Fibrillation of the major curli subunit CsgA under a wide range of conditions implies a robust design of aggregation

Abstract

The amyloid fold is usually considered a result of protein misfolding. However, a number of studies have recently shown that the amyloid structure is also used in nature for functional purposes. CsgA is the major subunit of Escherichia coli curli, one of the most well-characterized functional amyloids. Here we show, using a highly efficient approach to prepare monomeric CsgA, that in vitro fibrillation of CsgA occurs under a wide variety of environmental conditions and that the resulting fibrils exhibit similar structural features. This highlights how fibrillation is "hardwired" into amyloid that has evolved for structural purposes in a fluctuating extracellular environment and represents a clear contrast to disease-related amyloid formation. Furthermore, we show that CsgA polymerization in vitro is preceded by the formation of thin needlelike protofibrils followed by aggregation of the amyloid fibrils.

Figure 1

Effect of environmental conditions on the fibrillation of CsgA. (A, E, G, and J) Fibrillation of CsgA followed by ThT at various pH values (A), NaCl concentrations (E), protein concentrations (G), and CsgA fibril seed concentrations (J). The standard conditions for the fibrillations were 20 µM CsgA in 20 mM sodium phosphate (pH 7) without addition of NaCl or seeds. (B, F, I, and K) Effect of pH (B), ionic strength (F), protein concentration (I), and CsgA fibril seeds (concentrations in weight percentage) (K) on the kinetic parameters t₅₀ and lag time. (C) Theoretical overall charge of mature CsgA as a function of pH calculated using CLC DNA Workbench version 5.7.1. (D and E) Effect of pH (C) and protein concentration (H) on the ThT maximum. (L) Effect of seeds on ThT start level and maximum.

Figure 2

Morphology of the CsgA fibrils. TEM image of CsgA fibrils formed in 20 mM sodium phosphate (pH 7).

Figure 3

Biophysical characterization of CsgA fibrils formed under various environmental conditions. (A and B) CD spectra of CsgA fibrils formed at various pH values (A) and at pH 7 with varying NaCl concentrations (B). (C and D) FTIR (C) and second-derivative (D) spectra of CsgA fibrils formed at various pH values. (E and F) FTIR (E) and second-derivative (F) spectra of CsgA fibrils formed at pH 7 with varying NaCl concentrations. (G) X-ray fiber diffraction pattern of CsgA fibrils formed at pH 7. (H) Radial averaged X-ray fiber diffraction spectra of fibrils formed at various pH values.

Figure 4

Fibrillation of CsgA followed by TEM. Samples was collected at various time points during the fibrillation of 40 µM CsgA at pH 7 and examined by TEM. The fibrillation curve measured by ThT is shown at the top, with circles corresponding to the time points shown in the bottom panels.

Figure 5

Fibrillation of CsgA followed by ANS fluorescence and SDS–PAGE. (A) Fibrillation of 20 µM CsgA followed by ThT and ANS fluorescence. (B) Fibrillation of 20 µM CsgA followed by ThT and SDS–PAGE. SDS–PAGE data were expressed as the CsgA monomer concentration calculated from the CsgA monomer band intensity using the ImageJ gel analysis tool (http://rsbweb.nih.gov/ij/).

Figure 6

Fibrillation of CsgA followed by circular dichroism. (A) Fibrillation of 20 µM CsgA followed by ThT and CD signal intensity at 197 nm. (B) CD wavelength spectra of samples collected during fibrillation.

Figure 7

Proposed CsgA fibrillation mechanism. Monomeric CsgA initially forms thin needlelike protofibrils. These protofibrils rearrange to form amyloid fibrils, which subsequently aggregate into dense clumps.