The essential forces stabilizing membrane proteins and governing their folding and unfolding are difficult to decipher. Single-molecule atomic force spectroscopy mechanically unfolds individual membrane proteins and quantifies their dynamics and energetics. However, it remains challenging to structurally assign unfolding intermediates precisely and to deduce dominant interactions between specific residues that facilitate either the localized stabilization of these intermediates or the global assembly of membrane proteins. Here, we performed force spectroscopy experiments and multiscale molecular dynamics simulations to study the unfolding pathway of diacylglycerol kinase (DGK), a small trimeric multispan transmembrane enzyme. The remarkable agreement between experiments and simulations allowed precise structural assignment and interaction analysis of unfolding intermediates, bypassing existing limitations on structural mapping, and thus provided mechanistic explanations for the formation of these states. DGK unfolding was found to proceed with structural segments varying in size that do not correlate with its secondary structure. We identified intermolecular side-chain packing interactions as one of the major contributions to the stability of unfolding intermediates. Mutagenesis creating packing defects induced a dramatic decrease in the mechano-stability of corresponding intermediates and also in the thermo-stability of DGK trimer, in good agreement with predictions from simulations. Hence, the molecular determinants of the mechano- and thermo-stability of a membrane protein can be identified at residue resolution. The accurate structural assignment established and microscopic mechanism revealed in this work may substantially expand the scope of single-molecule studies of membrane proteins.
© 2024 The Authors. Published by American Chemical Society.