The determination of binding constants of micellar-packaged gramicidin A by 13C-and 23Na-NMR

摘要

Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, β6.3-helix in dodecyl phosphocholine (DPC) lipid micelles (Jing et al. (1994) Biophys. J. 66, A353), the determination of cation binding sites for gramicidin A (GA) in DPC micelles becomes a significant step in the study of ion transport through the model channel. First, the investigation of cation binding sites in DPC micellar packaged gramicidin A was achieved by 13C-NMR experiments at 30°C using four C-13 labeled GA samples. Then, the analyses based on two different equations, one for single and one for double occupancy, were employed to evaluate the correct occupancy model for GA in DPC micelles. The results clearly indicate double occupancy to be correct for Na+ ion as well as for K+, Rb+, Cs+ and Tl+ ions. Finally, the binding constants for Na+ ion were also estimated by the measurement of the longitudinal relaxation time (T1) using 23Na-NMR of the same sample at the same temperature as used for the 13C-NMR study. The binding constants obtained from 23Na-NMR are essentially equivalent to those determined from the 13C-chemical shifts.