View source for 6wrk


==Crystal structure of 3rd-generation Mj 3-nitro-tyrosine tRNA synthetase ("A7") bound to 3-nitro-tyrosine==
<StructureSection load='6wrk' size='340' side='right'caption='[[6wrk]], [[Resolution|resolution]] 1.95&Aring;' scene=''>
== Structural highlights ==
<table><tr><td colspan='2'>[[6wrk]] is a 1 chain structure with sequence from [https://en.wikipedia.org/wiki/Methanocaldococcus_jannaschii_DSM_2661 Methanocaldococcus jannaschii DSM 2661]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=6WRK OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=6WRK FirstGlance]. <br>
</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.95&#8491;</td></tr>
<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=GOL:GLYCEROL'>GOL</scene>, <scene name='pdbligand=NA:SODIUM+ION'>NA</scene>, <scene name='pdbligand=NIY:META-NITRO-TYROSINE'>NIY</scene></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=6wrk FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=6wrk OCA], [https://pdbe.org/6wrk PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=6wrk RCSB], [https://www.ebi.ac.uk/pdbsum/6wrk PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=6wrk ProSAT]</span></td></tr>
</table>
== Function ==
[https://www.uniprot.org/uniprot/SYY_METJA SYY_METJA] Catalyzes the attachment of tyrosine to tRNA(Tyr) in a two-step reaction: tyrosine is first activated by ATP to form Tyr-AMP and then transferred to the acceptor end of tRNA(Tyr).<ref>PMID:10585437</ref> 
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
Genetic Code Expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of E. coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a 2(nd)-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.

Overcoming near-cognate suppression in a Release Factor 1-deficient host with an improved nitro-tyrosine tRNA synthetase.,Beyer J, Hosseinzadeh P, Gottfried-Lee I, Van Fossen EM, Zhu P, Bednar RM, Karplus PA, Mehl RA, Cooley RB J Mol Biol. 2020 Jun 19. pii: S0022-2836(20)30410-1. doi:, 10.1016/j.jmb.2020.06.014. PMID:32569745<ref>PMID:32569745</ref>

From MEDLINE&reg;/PubMed&reg;, a database of the U.S. National Library of Medicine.<br>
</div>
<div class="pdbe-citations 6wrk" style="background-color:#fffaf0;"></div>
== References ==
<references/>
__TOC__
</StructureSection>
[[Category: Large Structures]]
[[Category: Methanocaldococcus jannaschii DSM 2661]]
[[Category: Beyer JN]]
[[Category: Cooley RB]]
[[Category: Hosseinzadeh P]]
[[Category: Karplus PA]]
[[Category: Mehl RA]]