Structure formation during translocon-unassisted co-translational membrane protein folding

Nicola J Harris,Ramona Schlesinger,Lucjan Grzegorzewski,Xia Liu,Paula J Booth,Joachim Heberle,Kenichi Ataka,Kalypso Charalambous,Eamonn Reading

doi:10.1038/s41598-017-08522-9

Nicola J Harris, Ramona Schlesinger + Show 7 more

Open Access

https://doi.org/10.1038/s41598-017-08522-9

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Abstract

Correctly folded membrane proteins underlie a plethora of cellular processes, but little is known about how they fold. Knowledge of folding mechanisms centres on reversible folding of chemically denatured membrane proteins. However, this cannot replicate the unidirectional elongation of the protein chain during co-translational folding in the cell, where insertion is assisted by translocase apparatus. We show that a lipid membrane (devoid of translocase components) is sufficient for successful co-translational folding of two bacterial α-helical membrane proteins, DsbB and GlpG. Folding is spontaneous, thermodynamically driven, and the yield depends on lipid composition. Time-resolving structure formation during co-translational folding revealed different secondary and tertiary structure folding pathways for GlpG and DsbB that correlated with membrane interfacial and biological transmembrane amino acid hydrophobicity scales. Attempts to refold DsbB and GlpG from chemically denatured states into lipid membranes resulted in extensive aggregation. Co-translational insertion and folding is thus spontaneous and minimises aggregation whilst maximising correct folding.

Highlights

The folding of proteins from an amino acid sequence to their correct functional state is a fundamental process that is essential to the biological activity of living systems
We address several key questions: is insertion and folding directly into the lipid membrane, without any translocon apparatus, co-translational? Does the lipid composition, and associated headgroup charge and bilayer mechanical properties, modulate membrane insertion yield? Do membrane proteins fold through different co-translational pathways, and if so, is it linked to their amino acid sequence? Can the thermodynamic free energy alone produce folded and functional membrane proteins akin to those produced in vivo? Is co-translational folding advantageous in avoiding aggregation that hinders folding from chemically denatured states?
We have shown that the membrane proteins GlpG and DsbB can insert spontaneously into lipid membranes during co-translational synthesis in the absence of cellular translocase machinery, and can fold efficiently into functional membrane proteins

Summary

Results

Cell-free synthesis in the presence of a lipid membrane can produce spontaneously inserted GlpG and DsbB. When attempting to transfer GlpG and DsbB from a SDS solubilised state into liposomes (final SDS concentration 0.5 mM), most of the protein remained in the bottom fraction of the sucrose gradient, indicating poor membrane insertion and aggregation during this process, despite variations in lipid composition (Fig. S6). This lack of insertion is in line with previous reports for DsbB28 which, despite its insignificant structural loss in SDS, did not efficiently transfer into PC lipid vesicles. The results for chemically denatured proteins are in stark contrast to our cell-free inserted GlpG and DsbB into lipid membranes (Fig. 2), which produced significant amounts of homogenous folded proteins possessing a specific activity comparable to the proteins isolated from E. coli

Discussion

Materials and Methods

Methods