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Genetic Encoding of 3-iodo-L-tyrosine in Escherichia Coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography

Genetic Encoding of 3-iodo-L-tyrosine in Escherichia Coli for Single-Wavelength Anomalous Dispersion Phasing in Protein Crystallography

Kensaku Sakamoto, Kazutaka Murayama, Kenji Oki, Fumie Iraha, Miyuki Kato-Murayama, Masahiro Takahashi, Kazumasa Ohtake, Takatsugu Kobayashi, Seiki Kuramitsu, Mikako Shirouzu, Shigeyuki Yokoyama

Structure. 2009 Mar 11;17(3):335-44.

PMID: 19278648

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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP70780	3-Iodo-L-tyrosine		70-78-0	Price

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