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PDBsum entry 6wrn
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References listed in PDB file
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Key reference
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Title
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Overcoming near-Cognate suppression in a release factor 1-Deficient host with an improved nitro-Tyrosine tRNA synthetase.
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Authors
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J.N.Beyer,
P.Hosseinzadeh,
I.Gottfried-Lee,
E.M.Van fossen,
P.Zhu,
R.M.Bednar,
P.A.Karplus,
R.A.Mehl,
R.B.Cooley.
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Ref.
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J Mol Biol, 2020,
432,
4690-4704.
[DOI no: ]
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PubMed id
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Abstract
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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 Escherichia 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
second-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.
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