Protein Folding Stability Changes Across the Proteome Reveal Targets of Cu Toxicity in E. coli .

Journal Article

The ability of metal ionophores to induce cellular metal hyperaccumulation endows them with potent antimicrobial activity; however, the targets and mechanisms behind these outcomes are not well understood. This work describes the first utilization of proteome-wide measurements of protein folding stability in combination with protein expression level analysis to identify protein targets of copper, thereby providing new insight into ionophore-induced copper toxicity in E. coli . The protein folding stability analysis employed a one-pot protocol for p ulse p roteolysis (PP) in combination with a s emi-t ryptic peptide e nrichment strategy for p roteolysis p rocedures (STEPP) to generate stability profiles for proteins in cell lysates derived from E. coli exposed to copper with and without two ionophores, the antimicrobial agent pyrithione and its β-lactamase-activated prodrug, PcephPT. As part of this work, the above cell lysates were also subject to protein expression level analysis using conventional quantitative bottom-up proteomic methods. The protein folding stability and expression level profiles generated here enabled the effects of ionophore vs copper to be distinguished and revealed copper-driven stability changes in proteins involved in processes spanning metabolism, translation, and cell redox homeostasis. The 159 differentially stabilized proteins identified in this analysis were significantly more numerous (∼3×) than the 53 proteins identified with differential expression levels. These results illustrate the unique information that protein stability measurements can provide to decipher metal-dependent processes in drug mode of action studies.

Full Text

Duke Authors

Cited Authors

  • Wiebelhaus, N; Zaengle-Barone, JM; Hwang, KK; Franz, KJ; Fitzgerald, MC

Published Date

  • January 2021

Published In

Volume / Issue

  • 16 / 1

Start / End Page

  • 214 - 224

PubMed ID

  • 33305953

Pubmed Central ID

  • 33305953

Electronic International Standard Serial Number (EISSN)

  • 1554-8937

International Standard Serial Number (ISSN)

  • 1554-8929

Digital Object Identifier (DOI)

  • 10.1021/acschembio.0c00900

Language

  • eng