Genetic Encoding of 3-Iodo-l-Tyrosine in Escherichia coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography

Abstract

We developed an Escherichia coli cell-based system to generate proteins containing 3-iodo-L-tyrosine at desired sites, and we used this system for structure determination by single-wavelength anomalous dispersion (SAD) phasing with the strong iodine signal. Tyrosyl-tRNA synthetase from Methanocaldococcus jannaschii was engineered to specifically recognize 3-iodo-L-tyrosine. The 1.7 A crystal structure of the engineered variant, iodoTyrRS-mj, bound with 3-iodo-L-tyrosine revealed the structural basis underlying the strict specificity for this nonnatural substrate; the iodine moiety makes van der Waals contacts with 5 residues at the binding pocket. E. coli cells expressing iodoTyrRS-mj and the suppressor tRNA were used to incorporate 3-iodo-L-tyrosine site specifically into the ribosomal protein N-acetyltransferase from Thermus thermophilus. The crystal structure of this enzyme with iodotyrosine was determined at 1.8 and 2.2 Angstroms resolutions by SAD phasing at CuK alpha and CrK alpha wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.

High-Yield, Zero-Leakage Expression System with a Translational Switch Using Site-Specific Unnatural Amino Acid Incorporation

“…We used here a specific method for 3-iodo-L-Tyr (IY) incorporation in E. coli ( Fig. 1A) (27). With this method, the extracellular IY enters the intracellular space and forms an aminoacyltRNA by the use of a pair of plasmid-encoding genes, an amber suppressor tRNA (tRNA CUA ) MJR1 and a variant of aminoacyltRNA synthetase (aaRS) IYRS that specifically recognizes IY and MJR1 from the archaebacterium Methanocaldococcus jannaschii, resulting in IY incorporating at AMBs inserted in target genes.…”

“…The amber suppressor plasmid pTYR MjIYRS2-1(D286) MJR1ϫ3 (p15A replicon) encoding IYRS (driven by the E. coli Tyr tRNA synthetase promoter P TyrRS ) and MJR1 (driven by the E. coli lpp promoter P lpp ) were kindly provided by K. Sakamoto, RIKEN (27). For conditional expression of either IYRS or MJR1, two derivatives were also constructed: either the E. coli lac promoter P lac was substituted for P TyrRS or the T7 promoter with lacO P T7 -lacO was substituted for P lpp .…”

“…Since Schultz and collaborators achieved the first breakthrough, various UAAs, including derivatives of Lys and Tyr, have been incorporated in vivo (19,20). This technique has been widely applied for protein engineering in applications such as structural analyses, functional analyses, protein therapeutics, and functional improvement (23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33).…”

Ultraviolet Action Spectroscopy of Iodine Labeled Peptides and Proteins in the Gas Phase

“…If multiple tyrosine residues were present in a protein, selective iodination at each tyrosine residue could be utilized to obtain site specific structural information from each tyrosine. 37 This is an advantage which is not easily realized in electron detachment experiments. Conclusions: Action spectra obtained from iodotyrosine residues are sensitive to side chain solvation, which is a molecule specific phenomenon.…”

Controlling Enzyme Inhibition Using an Expanded Set of Genetically Encoded Amino Acids

“…Residue-specific incorporation of selenomethionine into proteins using a methionine auxotrophic E. coli strain allows the determination of X-ray crystallographic structures of proteins by multiple (MAD) or single wavelength anomalous diffraction (SAD) [65,66]. Site-specific incorporation of p-iodophenylalanine and 3-iodotyrosine into bacteriophage T4 lysozyme and N-acetyltransferase, has also been reported to facilitate the SAD phasing [63,64]. Fourth, NAA has been used to modulate the spectral property of the proteins, such as fluorescence [67,68].…”