FK506-Binding Protein 2 Participates in Proinsulin Folding.

Apart from chaperoning, disulfide bond formation, and downstream processing, the molecular sequence of proinsulin folding is not completely understood. Proinsulin requires proline isomerization for correct folding. Since FK506-binding protein 2 (FKBP2) is an ER-resident proline isomerase, we hypothesized that FKBP2 contributes to proinsulin folding. We found that FKBP2 co-immunoprecipitated with proinsulin and its chaperone GRP94 and that inhibition of FKBP2 expression increased proinsulin turnover with reduced intracellular proinsulin and insulin levels. This phenotype was accompanied by an increased proinsulin secretion and the formation of proinsulin high-molecular-weight complexes, a sign of proinsulin misfolding. FKBP2 knockout in pancreatic ß-cells increased apoptosis without detectable up-regulation of ER stress response genes. Interestingly, FKBP2 mRNA was overexpressed in ß-cells from pancreatic islets of T2D patients. Based on molecular modeling and an in vitro enzymatic assay, we suggest that proline at position 28 of the proinsulin B-chain (P28) is the substrate of FKBP2's isomerization activity. We propose that this isomerization step catalyzed by FKBP2 is an essential sequence required for correct proinsulin folding.

Isolation and Crystallization of the D156C Form of Optogenetic ChR2.

Channelrhodopsins (ChRs) are light-gated ion channels that are receiving increasing attention as optogenetic tools. Despite extensive efforts to gain understanding of how these channels function, the molecular events linking light absorption of the retinal cofactor to channel opening remain elusive. While dark-state structures of ChR2 or chimeric proteins have demonstrated the architecture of non-conducting states, there is a need for open- and desensitized-state structures to uncover the mechanistic principles underlying channel activity. To facilitate comprehensive structural studies of ChR2 in non-closed states, we report a production and purification procedure of the D156C form of ChR2, which displays prolonged channel opening compared to the wild type. We demonstrate considerable yields (0.45 mg/g fermenter cell culture) of recombinantly produced protein using S. cerevisiae, which is purified to high homogeneity both as opsin (retinal-free) and as functional ChR2 with added retinal. We also develop conditions that enable the growth of ChR2 crystals that scatter X-rays to 6 Å, and identify a molecular replacement solution that suggests that the packing is different from published structures. Consequently, our cost-effective production and purification pipeline opens the way for downstream structural studies of different ChR2 states, which may provide a foundation for further adaptation of this protein for optogenetic applications.

Saccharomyces cerevisiae as a superior host for overproduction of prokaryotic integral membrane proteins.

Integral membrane proteins (IMPs) constitute ~30% of all proteins encoded by the genome of any organism and Escherichia coli remains the first-choice host for recombinant production of prokaryotic IMPs. However, the expression levels of prokaryotic IMPs delivered by this bacterium are often low and overproduced targets often accumulate in inclusion bodies. The targets are therefore often discarded to avoid an additional and inconvenient refolding step in the purification protocol. Here we compared expression of five prokaryotic (bacterial and archaeal) IMP families in E. coli and Saccharomyces cerevisiae. We demonstrate that our S. cerevisiae-based production platform is superior in expression of four investigated IMPs, overall being able to deliver high quantities of active target proteins. Surprisingly, in case of the family of zinc transporters (Zrt/Irt-like proteins, ZIPs), S. cerevisiae rescued protein expression that was undetectable in E. coli. We also demonstrate the effect of localization of the fusion tag on expression yield and sample quality in detergent micelles. Lastly, we present a road map to achieve the most efficient expression of prokaryotic IMPs in our yeast platform. Our findings demonstrate the great potential of S. cerevisiae as host for high-throughput recombinant overproduction of bacterial and archaeal IMPs for downstream biophysical characterization.