Interaction between TCS and ribosome was compromised by K173A/R174A/K177A triple-alanine substitutions

Abstract

<p><b>Copyright information:</b></p><p>Taken from "Interaction between trichosanthin, a ribosome-inactivating protein, and the ribosomal stalk protein P2 by chemical shift perturbation and mutagenesis analyses"</p><p></p><p>Nucleic Acids Research 2007;35(5):1660-1672.</p><p>Published online 18 Feb 2007</p><p>PMCID:PMC1865052.</p><p>© 2007 The Author(s).</p> () . Rat ribosome was loaded to NHS-Sepharose coupled with TCS or its triple-alanine (K173A/R174A/K177A) variants. After extensive washing, the bound proteins were eluted with 1 M NaCl, and detected by western blot using anti-P antibody. Ribosomal proteins P0, P1 and P2 were pull-down by wild-type TCS (lane 2), while the interaction between ribosome and the triple-alanine variants (lane 1) was greatly reduced to that similar to the control (lane 3), in which the faint band of P0 was due to non-specific interactions between ribosome and the uncoupled resins. () . After rat ribosome was incubated with TCS or the triple-alanine variants in room temperature for 20 min, DSS was added to induce cross-linking between TCS and ribosomal proteins, and cross-linking product was detected by western blot using anti-P or anti-TCS antibodies. A protein band at ∼66 kDa, corresponding to the size of TCS–P0 complex, was detected by both anti-P and anti-TCS antibodies when ribosome was cross-linked with wild-type TCS (lane 2), but not with the triple-alanine variants (lane 5) and in other negative controls (lanes 1 and 4: without addition of ribosome; lanes 3, 6 and 8: without addition of DSS; lanes 7 and 8: without addition of TCS or its variants)