Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site.
Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a GlySer insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.
Benz
R
R
Lehrstühl für Biotechnologie, Universität Würzburg, Germany.
Francis
G
G
Nakae
T
T
Ferenci
T
T
D000595:Amino Acid Sequence; D001425:Bacterial Outer Membrane Proteins; D001665:Binding Sites; D004926:Escherichia coli / Q000378:metabolism*; D008564:Membrane Potentials; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D018272:Porins; D011487:Protein Conformation; D015202:Protein Engineering; D011991:Receptors, Virus / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D011134:Polysaccharides; D018272:Porins; D011991:Receptors, Virus; C016532:maltoporins; C008315:maltodextrin