Precrystallization clusters of holoferritin and apoferritin at low temperature

The formation of small nanosized clusters of the proteins holoferritin and apoferritin at low temperature was studied using small angle x-ray scattering. A strikingly large temperature dependence for the average molecular spacing in the clusters was observed. Calculations of the scattered intensity for various cluster models were performed. Comparison of the data with the simulations revealed the presence of crystalline order in the clusters of size ranging from a few molecules to a few hundred molecules. The crystalline order was found to be preserved with the lattice spacing varying with temperature by up to 20%. The small clusters were observed to grow into large micron-sized crystals when they were annealed and under certain conditions, the small clusters were found to coexist with the large crystals. This suggests that these clusters are closely related to critical nucleation. The data are consistent with an isotropic nucleation pathway, but cannot completely rule out a smaller presence of planar nucleation.

Figure 1

(Color online) Two-dimensional SAXS data from ferritin (a) and apoferritin (b). The oscillations seen for apoferritin are washed away in holoferritin by the presence of the iron core. (c) SAXS pattern of ferritin to which Cd salt was added to produce many small crystals. (d) Solution of ferritin frozen to $-20°\mathrm{C}$.

Figure 2

(Color online) SAXS data at indicated temperatures showing the change in scattered intensity from holoferritin solution at $200\mathrm{mg}∕\mathrm{ml}$ upon freezing and as the temperature is varied.

Figure 3

(Color online) Top: Average spacing of the ferritin molecules (within the clusters) versus temperature. The {111} lattice spacing of fcc ferritin crystals is $106\mathrm{\AA }$. Thermal expansion of 30% is seen over the range of temperature. Bottom: Average size of the clusters obtained from inverting the width of the peaks plotted versus temperature.

Figure 4

(Color online) Structure factors at various temperature for holoferritin solutions at (a) $100\mathrm{mg}∕\mathrm{ml}$, (b) $50\mathrm{mg}∕\mathrm{ml}$, and (c) $35\mathrm{mg}∕\mathrm{ml}$. In each case, the first temperature measured was the lowest, shown on top. As the temperature is increased, the peaks get narrower indicating an annealing process creating larger crystals. The overall cluster size is smaller as the concentration increases as seen by the broader peaks at $100\mathrm{mg}∕\mathrm{ml}$.

Figure 5

(Color online) Comparison of the structure factor from large crystals (black), small clusters (blue), and the coexistence of the two (red). Sharp peaks arise from large crystals while broader features are associated with small nanoclusters.

Figure 6

(Color online) SAXS intensity from solutions containing (a) $50\mathrm{mg}∕\mathrm{ml}$ and (b) $150\mathrm{mg}∕\mathrm{ml}$ of apoferritin in $100\mathrm{mM}$ NaCl. The intensity changes significantly upon freezing indicating aggregation leading to a change in the structure factor.

Figure 7

(Color online) Structure factor measured from apoferritin at (a) $50\mathrm{mg}∕\mathrm{ml}$ and (b) $150\mathrm{mg}∕\mathrm{ml}$. The structure factor changes with temperature.

Figure 8

(Color online) Spacing of the apoferritin molecules versus temperature overlaid with the spacing from iron loaded ferritin.

Figure 9

(Color online) Simulated structure factors from fcc supermolecules made of 1, 2, 13, 19, 43, 55, 79, 87, 135, 141, and $177\text{molecules}$, corresponding to the monomer, the dimer, and filled shells up to [110], [200], [112], [220], [310], [222], [123], [400], and ([330] and [114]), respectively.

Figure 10

(Color online) Simulated structure factors from planar supermolecules made of 5 ⟨110⟩ rods of $1\text{to}12\text{molecules}$ each.

Figure 11

(Color online) Fits (solid lines) to the measured structure factors (symbols) at multiple temperature for (a) a $200\mathrm{mg}∕\mathrm{ml}$ holoferritin sample and (b) a $100\mathrm{mg}∕\mathrm{ml}$ sample. The temperature as well as the model used for the fit is indicated directly next to the data curve. The curves are offset for clarity.

Figure 12

(Color online) Fits (solid lines) to the measured structure factors (symbols) at multiple temperature for two separate $50\mathrm{mg}∕\mathrm{ml}$ apoferritin sample. The temperature as well as the model used for the fit is indicated directly next to the data curve. The curves are offset for clarity.