Bacterial proteins DsbB, VKOR, and the mammalian protein VKORC1 share similar functions involving electron transfer processes. While DsbB is not homologous to the bacterial and mammalian VKOR proteins, the three proteins share overall structural features. Based on the similarities between the three proteins and the finding that mtbVKOR can replace DsbB in E. coli, we considered the possibility of rat VKORC1 displaying similar functionality as mtbVKOR. Genetic selection and screening was done on an EMS mutagenized ΔdsbB strain expressing rat VKORC1wt from a plasmid for isolation of E.coli mutants that would facilitate complementation of the lack of DsbB by VKORC1wt. The principle for the genetic selection and screening is the restoration of disulfide bond forming pathway by replacement of DsbB. This phenotype of complementation can be assayed by restoration of motility, resistance to TCEP, and β-galactosidase inactivation on ∆dsbB strain. Results of the selection and screening process revealed mutations in the VKORC1 gene instead of the E.coli chromosome. On the other hand, we used a rational approach other than genetic screening. This approach involved targeting YidC hydrophilic groove, previously identified upon selection of mutants that facilitated functional expression of VKORC1∆AAR; a deletion of amino acids 31–33 (AAR), where other mutations inactivating protease HslV were also identified. For this approach, chromosomal mutations were introduced on selected residues in the YidC hydrophilic groove then functional expression of VKORC1wt or enhanced expression of VKORC1∆AAR in the new strains was assayed. We identified novel YidC mutants enhancing the expression of VKORC1∆AAR. From the analysis of these mutations and the VKORC1 mutations obtained from the screens, we concluded that the charge imbalance by VKORC1wt violates the positive-inside rule impeding its ability to substitute DsbB. The correct assembly of VKORC1∆AAR provided insight on the involvement of E.coli YidC in correct folding and insertion of foreign membrane proteins, with the hydrophilic groove being core for its membrane insertase functions. The improved functional expression of VKORC1∆AAR upon HslV inactivation provided insight on the mis-assembly of foreign membrane proteins as a quality control system. These findings suggested that the native VKORC1wt may not assemble properly in the E.coli inner membrane, and is degraded by proteases as a quality control mechanism.
| Identifer | oai:union.ndltd.org:oulo.fi/oai:oulu.fi:nbnfioulu-201510132059 |
| Date | 13 October 2015 |
| Creators | Mandela, E. (Eric) |
| Publisher | University of Oulu |
| Source Sets | University of Oulu |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/masterThesis, info:eu-repo/semantics/publishedVersion |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess, © Eric Mandela, 2015 |
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