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 (ScrYDelta3-72), 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.

Chitoporin from Vibrio harveyi, a Channel with Exceptional Sugar Specificity

“…This will be a subject of our further investigation. Translocation of chitohexaose particularly involved specific substrate-protein interactions, resulting in Michaelis-Menten transport kinetics resembling those of previously reported sugar-specific porins, including maltoporin (LamB) (31)(32)(33)(34), sucrose porin (ScrY) (25,35), glucoseinducible porin (OprB) (36,37), and cyclodextrin porin (CymA) (38, 39). The on-rate constant (k on ) was estimated from k on ϭ K⅐k off .…”

“…Our study revealed that chitohexaose is the most potent substrate of the VhChiP channel, as it blocked the ion flow even at nanomolar concentrations, and the monomeric subunit was already saturated below 1 M. In Table 2, we summarize the rate constants: the on-rate (k on ) is by far the highest for chitohexaose, whereas the off-rate (k off ) is the lowest. Consequently, the resultant binding constant (K) of 5.0 ϫ 10 6 M Ϫ1 is 1-5 orders stronger than the reported values for other analogs (25,31,(35)(36)(37)(38)(39)(40). According to the kinetic data in Table 2, VhChiP is the most active sugar-specific channel reported to date.…”

“…Estimation of Binding Constants-The equilibrium binding constant (K, M Ϫ1 ) was estimated from the reduction of the ion conductance in the presence of increasing concentrations of sugar using Equation 1 (24,25),…”

Molecular Basis of Bacterial Outer Membrane Permeability Revisited

“…A specific channel that is homologous to LamB (25% identity at the amino acid level), ScrY, is encoded by a plasmid that allows some strains of E. coli and Salmonella to utilize sucrose as a carbon source (256). This channel allows the rapid diffusion of a large variety of sugars, such as glucose, fructose, arabinose, maltose, lactose, raffinose, and maltodextrins, in addition to sucrose (13,256,591,595). The N-terminal segment of ScrY, about 70 residues long, has no equivalent part in LamB, and in the crystal structure of ScrY this portion was disordered.…”

Chitoporin from the Marine Bacterium Vibrio harveyi

“…Detailed assessment of the kinetic parameters suggested that VhChiP was most active with chitohexaose, its binding constant of K ϭ 500,000 M Ϫ1 being several orders of magnitude higher than that of its sugar-specific homologues, such as sucrose-specific porin, maltose-specific porin, glucose-inducible porin, and cyclodextrin-specific porin (12)(13)(14)(15)(16)(17)(18)(19). According to these data, VhChiP is the most active sugar-specific porin reported to date.…”