Monitoring Membrane Protein Folding Assisted by Insertases and Translocases Using AFM-Based Single-Molecule Force Spectroscopy.

In this review we discuss how atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) approaches can be applied to monitor the unfolding and folding pathways of individual membrane proteins. Particularly, we focus on the insertion and folding of prokaryotic α-helical and β-barrel membrane proteins and compare their unassisted insertion and folding pathways with those assisted by insertases, translocases, and chaperones. We highlight examples in which SMFS is applied to detect the misfolding of membrane proteins such as induced by the lipid composition of the membrane or resulting from unassisted folding. While SMFS can monitor how soluble and transmembrane chaperones reduce misfolding of structural segments, it can also monitor how insertases and translocases guide their stepwise insertion and folding into membranes until the membrane protein has completed folding. Examples show that the inner membrane insertase YidC inserts structural segments in a random order, whereas the SecYEG translocon inserts transmembrane α-helices sequentially. However, when acting together, SecYEG dominates over YidC, consistent with the role of the translocon in directing membrane protein insertion and folding. Finally, we discuss β-barrel membrane protein folding in the bacterial outer membrane, including the β-barrel assembly machinery (BAM) complex, and how SMFS applied to native outer membrane vesicles provides access to monitor the insertion and folding of membrane proteins in the native-like membrane environment.