Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions - PubMed (original) (raw)

Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions

Sarah L Shammas et al. Biophys J. 2011.

Abstract

The self-assembly of proteins and peptides into polymeric amyloid fibrils is a process that has important implications ranging from the understanding of protein misfolding disorders to the discovery of novel nanobiomaterials. In this study, we probe the stability of fibrils prepared at pH 2.0 and composed of the protein insulin by manipulating electrostatic interactions within the fibril architecture. We demonstrate that strong electrostatic repulsion is sufficient to disrupt the hydrogen-bonded, cross-β network that links insulin molecules and ultimately results in fibril dissociation. The extent of this dissociation correlates well with predictions for colloidal models considering the net global charge of the polypeptide chain, although the kinetics of the process is regulated by the charge state of a single amino acid. We found the fibrils to be maximally stable under their formation conditions. Partial disruption of the cross-β network under conditions where the fibrils remain intact leads to a reduction in their stability. Together, these results support the contention that a major determinant of amyloid stability stems from the interactions in the structured core, and show how the control of electrostatic interactions can be used to characterize the factors that modulate fibril stability.

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Figures

Figure 1

pH dependence of insulin fibril dissociation. (Left panel) The extent of fibril dissociation at steady state was determined by measuring the protein concentration of the supernatants from ultracentrifuged solutions of fibrils that had been incubated at various pH values for 48 h. (Right panels) Representative fibril TEM images are from samples at various pH values as indicated in the figure. Scale bars represent 200 nm.

Figure 2

Dependence of insulin fibril dissociation on polypeptide net charge. (A) The amino-acid sequence of insulin is shown including the position of disulfide bonds (black lines). Residues with acidic and basic ionizable moieties are highlighted in magenta and blue, respectively. (B) The correlation between the predicted net charge (Q) of the insulin molecule and the fraction of soluble protein liberated from the fibrils in the pH range 2.0–12.5. Where Q ≤ 0, the data were fitted to a linear polymerization model (34), modified such that the free energy difference between the soluble and fibrillar state has been split into an electrostatic component and a contribution to fibril stability in the absence of charge (see main text). Errors were assigned to each value of Q to account for the deviations of the _pK_a values of ionizable moieties in the folded environment of the fibril, based on the variability of _pK_a values observed for folded proteins (29). (Shaded region overlaying the data) Corresponding error in the line of best fit.

Figure 3

FTIR spectra and thermodynamic stability of insulin fibrils. (A) FTIR spectra of insulin fibrils formed at pH∗ 1.6 (solid line) and adjusted to pH∗ 3.6 (dashed dotted line), pH∗ 5.6 (dashed line), and pH∗ 7.6 (dotted line) by addition of NaOD. The secondary structure contributions determined from deconvolution of the spectra of the fibrils at pH∗ 1.6 and pH∗ 7.6 are given in the table panel in Fig. S1 in the Supporting Material. (B) Insulin fibrils were incubated in increasing concentrations of guanidinium thiocyanate at pH 2.0 (dark blue squares), pH 4.0 (light blue squares), pH 6.0 (purple squares), and pH 8.0 (red squares) for 48 h and the subsequent extent of dissociation determined. The data were fit to the linear polymerization model.

Figure 4

pH and temperature dependence of the kinetics of insulin fibril dissociation. (A) Insulin fibril dissociation kinetics were followed by turbidity. Representative kinetic traces are shown for insulin solutions adjusted to pH 10.6 (red diamonds), 11.0 (green squares), 11.4 (blue triangles), and 12.0 (yellow circles). The lines represent the double-exponential functions that best fit the data. (B) Dependence of the fitted rate constants on pH. (C) Dependence of the fitted rate constants for fibril dissociation at pH 11.3 on inverse temperature in the range 15–50°C. These data represent the average of three independent experiments. (Solid lines, panels B and C) Linear fits to the data. (D) The cartoon illustrates the strong influence of a single ionization event on the kinetics of fibril dissociation. (Left) When Arg-B22 forms a stabilizing intra- or interchain salt bridge with a negatively charged moiety, dissociation is a rare event (_k_1). When this salt bridge is disrupted (right), the rate of dissociation increases dramatically (_k_2).

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Perturbation of the stability of amyloid fibrils through alteration of electrostatic interactions - PubMed (original) (raw)