Single-Molecule Force Spectroscopy of Transmembrane β-Barrel Proteins

Single-molecule force spectroscopy (SMFS) has been widely applied to study the mechanical unfolding and folding of transmembrane proteins. Here, we review the recent progress in characterizing bacterial and human transmembrane β-barrel proteins by SMFS. First, we describe the mechanical unfolding of transmembrane β-barrels, which follows a general mechanism dictated by the sequential unfolding and extraction of individual β-strands and β-hairpins from membranes. Upon force relaxation, the unfolded polypeptide can insert stepwise into the membrane as single β-strands or β-hairpins to fold as the native β-barrel. The refolding can be followed at a high spatial and temporal resolution, showing that small β-barrels are able to fold without assistance, whereas large and complex β-barrels require chaperone cofactors. Applied in the dynamic mode, SMFS can quantify the kinetic and mechanical properties of single β-hairpins and reveal complementary insight into the membrane protein structure and function relationship. We further outline the challenges that SMFS experiments must overcome for a comprehensive understanding of the folding and function of transmembrane β-barrel proteins.