The Hofmeister effect on amyloid formation using yeast prion protein

Abstract

A variety of proteins are capable of converting from their soluble forms into highly ordered fibrous cross-beta aggregates (amyloids). This conversion is associated with certain pathological conditions in mammals, such as Alzheimer disease, and provides a basis for the infectious or hereditary protein isoforms (prions), causing neurodegenerative disorders in mammals and controlling heritable phenotypes in yeast. The N-proximal region of the yeast prion protein Sup35 (Sup35NM) is frequently used as a model system for amyloid conversion studies in vitro. Traditionally, amyloids are recognized by their ability to bind Congo Red dye specific to beta-sheet rich structures. However, methods for quantifying amyloid fibril formation thus far were based on measurements linking Congo Red absorbance to concentration of insulin fibrils and may not be directly applicable to other amyloid-forming proteins. Here, we present a corrected formula for measuring amyloid formation of Sup35NM by Congo Red assay. By utilizing this corrected procedure, we explore the effect of different sodium salts on the lag time and maximum rate of amyloid formation by Sup35NM. We find that increased kosmotropicity promotes amyloid polymerization in accordance with the Hofmeister series. In contrast, chaotropes inhibit polymerization, with the strength of inhibition correlating with the B-viscosity coefficient of the Jones-Dole equation, an increasingly accepted measure for the quantification of the Hofmeister series.

Figure 1

The Hofmeister salts are listed along with their corresponding B-viscosity coefficients. The relative kosmotropicity and chaotropicity of each salt can be followed by its position on the arrow.

Figure 2

Spectral characteristics of Congo Red (CR) and Congo Red with amyloid fibrils. The legends in panel A and B indicates the concentration of CR. (A) Absorbance spectra of free CR at varying CR concentrations in (μM). (B) Absorbance spectra of a CR and varying amyloid concentrations (μM), including pure scattering without CR. (C) Molar absorptivity of free CR at the isosbestic point (415 nm) and point of maximum difference (543 nm). (D) Molar absorptivity of bound CR at isosbestic point (415 nm) and point of maximum difference (543 nm).

Figure 3

The effect of the Hofmeister salts on the polymerization of Sup35 NM under seeded and unseeded condition. Sodium salt anions are listed to the right. Seeded reactions contained 2% by volume sonicated preformed fibril samples. (A) Seeded condition and kosmotropic salts. (B) Unseeded condition and kosmotropic salts. (C) Seeded condition and chaotropic salts. (D) Unseeded condition and chaotropic salts. Units of CR[bound] are (M cm⁻¹).

Figure 4

The effect of the Hofmeister salts on agitated Sup35 NM polymerization. (A) Agitated polymerization of 10 μM Sup35 NM in kosmotropic solutions. (B) Agitated polymerization of 10 μM Sup35NM in chaotropic solutions. (C) In a separately conducted agitated experiment with 15 μM Sup35 NM focusing on the chaotropic samples, a negative linear relationship was found between the lag phase of the solution (in hours) to the B-viscosity coefficient of the Hofmeister salt. The Figure with original data can be found in the Supporting Information. (D) In the same experiment as panel C, a linear correlation was found between the initial rate of aggregation to the B-viscosity coefficient of the Hofmeister salt.

Figure 5

Concentration dependence of the inhibition effect of NaNO₃ on Sup35NM polymerization. (A) Various concentrations of NaNO₃ were tested under the agitated condition. The concentrations used were 0 M, 0.15 M, 0.313 M, and 0.994 M NaNO₃ with 5 mM NaH₂PO₄ at pH 7.4. (B) Negative correlation between the initial polymerization rate and concentration of NaNO₃ were observed. (C) Linear correlation between the length of polymerization lag phase and the concentration of NaNO₃ was observed.

Figure 6

Congo Red binding to an amyloid in the presence of Hofmeister salts. The amyloids were formed in the absence of salts, but CR assay was conducted in the presence of salt as described in “Material and Methods” section. No inhibitory effect of any salt on CR binding is detected.