The bacterium Saccharopolyspora erythraea is utilized to produce the widely used antibiotic erythromycin. With the increasing problem of antibiotic resistance, the development of derivatives of the drug is of great importance. To produce antibiotics biosynthetically, it is crucial to understand the metabolic pathways utilized by the bacterium. It is key to clarify the behaviour of the proteins that sense the environment and regulate the gene expression accordingly. The iron-dependent regulator IdeR utilizes iron as a cofactor and is the main transcriptional regulator acting on genes crucial for iron homeostasis. Its main function is to act as a repressor for genes that are necessary for iron uptake, but it also functions as an activator for genes that promote iron storage. The role of the protein’s SH3-like domain in gene regulation is not understood. However, it might serve as a crosslinker between dimers bound to adjacent binding sites and be necessary for proper gene regulation. The aim of this paper was to examine this hypothesis. Cysteine residues with high probability of forming disulfide bonds between the SH3-like domain and the rest of the protein were introduced to IdeR and the constructed variants characterized. Methods used included electrophoretic mobility shift assay (EMSA), circular dichroism (CD) spectroscopy, and small-angle X-ray scattering (SAXS). Attempts to characterize the proteins using differential scanning fluorimetry (DSF) and X-ray crystallography were also performed. Four IdeR variants were produced and purified. The EMSA analysis indicated that theiraffinities for the consensus DNA sequence was similar to or a bit lower than that of the wild-type (wt). The CD spectroscopy measurements showed that the variants had, similar to the wt, mainly α-helical secondary structure. One of the variants had, however, less secondary structure content than the wt and was probably less stable. The secondary structure content of the wt did not change upon metal binding. The variants’ melting temperatures under oxidizing conditions were similar to or lower than that of the wt. The same experiment in reducing environment showed a loss of cooperativity for all examined variants. No data was gained from the DSF experiment and the IdeR variants did not crystallize under the investigated conditions during the project timeline. The results from the SAXS experiment show that the IdeR variants had the same overall structure as the wt as dimers and when bound to DNA. Conclusions that can be drawn from the experiments are that the introduced mutations did not affect the overall structure nor the thermal stability of the protein. However, since it cannot be concluded from the performed experiments if the crosslink between the protein domains formed as intended, another attempt to get crystal structures of the proteins is required.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:uu-481090 |
| Date | January 2022 |
| Creators | Hammar, Carita |
| Publisher | Uppsala universitet, Institutionen för kemi - BMC |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
| Relation | UPKEM E ; 343 |
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