DNA Facilitates Oligomerization and Prevents Aggregation via DNA Networks

Abstract

Previous studies have shown that nucleic acids can nucleate protein aggregation in disease-related proteins, but in other cases, they can act as molecular chaperones that prevent protein aggregation, even under extreme conditions. In this study, we describe the link between these two behaviors through a combination of electron microscopy and aggregation kinetics. We find that two different proteins become soluble under harsh conditions through oligomerization with DNA. These DNA/protein oligomers form "networks," which increase the speed of oligomerization. The cases of DNA both increasing and preventing protein aggregation are observed to stem from this enhanced oligomerization. This observation raises interesting questions about the role of nucleic acids in aggregate formation in disease states.

Figure 1

(A and C) TEM images of luciferase oligomers interacting with DNA. (A) Thermal denaturation or (C) chemical denaturation is shown. DNA can be seen bound to several oligomers as silver tendrils. Note that the protein concentration is different in the two different methods of denaturation, but the DNA/protein ratio is the same. (B and D) TEM of luciferase aggregates when no DNA is present. (B) Thermal denaturation or (D) chemical denaturation is shown. Note the difference scale bar size compared to the cases with DNA.

Figure 2

Light scattering profile of 65 nM luciferase under chemically denaturing conditions with varying concentrations of DNA. (A) The t_1/2 and maximum RALS signal are reduced in the presence of >0.65:0.065 DNA μM base pair/μM protein (purple, green, and cyan traces). Luciferase on its own aggregates at a slower rate (black) is shown. (B) At <0.65:0.065 DNA μM base pair/μM protein, the acceleration is still apparent (blue and red traces), but the maximum RALS signal is increased compared to luciferase (black) on its own.

Figure 3

(A) SDS-PAGE from spin-down assays with thermally or chemically denatured luciferase in the presence of increasing amounts of DNA are shown. (B and C) A pie chart of the size of luciferase oligomers in TEM images as a function of DNA concentration (DNA/protein ratios shown below each chart) when denatured (B) thermally or (C) chemically is shown. Oligomer sizes are given in nm². Note, actual concentrations are given in the ratios for the above assays. To see this figure in color, go online.

Figure 4

TEM images of luciferase oligomers at low DNA concentrations. Where DNA is evident, it is indicated with black arrows. (A) Thermally denatured luciferase pellet and soluble images are shown. (B) Chemically denatured luciferase pellet and soluble images are shown. Although generally larger, the pellets share similar homology to the soluble fraction oligomers. Note the DNA/protein ratios between denaturation methods are the same.

Figure 5

SDS-PAGE results of variable spin-down assay from thermal aggregation. From left to right, the rotor speed is increasing, and from top to bottom, there is increasing DNA concentration. The oligomers remain soluble at greater rotor speeds with increasing amounts of DNA.

Figure 6

TEM, CD, and SDS-PAGE of MDH-DNA complexes. (A) SDS-PAGE of MDH at the given condition is shown. (B) CD of 3.2 μM MDH with 90 μM bp DNA at several temperatures are shown. “For” indicates the forward melting experiment up to 80°C, and “Rev” indicates the reverse experiment back to room temperature. (C) Micrographs of 3.2 μM MDH and 360 μM bp DNA are shown. Zoomed out micrographs shown in Fig. S6. (D) A TEM micrograph of MDH aggregate in the absence of DNA is shown. Note the change in scale bar size. P, pellet fraction; S, soluble fraction; T, total fraction. To see this figure in color, go online.