Crowding Alone Cannot Account for Cosolute Effect on Amyloid Aggregation

Amyloid fiber formation is a specific form of protein aggregation, often resulting from the misfolding of native proteins. Aimed at modeling the crowded environment of the cell, recent experiments showed a reduction in fibrillation halftimes for amyloid-forming peptides in the presence of cosolutes that are preferentially excluded from proteins and peptides. The effect of excluded cosolutes has previously been attributed to the large volume excluded by such inert cellular solutes, sometimes termed “macromolecular crowding”. Here, we studied a model peptide that can fold to a stable monomeric β-hairpin conformation, but under certain solution conditions aggregates in the form of amyloid fibrils. Using Circular Dichroism spectroscopy (CD), we found that, in the presence of polyols and polyethylene glycols acting as excluded cosolutes, the monomeric β-hairpin conformation was stabilized with respect to the unfolded state. Stabilization free energy was linear with cosolute concentration, and grew with molecular volume, as would also be predicted by crowding models. After initiating the aggregation process with a pH jump, fibrillation in the presence and absence of cosolutes was followed by ThT fluorescence, transmission electron microscopy, and CD spectroscopy. Polyols (glycerol and sorbitol) increased the lag time for fibril formation and elevated the amount of aggregated peptide at equilibrium, in a cosolute size and concentration dependent manner. However, fibrillation rates remained almost unaffected by a wide range of molecular weights of soluble polyethylene glycols. Our results highlight the importance of other forces beyond the excluded volume interactions responsible for crowding that may contribute to the cosolute effects acting on amyloid formation

Aggregation lag times <i>t_lag</i> (in minutes) for different peptide stabilities ΔΔG (in kJ/mol).^a

a<p>value of <i>t_lag</i> in the absence of cosolutes was 340±80 min.</p

Kinetics of amyloid formation followed by CD spectroscopy.

<p>(<b><i>left column</i></b>) CD spectra measured at different times of the aggregation process in the absence (<i>top</i>) and presence of sorbitol (<i>center</i>) and PEG 4000 (<i>bottom</i>). (<b><i>right column</i></b>) Contribution of unfolded (<i>triangles</i>), β-sheet (<i>squares</i>) and amyloid (<i>circles</i>) formations to each of the CD spectra presented on the left column, as determined by CCA analysis, shown as a function of time for each of the aggregation reactions shown on the left. Lines are guides for the eyes.</p

Effects of cosolute addition on ThT fluorescence.

<p>Ratio of ThT emission values at λ = 485 nm before and after cosolute addition. A value close to 1.0 represents no change in emission upon dilution. Inset shows ThT fluorescence emission vs. time, with the point of dilution at <i>t</i> = 2600 minutes. The value of signal at the plateau prior to dilution (<i>f^*</i>) (Eq. 2) was divided by the average emission value of the hour following ThT addition to obtain the relative deviation values of the fluorescence at peak emission as a result of cosolute addition. Circles (<i>green</i>) show the emission of buffered ThT without the addition of MET16.</p

Length distribution analysis of fibrils imaged using TEM.

<p>Fibril lengths were measured in the absence of cosolutes, (<b>A</b>) at <i>t</i> = 0 (average length, as calculated directly from measurements, 263±114 nm), (<b>B</b>) at <i>t</i> = 500 min, (average length 458±146 nm); and in presence of 30% (w/w) sorbitol, (<b>C</b>) at <i>t</i> = 0 min (average length is 142±64 nm), (<b>D</b>) at <i>t</i> = 500 min (average length 265±95 nm). Errors in average length are standard deviation of length measurements.</p

Analyses of CD-resolved kinetics.

<p>(<b>A</b>) RMS deviation of simulated CD spectra based on the derived basis set of P spectra (<b>B</b>) Simulated CD curves of the three basic structures calculated by CCA. These correspond to pure β-sheet (solid black line), amyloid (dashed) and unfolded (dot-dashed). The solid cyan line is the experimental spectrum of the fully folded peptide in the presence of 55% (w/w) MeOH. (<b>C</b>) Ratio of β-sheet (φ_f) over unfolded (φ_u) mole fraction as a function of time for MET16 in water (<i>squares</i>), and enough sorbitol (<i>circles</i>) or PEG 4000 (<i>triangles</i>) to induce a stabilization of ΔΔG = −1.5 kJ/mol to the β-sheet conformation. The dotted lines represent theoretical values for the equilibrium constant for folding for the reaction in aqueous media (ΔΔG = 0) and in the presence of the cosolutes (ΔΔG = −1.5 kJ/mol).</p

Schematic of MET16 states and transitions.

<p>In buffered environment (pH 7) the peptide exists in two-state equilibrium between native (N) and unfolded (D) conformations. After ∼90 min a third, fibrillar aggregate conformation appears. The folded conformations appearing in the fibril need not be the same as N.</p