Osmolyte Induced Changes in Peptide Conformational Ensemble Correlate with Slower Amyloid Aggregation: A Coarse-Grained Simulation Study

Stabilizing osmolytes are known to impact the process of amyloid aggregation, often altering aggregation kinetics. Recent evidence further suggests that osmolytes modify the peptide conformational dynamics, as well as change the physical characteristics of assembling amyloid fibrils. To resolve how these variations emerge on the molecular level, we simulated the initial aggregation steps of an amyloid-forming peptide in the presence and absence of the osmolyte sorbitol, a naturally occurring polyol. To this end, a coarse-grained force field was extended and implemented to access larger aggregate sizes and longer time scales. The force field optimization procedure placed emphasis on calibrating the solution thermodynamics of sorbitol, the aggregating peptide in its monomeric form, and the interaction of both of these components with each other and with water. Our simulations show a difference in aggregation kinetics and structural parameters in the presence of sorbitol compared to water, which qualitatively agree well with our experimentally resolved aggregation kinetics of the same peptide. The kinetic changes induced by sorbitol can be traced in our simulations to changes in monomer conformations resulting from osmolyte presence. These translate into changes in peptide conformations within the aggregated clusters and into differences in rates of monomer nucleation and of association to formed fibrils. We find that, compared to pure water as solvent, the presence of sorbitol induces formation of more aggregates each containing fewer peptides, with an increased tendency toward parallel interpeptide contacts

Can Local Probes Go Global? A Joint Experiment–Simulation Analysis of λ_6–85 Folding

The process of protein folding is known to involve global motions in a cooperative affair; the structure of most of the protein sequences is gained or lost over a narrow range of temperature, denaturant, or pressure perturbations. At the same time, recent simulations and experiments reveal a complex structural landscape with a rich set of local motions and conformational changes. We couple experimental kinetic and thermodynamic measurements with specifically tailored analysis of simulation data to isolate local versus global folding probes. We find that local probes exhibit lower melting temperatures, smaller surface area changes, and faster kinetics compared to global ones. We also see that certain local probes of folding match the global behavior more closely than others. Our work highlights the importance of using multiple probes to fully characterize protein folding dynamics by theory and experiment

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

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

Effects of high viscosity on amyloid formation kinetic constants.

<p>Relative lag times <i>t_lag</i> (<b>A</b>) and elongation lifetimes <i>τ</i>_el (<b>B</b>) for 50% (w/w) glycerol, TEG and PEG 400, and 30% (w/w) for sorbitol. N.C. corresponds to the reaction in the absence of cosolute, to which the other reactions are compared. (<b>C</b>) The viscosity of each solution, in cP.</p

Effect of cosolutes on monomeric peptide structure and stability.

<p>(<b>A</b>) Representative CD spectra of MET16 with increasing PEG 4000 concentrations (0, 8, 12, 17, 21, and 25% (w/w)). Inset shows CD spectra for fully folded MET16 in the presence of 55% (w/w) methanol (solid line) and the calculated unfolded peptide spectra (dashed line, see text for details). (<b>B</b>) Peptide folding free energy (ΔΔG) as a function of cosolute concentration. The dashed lines delineate cosolute concentration corresponding to ΔΔG = −0.5 kJ/mol and −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

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