No abstract

Escherichia coli K-12 strain PS1-28-37 carries the multicopy plasmid pPSO28-37 containing a DNA fragment coding for two of the proteins that enable bacteria to utilize sucrose as sole carbon source. One of the different gene products of the plasmid is the outer membrane protein, ScrY. This protein was isolated and purified by chromatography across a gel filtration column. Reconstitution experiments with lipid bilayer membrane demonstrated that ScrY formed ion-permeable channels with properties very similar to those of general diffusion pores of enteric bacteria. The presence of sugars in the aqueous phase led to a dose-dependent block of ion transport through the channel, like the situation found with LamB (maltoporin) of Escherichia coli and Salmonella typhimurium. The binding constants of a variety of different sugars were determined. The stability constant for malto-oligosaccharide binding increased with increasing numbers of glucose residues. Disaccharides generally had a larger binding constant than monosaccharides. The binding of different sugars to ScrY and LamB of E. coli is discussed with respect to the kinetics of sugar movement through the channel.

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.

LamB (maltoporin) of Salmonella typhimurium was found to be more strongly associated with the murein than OmpF. It was purified in one step using a hydroxyapatite (HTP) column. Reconstitution of the pure protein with lipid bilayer membrane showed that LamB of S. typhimurium formed small ion-permeable channels with a single channel conductance of about 90 pS in 1 M KCl and some preference for cations over anions. The conductance concentration curve was linear, which suggested that LamB of S. typhimurium does not contain any binding site for ions. Pore conductance was completely inhibited by the addition of 20 mM maltotriose. Titration of the LamB-induced membrane conductance with different sugars, including all members of the maltooligosaccharide series up to seven glucose residues, suggested that the channel contains, like LamB (maltoporin) of Escherichia coli, a binding site for sugars. The binding constant of sugars of the maltooligosaccharide series increased with increasing number of glucose residues up to five (saturated). Small sugars had a higher stability constant for sugar binding relative to LamB of E. coli. The advantage of a binding site inside a specific porin for the permeation of solutes is discussed with respect to the properties of a general diffusion porin.