Study of Sugar Binding to the Sucrose-specific ScrY Channel of Enteric Bacteria Using Current Noise Analysis

Abstract.

ScrY, an outer membrane channel of enteric Gram-negative bacteria, which confers to the bacteria the rapid uptake of sucrose through the outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated in the open and in the carbohydrate-induced closed state of the channel. The open state of the channel exhibited up to about 200 Hz 1/f-noise with a rather small spectral density. Upon addition of carbohydrates to the aqueous phase the current through the ScrY channels decreased in a dose-dependent manner. Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the carbohydrates with the binding site inside the channel and its reversible block. The frequency dependence of the spectral density was of the Lorentzian type but very often two Lorentzians were observed, from which the slow one may not be related to carbohydrate binding. Analysis of the power density spectra of the second Lorentzian using a previously proposed simple model of carbohydrate binding allowed the evaluation of the on- and the off-rate constants for the carbohydrate association with the binding site inside the ScrY channel and of a mutant (ScrYΔ3-72), in which 70 amino acids at the N-terminus are deleted. The binding of carbohydrates to ScrY was compared to those of the closely related maltoporin channels of Escherichia coli and Salmonella typhimurium by assuming that only the time constant and spectral density of the high frequency Lorentzian is related to carbohydrate transport.