Open AccessProtein Folding Stability Changes Across the Proteome Reveal Targets of Cu Toxicity in E. coli
Author(s) -
Nancy Wiebelhaus,
Jacqueline M. Zaengle-Barone,
Kevin K Hwang,
Katherine J. Franz,
Michael C. Fitzgerald
Publication year - 2020
Publication title -
acs chemical biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.899
H-Index - 111
eISSN - 1554-8937
pISSN - 1554-8929
DOI - 10.1021/acschembio.0c00900
Subject(s) - proteome , proteolysis , biochemistry , chemistry , protein folding , proteomics , folding (dsp implementation) , biology , biophysics , enzyme , electrical engineering , gene , engineering
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.