Single Molecule Force Spectroscopy to Probe Intermediates and Energetics of Membrane Protein Folding

Beyond structure, understanding membrane-protein folding and dynamics requires precise dissection of interaction energetics and mechanistic insight into the in vivo folding process. Over the past 25 years, single-molecule force spectroscopy (SMFS) measurements, in which individual membrane proteins are probed by application of mechanical force, have emerged as a new way of probing these aspects of membrane proteins. The field has advanced both by focusing attention on increasingly biologically realistic (or increasingly ingeniously experimentally contrived) systems and by expanding the technical capabilities of single-molecule manipulation experiments. This review explores key developments along both lines. It begins with a discussion of SMFS experimental principles as applied to membrane proteins and how assay-design and instrumentation advances have enabled higher quality and novel measurements. The review then explores how these advances have led to─and will continue to enable─progress in understanding key questions in membrane-protein folding: the formation of folded structures, the adoption of topology, the individual contributions to thermodynamic stability, and the interactions between proteins.