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

Research output: Contribution to journalArticlepeer-review

46 Citations (Scopus)

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 Å 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 Å resolutions by SAD phasing at CuKα and CrKα wavelengths, respectively. The native structure, determined by molecular replacement, revealed no significant structural distortion caused by iodotyrosine incorporation.

Original languageEnglish
Pages (from-to)335-344
Number of pages10
JournalStructure
Volume17
Issue number3
DOIs
Publication statusPublished - 2009 Mar 11

Keywords

  • PROTEINS

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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