Time-resolved small-angle x-ray scattering study of the early stage of amyloid formation of an apomyoglobin mutant

The description of the fibrillogenesis pathway and the identification of “on-pathway” or “off-pathway” intermediates are key issues in amyloid research as they are concerned with the mechanism for onset of certain diseases and with therapeutic treatments. Recent results on the fibril formation process revealed an unexpected complexity both in the number and in the types of species involved, but the early aggregation events are still largely unknown, mainly because of their experimental inaccessibility. To provide information on the early stage events of self-assembly of an amyloidogenic protein, during the so-called lag phase, stopped-flow time-resolved small angle x-ray scattering (SAXS) experiments were performed. Using a global fitting analysis, the structural and aggregation properties of the apomyoglobin W7FW14F mutant, which is monomeric and partly folded at acidic pH but forms amyloid fibrils after neutralization, were derived from the first few milliseconds onward. SAXS data indicated that the first aggregates appear in less than 20 ms after the pH jump to neutrality and further revealed the simultaneous presence of diverse species. In particular, worm-like unstructured monomers, very large assemblies, and elongated particles were detected, and their structural features and relative concentrations were derived as a function of time on the basis of our model. The final results show that, during the lag phase, early assembling occurs due to the presence of transient monomeric species very prone to association and through successive competing aggregation and rearrangement processes leading to coexisting on-pathway and off-pathway transient species.

Figure 1

Time evolution of SAXS intensity measured on the apomyoglobin W7FW14F mutant solution after the pH jump from 4 to 7. Curves, obtained by averaging at least 10 different runs, are shifted by a factor of 5 cm${}^{-1}$ for clarity. For each curve, a label indicates the starting time of the measurement after mixing, while the superimposed continuous line represents the theoretical fit to the data.

Figure 2

Kratky plots relative to apomyoglobins in solution: wild-type at pH 7 ($\square$) and W7FW14F mutant at pH 4 ($◯$). The superimposed continuous lines represent the theoretical fit to the data. Fitted parameters were as follows: wild-type, $\delta =(3.0\pm 0.2)$ $\AA$, ${\rho }_s=(0.340\pm 0.002)$ e$/\AA$${}^3$; W7FW14F mutant, $x_{\mathrm{wlk}}=(0.980\pm 0.005)$, $L=(370\pm 15)$ $\AA$, $b=(67\pm 4)$ $\AA$, $R_{\mathrm{apo}}=(4.2\pm 0.2)$ $\AA$, $r_0=(14\pm 1)$ $\AA$, $D=(2.3\pm 0.1)$, $\xi =(55\pm 3)$ $\AA$. Note that for W7FW14F mutant, $\delta$ was fixed to 3.5 $\AA$ and ${\rho }_s$ to 0.340 e/${\AA }^3$

Figure 3

Time course of W7FW14F mutant assembling after pH neutralization. (Top) Molar concentration of monomers occurring as large aggregates, worm-like species, and elongated particles. (Middle) Molar concentration of the cylindrical particles, separated according to their dimensions in fibril-like and early protofibril particles. (Bottom) Percentage of monomers incorporated into the cylindrical particles. Lines are guides for the eyes. Error bars are inside the symbol sizes. Because continuity is questionable, points at 10 ms are shown using solid symbols.

Figure 4

Time course of the electron density of the different aggregate particles after pH neutralization. The solid line indicates the nominal protein density, while dotted lines are guides for the eyes. Error bars are inside the symbol sizes. Because continuity is questionable, points at 10 ms are shown using solid symbols.

Figure 5

Time course of the structural properties of the fibril-like and early protofibril particles after pH neutralization. (Top) Particle length. (Middle) Particle radius. (Bottom) Particle aggregation number. Lines are guides for the eyes. Error bars are inside the symbol sizes. Because continuity is questionable, points at 10 ms are shown using solid symbols.