A general and Efficient All-Atom Simulation Method to Determine the Equilibrium Orientation of Transmembrane Proteins in Membranes

Abstract

Knowledge of the spatial orientation of transmembrane (TM) proteins is broadly useful in both in-vitro and in-silico approaches that aim to elucidate the dynamic and functional properties of TM proteins. Such approaches include site-directed spin labeling, cys-scanning mutagenesis, and atomistic molecular dynamics (MD) simulations using lipid bilayers. Because three-dimensional structures of TM proteins are generally obtained in a detergent solvent, these structures do not reveal the native orientation of the protein in the lipid membrane. For this reason, we aimed to develop a general and computationally-efficient MD approach to predict the most favorable orientation of TM proteins in a lipid membrane. In order to avoid the long relaxation time scales characterizing protein and lipid bilayer dynamics, our method treats the TM protein as a rigid body in a membrane-mimetic hydrated octane slab, allowing the protein to reach a stable orientation within 10 ns. The method was systematically tested on alpha-helical and beta-barrel TM proteins, each with different starting orientations in the hydrated octane slab. Each protein attained a consistent orientation irrespective of its starting orientation. Furthermore, the converged orientations are in good agreement with the known orientations of these test proteins in lipid bilayers. These results indicate that this method is reliable as a general protocol that can be used to determine the orientation of TM proteins of known structure.