Membrane Protein Folding and Biogenesis: Insights from Single-Molecule Force Spectroscopy.

Membrane proteins perform essential cellular functions, and their remarkable versatility arises from the precise assembly of complex folded structures within the dynamic lipid bilayer environment. Deciphering their folding mechanisms and pathways requires capturing transient intermediate states that are often obscured in conventional ensemble measurements. Single-molecule force spectroscopy (SMFS) has emerged as a powerful, high-resolution approach for probing membrane protein folding, providing access to detailed folding trajectories and the underlying thermodynamic and kinetic principles. After outlining the biogenesis of membrane proteins─including targeting, insertion, folding, and oligomerization─we discuss how SMFS approaches can resolve transient folding intermediates, quantify their transition rates, and reconstruct detailed folding energy landscapes. We further highlight emerging SMFS modalities, including those capable of mapping multistep oligomerization processes, directly linking folding dynamics to functional outputs, and probing folding events within cellular extracts. These advanced SMFS methods for membrane proteins offer promising opportunities for integrating molecular folding landscapes with cellular-scale biological complexity.