Voltage-sensor domains (VSDs), found in many voltage-sensitive ion channels and enzymes, are composed of four transmembrane helices (TMHs), including the atypical, highly positively charged S4 helix. VSDs are cotranslationally inserted into the membrane, raising the question of how the highly charged S4 helix is integrated into the lipid bilayer as it exits the ribosome. Here, we have used force profile analysis (FPA) to follow the cotranslational insertion of the six-TMH KvAP voltage-sensitive ion channel into the Escherichia coli inner membrane. We find that the insertion process proceeds through three semi-independent steps: i) insertion of the S1-S2 helix hairpin, ii) insertion of the S3-S5 helices, and iii) insertion of the Pore and S6 helices. Our analysis highlights the importance of the concerted insertion of helical hairpins, the dramatic influence of the positively charged residues in S4, and the unexpectedly strong forces and effects on downstream TMHs elicited by amphipathic and re-entrant helices.

Solution-state NMR can be used to study protein–lipid interactions, in particular, the effect that proteins have on lipids. One drawback is that only small assemblies can be studied, and therefore, fast-tumbling bicelles are commonly used. Bicelles contain a lipid bilayer that is solubilized by detergents. A complication is that they are only stable at high concentrations, exceeding the CMC of the detergent. This issue has previously been addressed by introducing a detergent (Cyclosfos-6) with a substantially lower CMC. Here, we developed a set of bicelles using this detergent for studies of membrane-associated mycobacterial proteins, for example, PimA, a key enzyme for bacterial growth. To mimic the lipid composition of mycobacterial membranes, PI, PG, and PC lipids were used. Diffusion NMR was used to characterize the bicelles, and spin relaxation was used to measure the dynamic properties of the lipids. The results suggest that bicelles are formed, although they are smaller than “conventional” bicelles. Moreover, we studied the effect of MTSL-labeled PimA on bicelles containing PI and PC. The paramagnetic label was shown to have a shallow location in the bicelle, affecting the glycerol backbone of the lipids. We foresee that these bicelles will be useful for detailed studies of protein–lipid interactions. 

We follow the cotranslational biosynthesis of three multispanning Escherichia coli inner membrane proteins in vivo using high-resolution force profile analysis. The force profiles show that the nascent chain is subjected to rapidly varying pulling forces during translation and reveal unexpected complexities in the membrane integration process. We find that an N-terminal cytoplasmic domain can fold in the ribosome exit tunnel before membrane integration starts, that charged residues and membrane-interacting segments such as re-entrant loops and surface helices flanking a transmembrane helix (TMH) can advance or delay membrane integration, and that point mutations in an upstream TMH can affect the pulling forces generated by downstream TMHs in a highly position-dependent manner, suggestive of residue-specific interactions between TMHs during the integration process. Our results support the 'sliding' model of translocon-mediated membrane protein integration, in which hydrophobic segments are continually exposed to the lipid bilayer during their passage through the SecYEG translocon.