [Full Text] [PDF]

				B. W. McLean, M. R. Bray, A. B. Boraston, N. R. Gilkes, C. A. Haynes, and D. G. Kilburn Analysis of binding of the family 2a carbohydrate-binding module from Cellulomonas fimi xylanase 10A to cellulose: specificity and identification of functionally important amino acid residues Protein Eng., November 1, 2000; 13(11): 801 - 809. [Abstract] [Full Text] [PDF]

				P. J. Simpson, H. Xie, D. N. Bolam, H. J. Gilbert, and M. P. Williamson The Structural Basis for the Ligand Specificity of Family 2 Carbohydrate-binding Modules J. Biol. Chem., December 22, 2000; 275(52): 41137 - 41142. [Abstract] [Full Text] [PDF]

				G. Carrard, A. Koivula, H. Soderlund, and P. Beguin Cellulose-binding domains promote hydrolysis of different sites on crystalline cellulose PNAS, September 12, 2000; 97(19): 10342 - 10347. [Abstract] [Full Text] [PDF]

				M. Hashimoto, T. Ikegami, S. Seino, N. Ohuchi, H. Fukada, J. Sugiyama, M. Shirakawa, and T. Watanabe Expression and Characterization of the Chitin-Binding Domain of Chitinase A1 from Bacillus circulans WL-12 J. Bacteriol., June 1, 2000; 182(11): 3045 - 3054. [Abstract] [Full Text]

				T. Ikegami, T. Okada, M. Hashimoto, S. Seino, T. Watanabe, and M. Shirakawa Solution Structure of the Chitin-binding Domain of Bacillus circulans WL-12 Chitinase A1 J. Biol. Chem., April 28, 2000; 275(18): 13654 - 13661. [Abstract] [Full Text] [PDF]

				G. Carrard and M. Linder Widely different off rates of two closely related cellulose-binding domains from Trichoderma reesei Eur. J. Biochem., June 15, 1999; 262(3): 637 - 643. [Abstract] [Full Text]