Evolution of Amber Suppressor tRNAs for Efficient Bacterial Production of Proteins Containing Nonnatural Amino Acids

K+ channels distinguish K + from Na + in the selectivity filter, which consists of four ion-binding sites (S1-S4, extracellular to intracellular) that are built mainly using the carbonyl oxygens from the protein backbone. In addition to ionic discrimination, the selectivity filter regulates the flow of ions across the membrane in a gating process referred to as C-type inactivation. A characteristic of Ctype inactivation is a dependence on the permeant ion, but the mechanism by which permeant ions modulate C-type inactivation is not known. To investigate, we used amide-to-ester substitutions in the protein backbone of the selectivity filter to alter ion binding at specific sites and determined the effects on inactivation. The amide-to-ester substitutions in the protein backbone were introduced using protein semisynthesis or in vivo nonsense suppression approaches. We show that an ester substitution at the S1 site in the KcsA channel does not affect inactivation whereas ester substitutions at the S2 and S3 sites dramatically reduce inactivation. We determined the structure of the KcsA S2 ester mutant and found that the ester substitution eliminates K + binding at the S2 site. We also show that an ester substitution at the S2 site in the K v AP channel has a similar effect of slowing inactivation. Our results link C-type inactivation to ion occupancy at the S2 site. Furthermore, they suggest that the differences in inactivation of K + channels in K + compared with Rb + are due to different ion occupancies at the S2 site.

Section: Resultsmentioning

confidence: 99%

“…The ester mutants of the KcsA and K v AP channels were generated either by semisynthesis or by the in vivo nonsense suppression approach (29,30,32,33). Crystals of the KcsA Y78-ester were grown as a complex with a Fab fragment as described (5).…”

Section: Methodsmentioning

confidence: 99%

Using protein backbone mutagenesis to dissect the link between ion occupancy and C-type inactivation in K ⁺ channels

Matulef

Komarov

Costantino

et al. 2013

“…In addition, because a high-resolution structure is available that depicts the ternary complex just prior to release (18), detailed structure-function studies are now possible. By stabilizing binding to EF-Tu, the T1 chimeras may also be helpful for improving the efficiency of incorporating certain nonnatural amino acids into protein (41,42). Finally, the decoding properties of the T1 chimera of Tyr-tRNA Tyr are interesting to compare with the highly specialized system that has evolved to introduce selenocysteine into proteins.…”

Section: Discussionmentioning

confidence: 99%

Tuning the affinity of aminoacyl-tRNA to elongation factor Tu for optimal decoding

Schrader

Chapman

Uhlenbeck

2011

123

To better understand why aminoacyl-tRNAs (aa-tRNAs) have evolved to bind bacterial elongation factor Tu (EF-Tu) with uniform affinities, mutant tRNAs with differing affinities for EF-Tu were assayed for decoding on Escherichia coli ribosomes. At saturating EF-Tu concentrations, weaker-binding aa-tRNAs decode their cognate codons similarly to wild-type tRNAs. However, tighter-binding aa-tRNAs show reduced rates of peptide bond formation due to slow release from EF-Tu•GDP. Thus, the affinities of aa-tRNAs for EF-Tu are constrained to be uniform by their need to bind tightly enough to form the ternary complex but weakly enough to release from EF-Tu during decoding. Consistent with available crystal structures, the identity of the esterified amino acid and three base pairs in the T stem of tRNA combine to define the affinity of each aa-tRNA for EF-Tu, both off and on the ribosome.T he ternary complex of bacterial elongation factor Tu (EF-Tu), GTP and aminoacyl-tRNA (aa-tRNA) binds to the ribosome and participates in a multistep decoding pathway in which GTP is hydrolyzed, EF-Tu•GDP is released, and the aa-tRNA enters the ribosomal A site (1-6). Although all elongator aa-tRNAs bind EF-Tu•GTP with similar affinities (7-9), studies with misacylated tRNAs reveal that the protein shows substantial specificity for both the esterified amino acid and the tRNA body (10-12). The nearly uniform EF-Tu binding affinity observed for tRNAs acylated with their correct (cognate) amino acid occurs because the sequence of each tRNA has evolved to compensate for the variable thermodynamic contribution of the esterified amino acid. Thus, weak-binding esterified amino acids such as glycine and alanine have corresponding tRNAs that bind the protein tightly, while tight-binding amino acids such as tyrosine or glutamine have corresponding tRNAs that bind poorly. The crystal structure of Thermus aquaticus EF-Tu•GTP bound to Saccharomyces cerevisiae Phe-tRNA Phe (13) reveals that the protein primarily forms extensive interactions with the helical phosphodiester backbone of the acceptor and T stems of tRNA Phe . Recent protein (14) and tRNA (15, 16) mutagenesis experiments indicate that much of the specificity is the result of interactions made between three amino acids of EF-Tu and three adjacent base pairs in the T stem (16). Additional mutagenesis experiments indicate that the thermodynamic contribution of each of the three base pairs is independent of the others, making it possible to adjust the affinity of aa-tRNAs to EF-Tu in a predictable manner.Although a detailed structural and thermodynamic understanding of how EF-Tu achieves uniform binding with different aa-tRNAs is beginning to emerge, the underlying selective pressures that lead to uniform binding are less clear. While aa-tRNAs must bind EF-Tu tightly enough to participate in translation, the high intracellular concentration of EF-Tu (17) ensures that they do not significantly compete with one another for the protein.It therefore seems unlikely that a minimum threshold binding af...

“…After the negative selection, the surviving pool was subjected to one more round of positive selection before individual clones were characterized. Active but orthogonal tRNA variants were selected as described previously (31).…”

Section: Construction Of Librariesmentioning

confidence: 99%

Evolution of multiple, mutually orthogonal prolyl-tRNA synthetase/tRNA pairs for unnatural amino acid mutagenesis in Escherichia coli

Chatterjee

Xiao

Schultz

2012

The site-specific incorporation of unnatural amino acids (UAAs) into proteins in living cells relies on an engineered tRNA/aminoacyl-tRNA synthetase (tRNA/aaRS) pair, orthogonal to the host cell, to deliver the UAA of choice in response to a unique nonsense or frameshift codon. Here we report the generation of mutually orthogonal prolyl-tRNA/prolyl-tRNA synthase (ProRS) pairs derived from an archaebacterial ancestor for use in Escherichia coli . By reprogramming the anticodon-binding pocket of Pyrococcus horikoshii ProRS (PhProRS), we were able to identify synthetase variants that recognize engineered Archaeoglobus fulgidus prolyl-tRNAs (Af-tRNA Pro ) with three different anticodons: CUA, AGGG, and CUAG. Several of these evolved PhProRSs show specificity toward a particular anticodon variant of Af-tRNA Pro , whereas others are promiscuous. Further evolution of the Af-tRNA Pro led to a variant exhibiting significantly improved amber suppression efficiency. Availability of a prolyl-tRNA/aaRS pair should enable site-specific incorporation of proline analogs and other N-modified UAAs into proteins in E. coli . The evolution of mutually orthogonal prolyl-tRNA/ProRS pairs demonstrates the plasticity of the tRNA–aaRS interface and should facilitate the incorporation of multiple, distinct UAAs into proteins.