Three-stage model of helical membrane protein folding: Role of membrane-water interface as the intermediate stage vestibule for TM helices during their in membrano assembly

Integral membrane proteins (MPs) are dominated by transmembrane α-helical (TMH) proteins playing critical roles in cellular signaling processes. These proteins display a wide range of sizes from one TMH domain to at least 26 TMH domains and diverse structural folds. A common feature of most of these folds is the TM orientation of the helical domains and the approximately parallel packing of these domains into helical bundles of varying stability, however, it has been challenging to study the folding of these proteins experimentally. The contribution of helix stabilization in membrane and interface to the folding energy landscape are investigated here for the full range of TMH protein sizes containing 1 TM domain (1-TMH protein) to 24 TM domains (24-TMH protein) for all TMH proteins with available structures using structural bioinformatics based hydropathy analysis. The TM helix insertion stabilization energies from Water to membrane-water Interface (WAT→INT energies) are on average half of those insertion energies from water to transmembrane orientation (WAT→TM energies) for the whole polytopic helical membrane proteome (1-TMH to 24-TMH proteins). This suggests a potentially dominant role of the membrane-water interface as a viable holding vestibule for the TM helices during their release from the translocon. This provides proteome-level evidence for the broadly applicable four-step thermodynamic framework by White and co-workers as well as a natural extension of Popot and Engelman's original two-stage model of helical MP folding to a three-stage model, where, in the new intermediate stage, the membrane-water interface acts as a holding vestibule for the translated TM helices, reconciling the interface's critical role in MP folding seen in many previous studies. Support for this model is provided by showing the stability of hydrophobic TM helices at the membrane-water interface through several microsecond long molecular dynamics simulations of five hydrophobic helical domains and a helical hairpin pre-folded from the ribosomal exit vestibule.