Revision as of 03:38, 22 October 2019

Arsenic metallothionein

Human arsenic targeting metallothionein

Sequence and Features

Introduction

Metallothionein (MT) is a class of small metal-binding proteins that exists in bacteria, plants and animals. These proteins depending on their amino acid compositions have a high binding affinity with different bivalent metal ions. Once MT detects the corresponding metal, it binds the goal through covalent bonds, which are composed of sulfhydryl cysteine residues and stores the metal by tightly chelating the metal. Typically, it is assumed that MTs have two binding domains, one of which is the C-terminal part (α-domain) with three binding sites. The other one is the N-terminal part (β-domain) with four divalent binding sites ^[1]. Therefore, MTs are important for protecting the cell against heavy metal toxicity and maintaining cellular homeostasis.

Arsenic Metallothionein

As(V) can be reduced to As(III) by arsenate reductase, and then the arsenite can bind to thiol groups easily ^[2].Metallothionein is a great tool for E.coli to accumulate arsenic because it has a great amount of cysteine, which makes it a thiol-rich protein. part:BBa_K3275000 is from human metallothionein originally, which is called Metallothionein-1A (MT-1A). There are two metal binding domains in MT-1A: the α domain and the β domain. In the α domain, cysteinyl thiolate bridges let 11 cysteine ligands to coordinate with 4 divalent ions, and these ions are chelated with cluster A. In the β domain, the corresponding region, cluster B, helps ligate 3 divalent ions to 9 cysteines ^[3]. Figure 1 shows the structure of MT-1A ^[4]. The figure shows that MT-1A can bind with 2 zinc ions and 5 cadmium ions.

Figure 1. 3-D structure of MT-1A find more here

For part:BBa_K3275000, some bases from the original sequence are changed due to synthesis demands.

Characterization

The culmination of testing for this project was to examine how well both constructs (A1 and A2) could handle the bioremediation of arsenic. The protocol can be found in the last second page of the Protocol section in theRHIT|Experiment or in the RHIT|Notebook Details on the parts used in the design section of the wiki. Our results show that both A1 and A2 were successful at removing arsenite ions from the aqueous solution, and as the total amount of arsenite continued to move down as time increased, cells were able to survive as well. Small decreases were noticed in the negative control as well, as arsenite is naturally transported in and out of cells through aquaglyceroporins (AQPs). In the shorter term (8 hours), A2, which produces metallothionein constantly due to its constitutive promoter, showed better bioremediation of the arsenite. However, at the longer time scale, the arsenic controlled (through ArsR) A1 ended up proving more effective at removing arsenic.

References

1. ↑ Ruttkay-Nedecky, B., Nejdl, L., Gumulec, J., Zitka, O., Masarik, M., Eckschlager, T., … Kizek, R. (2013). The role of metallothionein in oxidative stress. International journal of molecular sciences, 14(3), 6044–6066. doi:10.3390/ijms14036044
2. ↑ Ngu, T. and Stillman, M. (2006). Arsenic Binding to Human Metallothionein. Journal of the American Chemical Society, 128(38), pp.12473-12483.
3. ↑ MT1A - Metallothionein-1A - Homo sapiens (Human) - MT1A gene & protein. (2019). Retrieved from https://www.uniprot.org/uniprot/P04731
4. ↑ SWISS-MODEL Repository | P04731. (2019). Retrieved from https://swissmodel.expasy.org/repository/uniprot/P04731?csm=8FBA7C54EE8B6A13