Structure and Energetics of Channel-Forming Protein−Polysaccharide Complexes Inferred via Computational Statistical Thermodynamics

The ion channel protein α-hemolysin (αHL) forms supramolecular complexes with the polysaccharide β-cyclodextrin (βCD). This system has potential uses in nanoscale device engineering. It has been found recently that βCD formed longer- or shorter-lived complexes with some engineered αHL mutants then with a wild type protein (Gu et al. J. Gen. Physiol. 2001, 118, 481−493). However, how changes in the protein sequence affect complex lifetime was not completely understood in part due to the lack of knowledge of structures of these metastable complexes. In this paper, we present an extensive molecular modeling study of the βCD−αHL and selected mutant complexes to gain insights into the βCD−αHL interaction mechanisms and to predict possible structures and energetics of the complexes. Thermodynamic integration (TI) and umbrella sampling (US) techniques (with the weighted histogram analysis method (WHAM)) were used to calculate the relative binding affinities of the complexes formed with the wild type αHL and the M113N, M113E, M113A, and M113V mutants. Our results are in excellent agreement with experiment. While βCD−M113N and βCD−M113A complexes were stable in the configuration of the wild type complex, the equilibrium configuration of the βCD−M113V and βCD−M113E complexes was significantly different. In these cases, TI alone was insufficient to accurately calculate the corresponding free energy differences. By utilizing a TI/US combination in a novel manner, we were able to accurately calculate free energy changes in these flexible systems. The βCD−M113A and βCD−M113E complexes, which exhibited shorter lifetimes than other complexes in an experiment, in simulations exhibited greater flexibility and higher water solvation of the βCD adapter. MD simulations of the βCD−M113N complex with βCD in a downward orientation were also performed.