Monitoring the binding and insertion of a single transmembrane protein by an insertase

Cells employ highly conserved families of insertases and translocases to insert and fold proteins into membranes. How insertases insert and fold membrane proteins is not fully known. To investigate how the bacterial insertase YidC facilitates this process, we here combine single-molecule force spectroscopy and fluorescence spectroscopy approaches, and molecular dynamics simulations. We observe that within 2 ms, the cytoplasmic alpha-helical hairpin of YidC binds the polypeptide of the membrane protein Pf3 at high conformational variability and kinetic stability. Within 52 ms, YidC strengthens its binding to the substrate and uses the cytoplasmic alpha-helical hairpin domain and hydrophilic groove to transfer Pf3 to the membrane-inserted, folded state. In this inserted state, Pf3 exposes low conformational variability such as typical for transmembrane alpha-helical proteins. The presence of YidC homologues in all domains of life gives our mechanistic insight into insertase-mediated membrane protein binding and insertion general relevance for membrane protein biogenesis. The insertion and folding nascent or fully synthesized polypeptides into membranes is assisted by insertases. Here, the authors use a range of biophysical approaches to provide molecular details of how the transmembrane insertase YidC facilitates the insertion a protein into a phospholipid membrane.