Molecular dynamics force probe simulations of antibody/antigen unbinding: Entropic control and non-additivity of unbinding forces.

Unbinding of a spin-labeled dinitrophenyl (DNP) hapten from the monoclonal antibody AN02 Fab fragment has been studied by force probe molecular dynamics (FPMD) simulations. In our nanosecond simulations, unbinding was enforced by pulling the hapten molecule out of the binding pocket. Detailed inspection of the FPMD trajectories revealed a large heterogeneity of enforced unbinding pathways and a correspondingly large flexibility of the binding pocket region, which exhibited induced fit motions. Principal component analyses were used to estimate the resulting entropic contribution of ∼6 kcal/mol to the AN02/DNP-hapten bond. This large contribution may explain the surprisingly large effect on binding kinetics found for mutation sites that are not directly involved in binding. We propose that such “entropic control” optimizes the binding kinetics of antibodies. Additional FPMD simulations of two point mutants in the light chain, Y33F and I96K, provided further support for a large flexibility of the binding pocket. Unbinding forces were found to be unchanged for these two mutants. Structural analysis of the FPMD simulations suggests that, in contrast to free energies of unbinding, the effect of mutations on unbinding forces is generally nonadditive

Water permeation across biological membranes: Mechanism and dynamics of Aquaporin-1 and GlpF.

Real time” molecular dynamics simulations of water permeation through human aquaporin-1 (AQP1) and the bacterial glycerol facilitator GlpF are presented. We obtained time-resolved, atomic-resolution models of the permeation mechanism across these highly selective membrane channels. Both proteins act as two-stage filters: Conserved fingerprint [asparagine-proline-alanine (NPA)] motifs form a selectivity-determining region; a second (aromatic/arginine) region is proposed to function as a proton filter. Hydrophobic regions near the NPA motifs are rate-limiting water barriers. In AQP1, a fine-tuned water dipole rotation during passage is essential for water selectivity. In GlpF, a glycerol-mediated “induced fit” gating motion is proposed to generate selectivity for glycerol over water

Water permeation through gramicidin A: Desformylation and the double helix: A molecular dynamics study

Multinanosecond molecular dynamics simulations of gramicidin A embedded in a dimyristoylphosphatidylcholine bilayer show a remarkable structural stability for both experimentally determined conformations: the head-to-head helical dimer and the double helix. Water permeability was found to be much higher in the double helical conformation, which is explained by lower hydrogen bond-mediated enthalpic barriers at the channel entrance and its larger pore size. Free-energy perturbation calculations show that the double helical structure is stabilized by the positive charges at the N termini introduced by the desformylation, whereas the helical dimer is destabilized. Together with the recent experimental observation that desformyl gramicidin conducts water hundredfold better than gramicidin, this suggests that desformyl gramicidin A predominantly occurs in the double helical conformation

Progress in the analysis of membrane protein structure and function

Structural information on membrane proteins is sparse, yet they represent an important class of proteins that is encoded by about 30% of all genes. Progress has primarily been achieved with bacterial proteins, but efforts to solve the structure of eukaryotic membrane proteins are also increasing. Most of the structures currently available have been obtained by exploiting the power of X-ray crystallography. Recent results, however, have demonstrated the accuracy of electron crystallography and the imaging power of the atomic force microscope. These instruments allow membrane proteins to be studied while embedded in the bi-layer, and thus in a functional state. The low signal-to-noise ratio of cryo-electron microscopy is overcome by crystallizing membrane proteins in a two- dimensional protein-lipid membrane, allowing its atomic structure to be determined. In contrast, the high signal-to- noise ratio of atomic force microscopy allows individual protein surfaces to be imaged at subnanometer resolution, and their conformational states to be sampled. This review summarizes the steps in membrane protein structure determination and illuminates recent progress. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies