Purpose:
Our goal is to design a peptide of tandem-repeated sequence (PTRSs) to imitate the binding of CLEC5A to the E protein from the dengue virus. In order to ensure the PTRSs and the E protein interact, all the structure of PTRSs and proteins and their interactions were modeled using Rosetta.
Method and Results:
- The structure of E protein (Figure 1) from a local strain (PL046) was generated using RosettaCM (comparative modeling with Rosetta) based on the crystal structure (PDB: 1OAN).
- The structures of PTRSs were predicted purely based on their sequences using the ab initio method in Rosetta, and the representative structures are shown in Figure 2.
- The most probable structure of the PTRS were identified by clustering.
- The interactions between the PTRSs and the E protein were evaluated using global protein-protein docking. The 100 most frequent docking sites are shown in Figure 3.
Figure 1. The homologous structure of PL046 E protein (cyan) based on the deposited structure (PDB: 1OAN) magenta.
Figure 2. The representative structures of a PTRS.
Figure 3. The best 100 results (based on the Rosetta score) of PTRS docking to PL046 E protein (red). The space above the plane (grey) indicates the external surface of the virion, where the interactions occur.
Interaction between Gold Nanoparticles
Purpose:
A potential weakness of our design is that peptides on the gold nanoparticles and on the glass fiber compete for the same binding sites on the E protein. If the E proteins on the virus particles are fully covered by gold nanoparticles, there would be no sites available to interact with the peptides attached to the glass fiber. Hence, we need to understand if this situation would hinder development of our kit.
Method:
We used DLVO theory to calculate the repulsion between gold nanoparticles to estimate the number of gold nanoparticles that would bind to a virus particle. The structure of dengue virus is icosahedral, and there are nine E proteins on each surface. The distances between the potential binding sites can be obtained from the structure in the protein data bank (1K4R).
DLVO theory can be described as follows,
Wtotal(D) = Wa(D) + Wr(D) = -AR/12D + 2πεε0RΨδ2exp(-κD)
W
total(D): total energy
W
a(D): van der Waals interaction energy
W
r(D): electrostatic interaction energy
A: Hamaker constant = 2.5×10
-19 J
R: radius = 1.3x10
-8 m (based on the size of gold nanoparticles)
C: center to center distance
D: C-R
ε: permittivity of vacuum = 8.854x10
-12 F/m
ε
0: dielectric of solution = 80.4 (at 20°C)
Ψ
δ2: Stern layer potential (zeta potential) = 1.794 mV
κ
-1: diffuse layer thickness = 7.95x10
-10 m
We took several representative positions on adjoining faces of the icosahedron to calculate the interactions between the gold nanoparticles based on DLVO theory.
Results:
The total energies are all positive, indicating repulsive forces, (Table 1) and these total energies are also larger than the typical biological interactions (~0.5 kcal/mol or 3.49 x10-21 J). The results suggest that there will always be free faces on the virus particles to interact with the peptides conjugated on the test line.
Table 1. Energies of the gold nanoparticles between representative positions.