Difference between revisions of "Part:BBa K3381006:Design"

Latest revision as of 01:35, 24 October 2020

Improved Mst-CopC-CBM2a

Design Notes

We applied a rational protein design approach to choose mutations that might result in increased CopC-Cu(II) binding. We started out with several dozen possible candidates with single mutations and combinations of mutations. This list was further refined by calculating each candidate’s energy score using Rosetta, a protein modelling software. About ten proteins with the best energy scores were then evaluated using molecular dynamics. In order to test the binding affinity, a 10 nanosecond simulation was performed using GROMACS with the Charmm36 force field (Vanommeslaeghe, 2010). Molecular dynamics showed that a candidate with the mutations Y34F, F3H, S81D, and H85D bound Cu(II) the most strongly, and is therefore the best of our improved Mst-CopC candidates (see our engineering and model pages for more information).

After the determined residues were mutated in the original amino acid sequence, it was codon optimized for ‘‘E. coli’’ and all cut sites for RFc 25 and NdeI/BamHI (required for cloning into pET 11a) were removed. Lastly, the prefix and suffix for RFc 25 were appended.

Source

Sources of DNA sequences, in order of occurrence in the fusion protein from N' to C':

His-tag and TEV linker sequence: Retrieved from His-tag and TEV linker that occurs in bacterial expression vector pMCSG7: https://plasmid.med.harvard.edu/PlasmidRepository/file/sequence/pMCSG7.gb

Original sequence of Mst-CopC (copper-binding domain) retrieved from Genbank (Methylosinus trichosporium O3B): https://www.ncbi.nlm.nih.gov/protein/ATQ66671

Sequence of the CBM linker domain was back-translated from its amino acid sequence, which was retrieved from the following paper describing a natural occurrence of the linker in a protein with a cellulose-binding domain: https://www.jbc.org/content/293/34/13006.full

Sequence of CBM2a (cellulose-binding domain) retrieved from DNA sequence of BBa_K863101 (Cellulose-binding domain of Cellulomonas fimi exoglucanase): https://parts.igem.org/Part:BBa_K863101

References

Courtade, G., Forsberg, Z., Heggset, E. B., Eijsink, V. G., & Aachmann, F. L. (2018, August 24). The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation. Retrieved September 30, 2020, from https://www.jbc.org/content/293/34/13006.full

Koropatkin, N., Randich, A. M., Bhattacharyya-Pakrasi, M., Pakrasi, H. B., & Smith, T. J. (2007, September 14). The Structure of the Iron-binding Protein, FutA1, from Synechocystis 6803. Retrieved September 30, 2020, from https://www.jbc.org/content/282/37/27468.full

Vanommeslaeghe, K. Hatcher, E. Acharya, C. Kundu, S. Zhong, S. Shim, J. E. Darian, E. Guvench, O. Lopes, P. Vorobyov, I. and MacKerell, Jr. A.D. "CHARMM General Force Field (CGenFF): A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields," Journal of Computational Chemistry 31: 671-90, 2010, PMC2888302

Zhou, J., Chen, J., Zhuang, N., Zhang, A., Chen, K., Xu, N., . . . Jiang, M. (2020, May 12). Immobilization and Purification of Enzymes With the Novel Affinity Tag ChBD-AB From Chitinolyticbacter meiyuanensis SYBC-H1. Retrieved September 30, 2020, from https://www.frontiersin.org/articles/10.3389/fbioe.2020.00579/full