Analysis of copper-induced protein precipitation across the E. coli proteome

Abstract

Metal cations have been exploited for their precipitation properties in a wide variety of studies, ranging from differentiating proteins from serum and blood to identifying the protein targets of drugs. Despite widespread recognition of this phenomenon, the mechanisms of metal-induced protein aggregation have not been fully elucidated. Recent studies have suggested that copper's (Cu) ability to induce protein aggregation may be a main contributor to Cu-induced cell death. Here, we provide the first proteome-wide analysis of the relative sensitivities of proteins across the Escherichia coli proteome to Cu-induced aggregation. We utilize a metal-induced protein precipitation (MiPP) methodology that relies on quantitative bottom-up proteomics to define the metal concentration-dependent precipitation properties of proteins on a proteomic scale. Our results establish that Cu far surpasses other metals in promoting protein aggregation and that the protein aggregation is reversible upon metal chelation. The bulk of the Cu bound in the protein aggregates is Cu1+, regardless of the Cu2+ source. Analysis of our MiPP data allows us to investigate underlying biophysical characteristics that determine a protein's sensitivity to Cu-induced aggregation, which is independent of the relative concentration of protein in the lysate. Overall, this analysis provides new insights into the mechanism behind Cu cytotoxicity, as well as metal cation-induced protein aggregation.

Keywords: Escherichia coli, folding, protein; aggregation, copper; toxicity.

Graphical Abstract

Copper surpasses other metal cations in inducing protein precipitation. A quantitative proteomics strategy was used to define the Cu precipitation midpoint, or C_m value, of individual proteins across the E. coli proteome, providing insight into attributes that influence protein susceptibility, or tolerance, to Cu-induced protein misfolding. (Graphic created with BioRender.com)

Fig. 1

Cu induces reversible protein precipitation to a greater extent than other biorelevant metals. (A) Addition of Cu²⁺ precipitated significantly more proteins from E. coli than other metals at 10 mM. n = 3. (B) Precipitation curves indicated that Fe²⁺, Co²⁺, Ni²⁺, and Zn²⁺ did not induce complete precipitation even at 50 mM. n = 2 for all metals except Cu²⁺, for which n = 3. (C) Structures of glutathione (GSH), ethylenediaminetetraacetic acid (EDTA), dl-dithiothreitol, 1,4-dithiothreitol (DTT), and bathocuproinedisulfonic acid disodium salt (BCS). (D) EDTA (5 mM), BCS (50 mM), DTT (50 mM), and GSH (20 mM) allowed protein to return to the supernatant after treatment of E. coli lysate with 10 mM CuCl₂. Water did not rescue protein solubility, for which n = 3. After precipitation with heat, neither 5 mM EDTA nor water was able to rescue protein solubility. Lysate protein concentrations for all experiments were 12.5 mg/mL.

Fig. 2

Cu precipitates with proteins during Cu-induced protein precipitation and has a mixture of Cu¹⁺ and Cu²⁺ in the soluble and insoluble fractions. (A) The left y-axis shows a comparison of the amount of Cu measured by inductively coupled plasma–mass spectrometry (ICP–MS) found in the pellet vs. the supernatant at each Cu treatment condition for E. coli lysate with a protein concentration of 5 mg/mL. The right y-axis shows the relative fraction of protein in the supernatant for each Cu treatment condition, using the same biological replicates used for the ICP–MS data. Points represent mean and error bars represent ±1 SEM; n = 3. (B) Representative electron paramagnetic resonance (EPR) spectra of resuspended pellet (left) and supernatant (right) with and without hydrogen peroxide. Lysate treated with 6 mM Cu displayed a small Cu signal (red), which became more prominent after treatment with 100 mM hydroxylamine and 1 M hydrogen peroxide (blue) in both the supernatant and pellet. Replicates are shown in Supplementary Fig. S2. (C) Bar graph showing the percentage of reduced sulfhydryls in lysate treated with and without 6 mM Cu assayed by reaction with 5,5′-dithio-bis-(2-nitrobenzoic acid) (DTNB; Ellman's reagent). The bars represent the mean and error bars represent ±1 SEM; n = 3.

Fig. 3

Proteomic workflow employed in the large-scale metal-induced protein precipitation (MiPP) analysis across the soluble E. coli proteome. Created with BioRender.com.

Fig. 4

Cu midpoint values across identified E. coli proteins decrease as protein concentrations decrease. (A) Box and whisker plot of the C_m values recorded for E. coli proteins across the proteome in the MiPP experiments performed using E. coli lysate samples at 5 and 12.5 mg/mL concentrations of total protein. (B) Histogram showing the distribution of ΔC_m values (ΔC_m = C_m12.5–C_m5). (C, D) Venn diagrams showing the overlap of sensitive (C) and tolerant (D) proteins that were assayed in both the 5 and 12.5 mg/mL datasets.

Fig. 5

Copper precipitation midpoints lack strong correlation with other proteomic methods. (A) Linear regression comparing Cu midpoint values (C_m) to stability of proteins from rates of oxidation (SPROX) midpoint values (C_1/2). y = 0.02x + 1.2. R² = 0.002. (B) Linear regression comparing Cu C_m to thermal protein profiling (TPP) midpoint T_m values. y = 1x + 48. R² = 0.051. (C) Linear regression comparing Cu C_m values to chemical denaturation and protein precipitation (CPP) midpoint values (C1/2). y = 0.04x + 0.8. R² = 0.087. The regressions displayed here use the 5 mg/mL dataset. The same analyses were done on the 12.5 mg/mL dataset (Supplementary Fig. S3) and resulted in correlations nearly identical to those shown here.

Fig. 6

Amino acid analysis and secondary structure. (A) Heat map showing Welch's test P-values, which were found from comparing secondary structure composition of sensitive and tolerant protein groups identified from 5 mg/mL lysate samples. (B) Box and whisker plots showing distributions of secondary structure fraction for individual proteins in both the sensitive and tolerant categories. Each individual dot represents a single protein from the 5 mg/mL dataset. Only secondary structures that were found to have a Welch's test P-value <0.05 in both the 5 and 12.5 mg/mL dataset are displayed. (C) Heat map showing Welch's test P-values, which were found from comparing the amino acid composition of sensitive and tolerant protein groups identified from 5 mg/mL lysate samples. (D) Box and whisker plots showing distributions of amino acid compositions for individual proteins in both the sensitive and tolerant categories. Only amino acids that were found to have a Welch's test P-value <0.001 in both the 5 and 12.5 mg/mL dataset are displayed. For a full list of Welch's test P-values and analysis on every amino acid and secondary structure in both the 5 and 12.5 mg/mL datasets see Supplementary Tables S5 and S6.