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Biophys J, May 2002, p. 2466-2475, Vol. 82, No. 5

Site-Directed Mutagenesis of Tyrosine 118 within the Central Constriction Site of the LamB (Maltoporin) Channel of Escherichia coli. I. Effect on Ion Transport

Frank Orlik, Christian Andersen, and Roland Benz

Lehrstuhl für Biotechnologie, Theodor-Boveri-Institut (Biozentrum) der Universität Würzburg, Am Hubland, D-97074 Würzburg, Germany

The three-dimensional structure of the malto-oligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) is known from x-ray crystallography. The central constriction of the channel formed by the external loop 3 is controlled by a tyrosine residue (Y118). Y118 was replaced by site-directed mutagenesis by ten other amino acids (alanine, isoleucine, asparagine, serine, cysteine, aspartic acid, arginine, histidine, phenylalanine, and tryptophane) including neutral ones, negatively and positively charged amino acids to study the effect of their size, hydrophobicity, and charge on ion transport through LamB. The mutant proteins were purified to homogeneity. They were reconstituted into lipid bilayer membranes and single-channel conductance and ion selectivity were measured to get insight into the mechanism of ion transport through LamB. The mutation of Y118 to any other nonaromatic amino acid led to a substantial increase of the single-channel conductance by more than a factor of six at maximum. The highest effect was observed for Y118D. Additionally, a nonlinear relationship between the salt concentration in the aqueous phase and the channel conductance was observed for this mutant, indicating strong discrete charge effects on ion conductance. For all other mutants, with the exception of Y118R, linear relationships were found between single-channel conductance and bulk aqueous concentration. The individual hydrophobicity indices of the amino acids introduced inside the central constriction of the LamB channel had a somewhat smaller effect on the single-channel conductance as compared with the effect of their size and charge.

Biophys J, May 2002, p. 2466-2475, Vol. 82, No. 5
© 2002 by the Biophysical Society   0006-3495/02/05/2466/10  $2.00


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F. Orlik, C. Andersen, and R. Benz
Site-Directed Mutagenesis of Tyrosine 118 within the Central Constriction Site of the LamB (Maltoporin) Channel of Escherichia coli. II. Effect on Maltose and Maltooligosaccharide Binding Kinetics
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