THE DETERMINATION OF BINDING CONSTANTS OF MICELLAR-PACKAGED GRAMICIDIN-A BY C-13-NMR AND NA-23-NMR

Based on the malonyl gramicidin A structure of a single-stranded head-to-head hydrogen bonded right-handed, beta(6.3)-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 C-13-NMR experiments at 30 degrees 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 (T-1) using Na-23-NMR of the same sample at the same temperature as used for the C-13-NMR study. The binding constants obtained from Na-23-NMR are essentially equivalent to those determined from the C-13-chemical shifts.