Role of N-Terminal His6-Tags in Binding and Efficient Translocation of Polypeptides into Cells Using Anthrax Protective Antigen (PA)
Figure 1
Interaction of C2I with PA63 channels.
A: Titration of PA63 induced membrane conductance with His6-C2I. The membrane was painted from 1% (w/v) diphytanoyl phosphatidylcholine dissolved in n-decane. It contained about 300 PA63-channels. His6-C2I was added at the concentrations shown at the top of the panel to the cis-side of the membrane. Finally, about 83% of the PA63-channels were blocked. The aqueous phase contained 1 ng/ml activated PA63 (added only to the cis-side of the membrane), 150 mM KCl, 10 mM MES-KOH pH 6. The temperature was 20°C and the applied voltage was 20 mV. B: Lineweaver-Burk (double reciprocal) plot of the inhibition of the PA63-induced membrane conductance by His6-C2I using equation (2). The fit was obtained by linear regression of the data points taken from Figure 1A and corresponds to a stability constant K for His6-C2I binding to PA63 of (3.93±0.39)×107 M−1 (r = 0.955; half saturation constant Ks = 25 nM).