KOPS - The Institutional Repository of the University of Konstanz

Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli

Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli

Cite This

Files in this item

Checksum: MD5:5398724ad0a17b5bd8e775d51f634dd1

GERDING, Hanne R., Christiaan KARREMAN, Andreas DAIBER, Johannes DELP, Daniel HAMMLER, Martin MEX, Stefan SCHILDKNECHT, Marcel LEIST, 2019. Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli. In: Redox Biology. 26, 101251. eISSN 2213-2317. Available under: doi: 10.1016/j.redox.2019.101251

@article{Gerding2019-06Reduc-46296, title={Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli}, year={2019}, doi={10.1016/j.redox.2019.101251}, volume={26}, journal={Redox Biology}, author={Gerding, Hanne R. and Karreman, Christiaan and Daiber, Andreas and Delp, Johannes and Hammler, Daniel and Mex, Martin and Schildknecht, Stefan and Leist, Marcel}, note={Article Number: 101251} }

Leist, Marcel Reductive modification of genetically encoded 3-nitrotyrosine sites in alpha synuclein expressed in E.coli Leist, Marcel Mex, Martin Mex, Martin terms-of-use Gerding, Hanne R. Daiber, Andreas Delp, Johannes 2019-07-10T11:14:01Z Schildknecht, Stefan Gerding, Hanne R. Tyrosine nitration is a post-translational protein modification relevant to various pathophysiological processes. Chemical nitration procedures have been used to generate and study nitrated proteins, but these methods regularly lead to modifications at other amino acid residues. A novel strategy employs a genetic code modification that allows incorporation of 3-nitrotyrosine (3-NT) during ribosomal protein synthesis to generate a recombinant protein with defined 3-NT-sites, in the absence of other post-translational modifications. This approach was applied to study the generation and stability of the 3-NT moiety in recombinant proteins produced in E.coli. Nitrated alpha-synuclein (ASYN) was selected as exemplary protein, relevant in Parkinson's disease (PD). A procedure was established to obtain pure tyrosine-modified ASYN in mg amounts. However, a rapid (t<sub>1/2</sub> = 0.4 h) reduction of 3-NT to 3-aminotyrosine (3-AT) was observed. When screening for potential mechanisms, we found that 3-NT can be reduced enzymatically to 3-AT, whilst biologically relevant low molecular weight reductants, such as NADPH or GSH, did not affect 3-NT. A genetic screen for E.coli proteins, involved in the observed 3-NT reduction, revealed the contribution of several, possibly redundant pathways. Green fluorescent protein was studied as an alternative model protein. These data confirm 3-NT reduction as a broadly-relevant pathway in E.coli. In conclusion, incorporation of 3-NT as a genetically-encoded non-natural amino acid allows for generation of recombinant proteins with specific nitration sites. The potential reduction of the 3-NT moiety by E.coli, however, requires attention to the design of the purification strategy for obtaining pure nitrated protein. eng 2019-06 Hammler, Daniel Karreman, Christiaan Daiber, Andreas Karreman, Christiaan 2019-07-10T11:14:01Z Schildknecht, Stefan Hammler, Daniel Delp, Johannes

Downloads since Jul 10, 2019 (Information about access statistics)

Gerding_2-7pmht21lixib0.pdf 14

This item appears in the following Collection(s)

Search KOPS


Browse

My Account