RESUMEN
Employed for over half a century to study protein synthesis, cycloheximide (CHX, 1) is a small molecule natural product that reversibly inhibits translation elongation. More recently, CHX has been applied to ribosome profiling, a method for mapping ribosome positions on mRNA genome-wide. Despite CHX's extensive use, CHX treatment often results in incomplete translation inhibition due to its rapid reversibility, prompting the need for improved reagents. Here, we report the concise synthesis of C13-amide-functionalized CHX derivatives with increased potencies toward protein synthesis inhibition. Cryogenic electron microscopy (cryo-EM) revealed that C13-aminobenzoyl CHX (8) occupies the same site as CHX, competing with the 3' end of E-site tRNA. We demonstrate that 8 is superior to CHX for ribosome profiling experiments, enabling more effective capture of ribosome conformations through sustained stabilization of polysomes. Our studies identify powerful chemical reagents to study protein synthesis and reveal the molecular basis of their enhanced potency.
Asunto(s)
Productos Biológicos/farmacología , Cicloheximida/análogos & derivados , Extensión de la Cadena Peptídica de Translación/efectos de los fármacos , Amidas/química , Productos Biológicos/química , Cicloheximida/metabolismo , Cicloheximida/farmacología , Células HEK293 , Humanos , ARN de Transferencia/química , ARN de Transferencia/metabolismo , Ribosomas/metabolismoRESUMEN
Thioamides, single atom oxygen-to-sulfur substitutions of canonical amide bonds, can be valuable probes for protein folding and protease studies. Here, we investigate the fluorescence quenching properties of thioamides incorporated into the side-chains of amino acids. We synthesize and incorporate Fmoc-protected, solid-phase peptide synthesis building blocks for introducing Nε -thioacetyl-lysine and γ-thioasparagine. Using rigid model peptides, we demonstrate the distance-dependent fluorescence quenching of these thioamides. Furthermore, we describe attempts to incorporate of Nε -thioacetyl-lysine into proteins expressed in Escherichia coli using amber codon suppression.
Asunto(s)
Colorantes Fluorescentes/química , Tioamidas/química , Aminoácidos/química , Transferencia Resonante de Energía de Fluorescencia , Péptidos/síntesis química , Péptidos/química , Técnicas de Síntesis en Fase SólidaRESUMEN
Site-selective incorporation of thioamides into peptides and proteins provides a useful tool for a wide range of applications. Current incorporation methods suffer from low yields as well as epimerization. Here, we describe how the use of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) rather than piperidine in fluorenylmethyloxycarbonyl (Fmoc) deprotection reduces epimerization and increases yields of thioamide-containing peptides. Furthermore, we demonstrate that the use of DBU avoids byproduct formation when synthesizing peptides containing side-chain thioamides.