Cell-free expression tools to study co-translational folding of alpha helical membrane transporters

• Published in: Scientific reports Vol. 10; no. 1; p. 9125
• Main Authors: Harris, Nicola J., Pellowe, Grant A., Booth, Paula J.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 04-06-2020; Nature Publishing Group
• Subjects: 631/45/287; 631/45/470; 631/57/2270; 82/80; 82/83; Amino acid sequence; Cell-Free System; Databases, Protein; Escherichia coli - metabolism; Escherichia coli Proteins - chemistry; Escherichia coli Proteins - genetics; Escherichia coli Proteins - metabolism; Humanities and Social Sciences; Liposomes - metabolism; Membrane proteins; Membrane Transport Proteins - chemistry; Membrane Transport Proteins - genetics; Membrane Transport Proteins - metabolism; Monosaccharide Transport Proteins - chemistry; Monosaccharide Transport Proteins - genetics; Monosaccharide Transport Proteins - metabolism; multidisciplinary; Mutagenesis, Site-Directed; Protein biosynthesis; Protein Conformation, alpha-Helical; Protein Folding; Protein synthesis; Proteins; Science; Science (multidisciplinary); Symporters - chemistry; Symporters - genetics; Symporters - metabolism; Translation
• ISSN: 2045-2322; 2045-2322
• DOI: 10.1038/s41598-020-66097-4

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.