Abnormal SDS-PAGE migration of cytosolic proteins can identify domains and mechanisms that control surfactant binding

Abstract

The amino acid substitution or post-translational modification of a cytosolic protein can cause unpredictable changes to its electrophoretic mobility during SDS-PAGE. This type of "gel shifting" has perplexed biochemists and biologists for decades. We identify a mechanism for "gel shifting" that predominates among a set of ALS (amyotrophic lateral sclerosis) mutant hSOD1 (superoxide dismutase) proteins, post-translationally modified hSOD1 proteins, and homologous SOD1 proteins from different organisms. By first comparing how 39 amino acid substitutions throughout hSOD1 affected SDS-PAGE migration, we found that substitutions that caused gel shifting occurred within a single polyacidic domain (residues ~80-101), and were nonisoelectric. Substitutions that decreased the net negative charge of domain 80-101 increased migration; only one substitution increased net negative charge and slowed migration. Capillary electrophoresis, circular dichroism, and size exclusion chromatography demonstrated that amino acid substitutions increase migration during SDS-PAGE by promoting the binding of three to four additional SDS molecules, without significantly altering the secondary structure or Stokes radius of hSOD1-SDS complexes. The high negative charge of domain 80-101 is required for SOD1 gel shifting: neutralizing the polyacidic domain (via chimeric mouse-human SOD1 fusion proteins) inhibited amino acid substitutions from causing gel shifting. These results demonstrate that the pattern of gel shifting for mutant cytosolic proteins can be used to: (i) identify domains in the primary structure that control interactions between denatured cytosolic proteins and SDS and (ii) identify a predominant chemical mechanism for the interaction (e.g., hydrophobic vs. electrostatic).

Figure 1

Gel shifting of ALS variant hSOD1 during SDS-PAGE. A: SDS-PAGE and anti-SOD1 Western blotting of ALS mutant hSOD1. Black = no change in migration towards positive electrode; red = increase; green: decrease. Untransformed (UT) cells and cells expressing WT hSOD1 were loaded as controls. Asterisk denotes smearing artifact of Western blotting. Image represents composite of three Western blots (borders indicated by vertical dashed lines). B: Summary of migration of 39 ALS variants of hSOD1 with SDS-PAGE from this study, and published reports. Substitutions that decrease the net negative charge of hSOD1 (ΔZ = +) are highlighted in red; substitutions that increase the net negative charge (ΔZ = −) are green; isoelectric substitutions (ΔZ = 0) are black. C: Substitutions that cause gel shifting (indicated with red dashed lines) are clustered in a polyacidic domain (approximately residues 80–101) which has a high local net negative charge. D: Comparison of location of gel shifting domain with native 2° structure and number of known ALS amino acid substitutions at each residue in hSOD1.

Figure 2

ALS amino acid substitutions that increase migration during SDS-PAGE promote the binding of ∼3–4 SDS molecules without altering the gross 2° structure of SDS-saturated hSOD1. A: Capillary electropherograms of G93R, G85R, D90A, and WT apo-hSOD1 under native (black trace) and SDS-denatured conditions (red trace). Peak at μ = 0 in all electropherograms is neutral marker, dimethylformamide (DMF). The intense broad peak and shoulder at μ = 0–5 in red electropherograms is β-mercaptoethanol. The G85R, D90A, and G93R substitutions (which cause gel shifting) result in the binding of ∼3–4 additional SDS molecules per hSOD1 polypeptide after boiling in SDS-PAGE buffer. Note: the native G93R apo-SOD1 protein migrated as a doublet during CE (the smaller peak with a lower mobility has not yet been identified but is not due to the binding of a metal ion). B–C: Circular dichroism spectroscopy of WT, G85R, D90A, and G93R apo-hSOD1 proteins under (B) native conditions and (C) after boiling and disulfide reduction in SDS/TCEP). D: Table showing: (i) summary of changes in SDS stoichiometry (“ΔSDS”; standard deviation of three measurements listed in parentheses), and (ii) the abundance of secondary structure (% α-helix, β-sheet, random coil) in each SDS-denatured protein (standard deviation from at least 3 measurements listed in parentheses).

