Abstract
Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome. Studies thus far, however, have largely focussed on refolding full-length proteins from artificially induced denatured states that are far removed from the natural co-translational process. Cell-free translation offers opportunities to remedy this deficit in folding studies and has previously been used for membrane proteins. We exploit this cell-free approach to develop tools to probe co-translational folding. We show that two transporters from the ubiquitous Major Facilitator Superfamily can successfully insert into a synthetic bilayer without the need for translocon insertase apparatus that is essential in vivo. We also assess the cooperativity of domain insertion, by expressing the individual transporter domains cell-free. Furthermore, we manipulate the cell-free reaction to pause and re-start protein synthesis at specific points in the protein sequence. We find that full-length protein can still be made when stalling after the first N terminal helix has inserted into the bilayer. However, stalling after the first three helices have exited the ribosome cannot be successfully recovered. These three helices cannot insert stably when ribosome-bound during co-translational folding, as they require insertion of downstream helices.
Highlights
Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome
Liposomes composed of a 25:50:25 mol ratio of DOPC:DOPE:DOPG were supplied during synthesis, as we have found previously in studies of purified LacY that this lipid composition supports correct folding and function[17,37]
The sucrose gradient was fractionated into a top and bottom fraction, and the amount of cell-free synthesised protein in each detected by either western blotting or quantified by liquid www.nature.com/scientificreports scintillation counting (LSC) of incorporated [35S]-methionine
Summary
Most helical membrane proteins fold co-translationally during unidirectional polypeptide elongation by the ribosome. Manipulation of the lipid composition can increase the efficiency of insertion in the absence of a translocon, as demonstrated for GlpG, DsbB16, the human endothelin B receptor[21], the β1-adrenergic receptor[22], and a number of others[23,24,25,26,27,28] These studies set a precedent for extending our cell free, co-translational approach to larger membrane proteins to ascertain whether dynamic, multidomain proteins can insert spontaneously into lipid bilayers and fold correctly in the absence of translocon apparatus. We investigate the lipid dependence of LacY and XylE cell-free co-translational folding, by first using phosphocholine lipids as a neutral reference bilayer and measuring the insertion efficiency of each transporter in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). We found that changing the lipid composition of the liposomes alters the yield of LacY and XylE in the bilayer, with an increase in DOPE and DOPG improving the yield of both transporters
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