Team:SCAU-China/Contribution
New parts
We have built a number of parts as below.
Basic parts
Composite parts
Part Improvement
We optimized the DNA sequence of SmtA (BBa_K519010) in accordance with the codon bias of yeast. We also added a 6xHis tag at C terminal of the original and optimized sequences of SmtA. The whole sequences were respectively transformed into yeast. Finally, we observed differences in yeast by Western blot and stress treatments. Please go to improvement page for details.
Part Information Supplement
We consulted a large number of literatures and found more information about SmtA.
SmtA contains four zinc ions. The protein SmtA contains a cleft lined with Cys-sulfur and His-imidazole ligands that binds four zinc ions in a Zn4Cys9His2 cluster. The four Zn2+ ions and five bridging Cys thiolate sulfurs form two fused six-membered rings with distorted boat conformations.1 SmtA sequesters and detoxifies four zinc ions per molecule and contains a zinc finger structurally similar to eukaryotic GATA.2
Moreover, one of the two Cys coordination zinc ions in SmtA is easy to exchange with the exogenous metal (cadmium)(Fig.2), and the other is inert and hard to replace.
111Cd4SmtA prepared by reconstitution of apo-SmtA with 111Cd2+ (106.04 MHz,pH 7.0, 318 K, 10% D2O, 50 mM TriszHCl, 50 mM NaCl) showing four peaks with equal integrals. (b) Substitution of Zn2+ by Cd2+. Spectra recorded after mixing Zn4SmtA with 1 (bottom), 4 (middle), and 8 (top) mol equivalents of 111Cd2+ (pH7.0, 308 K, 10% D2O, 50 mM TriszHCl, 50 mM NaCl). Comparison with a shows that the first mol equivalent of 111Cd2+ selectively occupies binding site B, and that binding site A is not occupied at all. The various peaks reflect 111Cd2+ in mixed Cd, Zn clusters. The occupation of the sites is depicted by squares and circles in the order ABDC, clockwise from top left. Filled square, Cd in the site which gives rise to the respective peak; filled circle, Cd in an adjacent site; open circle, Zn; e.g., the peak at 656 ppm corresponds to111Cd2+ in site B, with Zn2+ in sites A and C and 111Cd2+ in site D. (c) Metal-to-ligand connectivities for SmtA (as deter-mined by 2D heteronuclear NMR experiments)
Fig.2. Proton-decoupled one-dimensional 111Cd NMR spectra of SMTA.
Molecular Simulation
In this year's project, molecular simulation has made an indelible contribution to the completion of the project. We hope that these experiences can help the iGEM community.
The experience of SCAU-CHINA's Drylab in molecular simulations is quite many twists and turns. We experience discovery problems, finding solutions, redesign, re-validation, to the full closed loop of discovering new problems.
Initially, we established the fusion proteins in the order of mCherry-CC-MT-ARR. Wetlab verified that the fusion protein could enter the vacuole, but the metallolic proteins did not bind cadmium.
We conjecture that the arrangement and combination of sequences of fusion proteins, and whether the linked peptide type is used, may all affect the conformation of fusion proteins and the role played by various functional domains. The fusion proteins have four domains, yielding a total of 24 permutation combinations by different domains. If Linker is added, 120 permutation combinations will be produced. We can't constructe them all within limited economic resources. We must infer the best combination of permutations from resources such as the existing literature, analyzing its structure and verifying its function.
By finding extensive literature and making summaries, we redesigned four appropriate fusion proteins, SpMTL-CC-mCherry-ARR,SpMTL-EAAAK-CC-mCherry-ARR,SpMTL- (EAAAK) 5-CC-mCherry-ARR, SpMTL-LRRRF-CC-mCherry-ARR.For the structure-analytical work, we consider two schemes, namely, the NMR and the molecular simulations.
We envisage NMR detection of fusion proteins at the experimental level.But the scheme was deliberate and abandoned.In order to arrive precisely out the frequency distribution in the spectrum of all groups, we had to highly purify the fusion protein, which would greatly increase our workload.Second, using instrumental analysis will lengthen our work cycle, in conflict with our plans that are now urgent.In addition, an NMR instrument resolving the fusion protein requires a minimum of 1000HMz of electromagnets and places the samples in a liquid nitrogen environment.But, in our current resources is lacking.Taken together, the scheme using NMR detection is not the optimal solution.
We turn our eyes to molecular simulations for structural analytical work.In the work on molecular simulations, our work is to perform modal homology modeling, modal-free heavy modeling using fusion protein sequences.ARR and mCherry are those with homologous modules that allow us to obtain their spatial structure efficiently.However, the Spmtl protein chelating cadmium ions is not modal, and we can only do heavy head modeling.Ultimately, we established a separate conformation of the four fusion proteins using trRosetta.
We successfully completed the experiment and obtained good results.We verified that the SpMTL-LRRRF-CC-mCherry-ARR can griddle cadmium ions, the ability to pull it at the same time into the liquid bubble.
However, we once again find the problem.The protein conformation obtained from the modeling is random and perhaps not at the lowest point of potential energy. With the suggestion of BUCT-CHINA, we used molecular dynamics simulations. However, based on the lack of theoretical knowledge at that time and very few computing resources, our work was very difficult at the beginning.
By learning and using tools such as Autodock and Gromacs step by step, we have solved the problem of insufficient theoretical knowledge. Later, Mr. Zhu Guohui provided financial support and Wang Weixu provided cluster servers, which gave us abundant computing resources. We use molecular dynamics simulation to continue to advance our MESEG project.
iGEM gives us the valuable experience of molecular simulation, and lets us do a lot of things that we have never tried before.We will write this experience into the above text and share it with you.It is hoped that future iGEMer can be inspired by our experiences and summaries, thus fewer detours from molecular simulations and achieving the goal more quickly.
References
- Blindauer C A , Harrison M D , Parkinson J A , et al. A metallothionein containing a zinc finger within a four-metal cluster protects a bacterium from zinc toxicity[J]. Proceedings of the National Academy of Sciences, 2001, 98(17):9593-9598.
- Blindauer, C.A., Harrison, M.D., Robinson, A.K., Parkinson, J.A., Bowness, P.W., Sadler, P.J. and Robinson, N.J. (2002), Multiple bacteria encode metallothioneins and SmtA‐like zinc fingers. Molecular Microbiology, 45: 1421-1432. doi:10.1046/j.1365-2958.2002.03109.x