Figure 3

ALS-linked amino acid substitutions that cause gel shifting do not significantly alter Stokes radius of hSOD1-SDS complexes. A: SDS-PAGE of fractions of SDS-denatured D90A and WT apo-hSOD1 after boiling in Laemlli buffer and simultaneous analysis with G-100 size exclusion chromatography. B: Analysis of same SDS-denatured D90A and WT apo-hSOD1 with high resolution SE-HPLC. C: Plot of intensity of hSOD1 in fractions from G-100 SE column demonstrates that D90A and WT hSOD1-SDS complexes co-elute. D: Illustration of gel shifting of D90A and WT apo-hSOD1 during SDS-PAGE.

Figure 4

Decreasing the net negative charge of domain ∼80–101 in SOD1 prevents ALS mutations from causing gel shifting. A: Sequence homology (% a.a.) and formal charge homology (%Z) of mouse and human SOD1; nonconserved charged residues are labeled +, 0, or −. B: Four chimeric forms of human and mouse hSOD1, two of which contain the ALS-linked G85R amino acid substitution that causes gel shifting in hSOD1. The h^N/m^C chimeric WT SOD1 contains N-terminal half (residues 1–80) from hSOD1 and C-terminal half (residues 81–153) from mSOD1 (and vice versa for m^N/h^C chimeric WT SOD1). The ALS-linked G85R substitution caused gel shifting with m^N/h^C but not with h^N/m^C. C: SDS-PAGE Western blot of mWT, hWT, and chimeric m/hSOD1 proteins expressed in cultured HEK293-FT cells. Faint bands in C (denoted **) represent endogenous hWT SOD1 expressed by HEK cells; faint bands at high molecular weight (*) represent nonspecific binding of immunoglobin or oligomeric SOD1.

Figure 5

Acetylation of multiple Lys-ε-NH₃⁺ in human SOD1 induces gel shifting during SDS-PAGE. A: Acetylation of WT hSOD1 (denoted Ac(∼N)) decreases migration during SDS-PAGE (N refers to the number of acetylated lysine in dimeric hSOD1). Inset shows side by side analysis of unmodified hSOD1-Ac(0) and peracetylated hSOD1-Ac(22) with SDS-PAGE. B: Increased migration of charge ladders during Native-PAGE (0 mM SDS). C,D: Plot of the magnitude of gel shifting of hSOD1 (in terms of molecular weight or migration distance) as a function of acetylated lysine residues (Lys-NHAc). Error bars represent the standard deviation from seven separate experiments.

Figure 6

Systematic neutralization of multiple Lys-ε-NH₃⁺ in human SOD1 decreases the number of bound SDS. A: Acetylation of native WT holo-hSOD1 increases capillary electrophoretic mobility in the absence of SDS (black), but reduces mobility after boiling and reduction in SDS (red). Peak at μ = 0 is neutral marker, DMF). The intense broad peak and shoulder present at μ = 0–5 in red electropherograms is β-mercaptoethanol. B: Decrease in number of SDS molecules bound to hSOD1 (ΔSDS per monomer) plotted as a function of number of acetylated lysine per hSOD1 monomer. C: Plot of number of SDS molecules dissociated from acetylated hSOD1 (ΔSDS) as a function of the magnitude of gel shifting. Error bars represent standard deviation from three separate experiments.

Figure 7

The acetylation of the single lysine in domain 80–101 of WT hSOD1 (Lys91) correlates with greatest magnitude of gel shifting. A–C: Nonrandom acetylation of Lys91 in hSOD1. A: Triply charged ion of peptide 87-Lys91(Ac)-115 detected in variably acetylated hSOD1. B: Doubly charged ion of peptide 80-Lys91(Ac)-115. C: Singly charged ion of peptide 7-Lys9-16; increase in signal intensity for acetylated Lys9 is greater than acetylated Lys91 at equal concentrations of acetic anhydride. D: Doubly charged ions of C-terminal peptide standard (residues 144–153) that does not contain lysine, and is cleaved at Arg143. E: Plot of the percent of acetylation for four different lysine in SOD1 as a function of the average degree of acetylation of all lysine residues (Ac(∼N)) in SOD1. F: Magnitude of gel shifting of hSOD1 plotted as a function of percent acetylation of four lysine residues from multiple hSOD1 domains; error bars represent standard deviation from five different enzymatic digests.