Dataset Information

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

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.

SUBMITTER: Harris NJ

PROVIDER: S-EPMC7272624 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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

Harris Nicola J NJ Pellowe Grant A GA Booth Paula J PJ

Scientific reports 20200604 1

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 p ...[more]

PMID: 32499529

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Project description:Intrinsically disordered proteins (IDPs) are abundant in eukaryotic proteomes and preform critical roles in many cellular processes, most often through the association with globular proteins. Despite lacking a stable three-dimensional structure by themselves, they may acquire a defined conformation upon binding globular targets. The most common type of secondary structure acquired by these binding motifs entails formation of an α-helix. It has been hypothesized that such disorder-to-order transitions are associated with a significant free energy penalty due to IDP folding, which reduces the overall IDP-target affinity. However, the exact magnitude of IDP folding penalty in α-helical binding motifs has not been systematically estimated. Here, we report the folding penalty contributions for 30 IDPs undergoing folding-upon-binding and find that the average IDP folding penalty is +2.0 kcal/mol and ranges from 0.7 to 3.5 kcal/mol. We observe that the folding penalty scales approximately linearly with the change in IDP helicity upon binding, which provides a simple empirical way to estimate folding penalty. We analyze to what extent do pre-structuring and target-bound IDP dynamics (fuzziness) reduce the folding penalty and find that these effects combined, on average, reduce the folding cost by around half. Taken together, the presented analysis provides a quantitative basis for understanding the role of folding penalty in IDP-target interactions and introduces a method estimate this quantity. Estimation and reduction of IDP folding penalty may prove useful in the rational design of helix-stabilized inhibitors of IDP-target interactions. STATEMENT: The α-helical binding motifs are ubiquitous among the intrinsically disordered proteins (IDPs). Upon binding their targets, they undergo a disorder-to-order transition, which is accompanied by a significant folding penalty whose magnitude is generally not known. Here, we use recently developed statistical-thermodynamic model to estimate the folding penalties for 30 IDPs and clarify the roles of IDP pre-folding and bound-state dynamics in reducing the folding penalty.

Dataset Information

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

Publications

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

Similar Datasets

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