Forces and energetics of hapten-antibody dissociation: A biased molecular dynamics simulation study

The unbinding of fluorescein from the single-chain Fv fragment of the 4D5Fl u antibody is investigated by biased molecular dynamics with an implicit so lvation model. To obtain statistically meaningful results, a large number o f unbinding trajectories are calculated; they involve a total simulation ti me of more than 200 ns. Simulations are carried out with a time-dependent p erturbation and in the presence of a constant force. The two techniques, wh ich provide complementary information, induce unbinding by favoring an incr ease in the distance between the ligand and the antibody. This distance is an appropriate progress variable for the dissociation reaction and permits direct comparison of the unbinding forces in the simulations with data from atomic force microscopy (AFM). The time-dependent perturbation generates u nfolding pathways that are close to equilibrium and can be used to reconstr uct the mean force; i.e. the derivative of the potential of mean force, alo ng the reaction coordinate. This is supported by an analysis of the overall unbinding profile and the magnitude of the mean force, which are similar t o those of the unbinding force (i.e. the external force due to the time-dep endent perturbation) averaged over several unbinding events. The multiple simulations show that unbinding proceeds along a rather well-d efined pathway for a broad range of effective pulling speeds. Initially, th ere is a distortion of the protein localized in the C-terminal region follo wed by the fluorescein exit from the binding site. This occurs in steps tha t involve breaking of specific electrostatic and van der Waals interactions . It appears that the simulations do not explore the same barriers as those measured in the AFM experiments because of the much higher unfolding speed in the former. The dependence of the force on the logarithm of the loading rate is linear and the slope is higher than in the AFM, in agreement with experiment in other systems, where different slopes were observed for diffe rent regimes. Based on the unbinding events, mutations in the 4D5Flu antige n binding site are predicted to result in significant changes in the unbind ing force. (C) 2001 Academic Press.