Difference between revisions of "Part:BBa K1724002"

Revision as of 17:18, 9 October 2022

MerR

The mercury –sensing regulatory protein, MerR(wild type), which regulates mercury resistance operons in Gram-negative bacteria, is subjected to directed evolution in an effect to generate a MerR mutant that responds to Cadmium ion but not mercury.That is, the MerR mutant is the cadmium-sensing regulatory protein. To get MerR mutant, Oligonucleotide-directed mutagenesis is used to introduce random mutations into the key metal-binding regions of MerR. Finally, Getting the generated Cd-specific MerR mutants appears to be unique.

The quantification data of MerR/CadA operon can be seen in BBa_K1724000.[[1]]

IMPROVEMENT: iGEM Peshawar 2017 has gotten this part synthesized and has submitted it to the registry for future use by teams.

Bioinformatic characterization – iGEM Edinburgh-UHAS_Ghana 2022

We characterized the structure and metal interaction of this part. We compared the metal binding properties of this mutated form of MerR (henceforth referred to as mut_MerR) to wild-type MerR, to see if the affinity for the heavy metal ion remains constant. We also generated a novel structure for mut_MerR, as well as a structure bound to pmerT, to show it can theoretically function in vivo.

Heavy Metal Docking

We performed docking simulations of AlphaFold generated structures of MerR and mut_MerR against Hg²⁺ and Cd²⁺ respectively. Docking was successful, and yielded the metal bound structures seen in Figure 1.

Figure 1: Structures of a) MerR bound to two Hg²⁺ ions b) mut_MerR bound to two Cd²⁺ ions.

As one can see in Figure 1, the predicted structures of MerR and mut_MerR are consistent with each other. The ions also dock at the same position, which indicates docking has been successful, as this is also consistent with known structures (PDB: 4ua1). There are some mild structural changes between MerR and mut_MerR, however they fundamentally remain quite similar, with coiled helices separating two helical bundles with an unstructured region, characteristic of transcription factors.

The docking simulations also produced values for the free energy of binding in both MerR and mut_MerR. These are -4.99 and -3.93 kcal/mol respectively. The value for mut_MerR’s affinity to Cd²⁺ is quite similar to MerR’s affinity for Hg²⁺, which indicates it could be effective in binding Cd²⁺. The affinity would be slightly lower than MerR’s affinity for Hg2+, but still very much spontaneous and high enough to justify using it.

DNA Docking

To confirm mut_MerR can still bind pmerT, we performed a docking simulation of mut_MerR against the pmerT sequence (Figure 2).

Figure 2: Structure of mut_MerR bound to DNA. a) overall structure shows typical transcription factor binding of residues in the major groove. b) closeup of binding residues shows hydrogen bonds, as well as pi-stacking interactions.

The successful docking of mut_MerR to pmerT indicates that mut_MerR will likely bind pmerT in vivo, and hence could be used for cadmium sensitive reporters.

Sequence and Features