The Achilles’ Heel of “Ultrastable” Hyperthermophile Proteins: Submillimolar Concentrations of SDS Stimulate Rapid Conformational Change, Aggregation, and Amyloid Formation in Proteins Carrying Overall Positive Charge

Abstract

Low concentrations (<3.0 mM) of the anionic surfactant sodium dodecyl sulfate (SDS) have been shown to induce the formation of amyloid fibers in more than 20 different mesophile-derived proteins in the cationic state. It is not known whether SDS has similar effects on hyperthermophile-derived proteins, which are otherwise thought to be "ultrastable" and inordinately resistant to structural perturbations at room temperature. Here, we show that low (<4.5 mM) concentrations of SDS rapidly induce the formation of aggregates and amyloid fibers in five different ultrastable Pyrococcus furiosus proteins in the cationic state. We also show that amyloid formation is accompanied by the development of a characteristic, negative circular dichroism band at ∼230 nm. These effects are not seen if the proteins have a net negative charge or when higher concentrations of SDS are used (which induce helix formation instead). Our results appear to reveal a potential weakness or "Achilles' heel" in ultrastable proteins from hyperthermophiles. They also provide very strong support for the view that SDS initially interacts with proteins through electrostatic interactions, and not hydrophobic interactions, eliciting similar effects entirely regardless of protein molecular weight, or structural features such as quaternary structure or tertiary structural stability.

Understanding anomalous mobility of proteins on SDS‐PAGE with special reference to the highly acidic extracellular domains of human E‐ and N‐cadherins

“…If the pH of a protein's environment is well below its pI, it protein possesses an overall positive charge, and is easily approached by SDS. Under these conditions, low (millimolar) concentrations of SDS bind to transform proteins into beta sheet‐based amyloids, as has now been shown with many proteins . When the pH is above the p I , and the protein possesses an overall negative charge, low millimolar concentrations of SDS are not sufficient to overcome the electrostatic repulsion, and transformation into amyloids is not observed.…”

“…Highly acidic proteins can be expected to repel SDS. In a recent series of papers examining the binding of SDS to proteins, it has been observed that the overall charged status of a protein is the primary determinant of the ease with which it can be approached by SDS molecules . If the pH of a protein's environment is well below its pI, it protein possesses an overall positive charge, and is easily approached by SDS.…”

“…When the pH is above the p I , and the protein possesses an overall negative charge, low millimolar concentrations of SDS are not sufficient to overcome the electrostatic repulsion, and transformation into amyloids is not observed. However, at higher SDS concentrations, (e.g., above 10 mM SDS), SDS binding transforms proteins into extended rigid helices which are held in the extended state by charge‐charge repulsions amongst bound SDS molecules . The SDS concentration used for SDS‐PAGE studies (0.4% SDS or about 14 mM SDS) falls in this higher range in which SDS binding occurs to transform proteins into extended rigid helices.…”

“…In a recent paper dealing with the effects of low concentrations of SDS upon proteins, we have provided detailed evidence and arguments to suggest that the initial interactions of SDS with proteins tend to be regulated mainly by electrostatic interactions, with hydrophobic interactions only playing subsequent (and secondary) role(s) . This suggests that proteins with very high contents of negatively charged residues, and an overall high net negative charge, must electrostatically repulse the initial approach of SDS molecules, since the latter happen to be negatively charged.…”

Monomeric Camelus dromedarius GSTM1 at low pH is structurally more thermostable than its native dimeric form

“…During thermal unfolding beyond 45˚C, a 222-nm peak shifted towards single a 225-nm peak ( Fig 12A). Thermal stress at pH 7.0 led to the conversion of CdGSTM1 from an alpha helical structure into a cross-beta sheeted structure, which aggregated beyond 75˚C [45]. However, thermal denaturation of monomeric CdGSTM1 led to the conversion of the same alpha-helical structure to a random-coil structure ( Fig 12B).…”