CymA of Klebsiella oxytoca Outer Membrane: Binding of Cyclodextrins and Study of the Current Noise of the Open Channel

摘要

CymA, the outer membrane component of the cyclodextrin (CD) uptake and metabolism system of Klebsiella oxytoca, was reconstituted into lipid bilayer membranes. The channel properties of this unusual porin were studied in detail. The binding of CDs to the channel resulted in its complete block for ion transport. This result allowed the detailed investigation of carbohydrate binding, and the stability constants for the binding of cyclic and linear carbohydrates to the binding site inside the channel were calculated from titration experiments of the membrane conductance with the carbohydrates. Highest stability constant was observed for α-cyclodextrin (α-CD; K = 32,000 1/M) followed by β-cyclodextrin (β-CD; K = 1970 1/M) and γ-cyclodextrin (γ-CD; K = 310 1/M). Linear maltooligosaccharides bound also to CymA but with much smaller stability constants as compared to cyclic ones. The noise of the current through CymA in multi- and single-channel experiments was investigated using fast Fourier transformation. The current through the open channels had a rather high spectral density, which was a Lorentzian function of the frequency up to 2000 Hz. Upon addition of cyclic dextrins to the aqueous phase the spectral density decreased in a dose-dependent manner, which made it impossible to evaluate the binding kinetics. Experiments with single CymA-channels demonstrated the channel is highly asymmetric concerning channel flickers and current noise.