1. Electrostatic Effects in Filamentous Protein Aggregation
Alexander K. Buell, Peter Hung, Xavier Salvatella, Mark E. Welland, Christopher M. Dobson, Tuomas P.J. Knowles 
Biophysical Journal 
Volume 104, Issue 5, Pages (March 2013)
DOI: /j.bpj
Copyright © 2013 Biophysical Society Terms and Conditions

2. Figure 1
AFM images of insulin amyloid fibrils on mica, acquired with a Pico Plus atomic force microscope (Molecular Imaging, San Diego, CA), using tapping mode in air. (a) Sonicated seed fibrils. (b) Elongated seed fibrils at the end of the light scattering experiment to monitor the kinetics of elongation.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

3. Figure 2
Results from experiments designed to study the influence of sodium chloride concentration on insulin amyloid fibril elongation rate. The elongation of added seed fibrils is monitored. Representative data are shown for experiments carried out in 0.01 M HCl, in the temperature range 35–45°C, using four different techniques: (a) Thioflavin T (ThT) fluorescence, 40°C; (b) dynamic light scattering (DLS), 45°C; (c) quartz crystal microbalance (QCM), 35°C; and (d) surface plasmon resonance (SPR), 40°C. Details on how the elongation rates were extracted from these data can be found in the Methods. (e) The logarithm of the insulin amyloid elongation rates from the experiments shown in panels a–d, as well as additional experiments at different temperatures, are plotted as a function of the square-root of solution ionic strength (0–20 mM NaCl added onto a background of 10 mM HCl), in a Debye-Hückel (DH) plot. (Inset) Slopes of linear fits to the data sets. The excellent agreement between the four techniques confirms that the process of fibril elongation along a surface is equivalent to the elongation in bulk solution. In addition, the plots illustrate that the observed screening effects have no detectable temperature dependence in the investigated temperature range. (f) A DH plot (obtained from QCM-experiments) is shown that extends to 100 mM NaCl, demonstrating the decrease in slope at higher ionic strength values. (g) The elongation rate of insulin amyloid fibrils was measured as a function of NaCl concentration up to 1 M. The behavior is nonmonotonic and likely to be due to a change in stability of the soluble protein.
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

4. Figure 3
Analysis of the effects of a wide variety of halide salts on the kinetics of amyloid fibril elongation of bovine insulin and bovine PI3K-SH3, obtained from QCM and SPR measurements. (a–c) Fits of three different models to the data sets (illustrated to the left). (a) Only Debye screening is included and this model is unable to reproduce the different behavior observed for the salts of monovalent and divalent cations. The only free parameter in this fit is Q, the effective charge for fibril elongation of the free monomer and the fibril-end. The fit yields z = 4.4 for insulin and z = 6.5 for PI3K-SH3. (b) Anion binding to the soluble and fibrillar protein is included in the model used for the fit; (c) binding of the divalent cations replaces anion binding. The fits to models that include ion binding to the protein have two free parameters: the effective charge z, and nKi, being the product of the number of binding sites and the binding constant of ions on these binding sites (see Appendix). The fitting parameters are as follows: Anion binding to insulin, z = 3.6, nK_ = 82.4 M−1; and to PI3K-SH3, z = 6.0, nK_ = M−1. Cation binding to insulin, z = 4.8, nK2 = 30.6 M−1; and to PI3K-SH3, z = 7.5, nK2 = M−1. Only the model involving cation binding can account quantitatively for the large differences in the effects of salts on fibril growth kinetics. The effect of the trivalent salt LaCl3 is also included in the figure, but has not been included in the fit (see text for details).
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions

5. Figure 4
Specific ion binding and its effects on the kinetics of amyloid fibril growth. QCM measurements of the elongation of glucagon and β2-microglobulin fibrils in the presence of different halide salts. (a and b) For these two polypeptides, chlorides of divalent cations are also less efficient in accelerating the fibril elongation rate than chlorides of monovalent cations, similar to the results shown in Fig. 3 for insulin and PI3K-SH3. (c and d) The data show that for these two proteins, specific electrostatic effects can be observed even for simple halide ions, as demonstrated by the difference in screening efficiency between chloride and bromide salts. (e) More pronounced specific ion effects due to strong association between the ions and the protein can be observed from the effect of nitrate and sulfate on the elongation rate of amyloid fibrils formed from PI3K-SH3. (Straight lines connecting the first two data points are shown as guides to the eye.) The effect of NaCl is shown for comparison; the fibril elongation rate is affected much more strongly by the salts of the more complex anions, compared to NaCl. (f) The measured increase in fibril growth rates is greater than would be expected from purely nonspecific screening effects, indicating that the nonelectrostatic parts of the free energy landscape are likely to be affected (see text).
Biophysical Journal  , DOI: ( /j.bpj )
Copyright © 2013 Biophysical Society Terms and Conditions