Probing the role of tryptophan residues in a cellulose-binding domain by chemical modification.
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 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 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.
Bray
M R
MR
Protein Engineering Network of Centres of Excellence, University of British Columbia, Vancouver, Canada.
Johnson
P E
PE
Gilkes
N R
NR
McIntosh
L P
LP
Kilburn
D G
DG
Warren
R A
RA
D001665:Binding Sites; D001974:Bromosuccinimide / Q000737:chemistry; D002482:Cellulose / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D010084:Oxidation-Reduction; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism*
D014364:Tryptophan; D002482:Cellulose; D001974:Bromosuccinimide