Figure 2. CDRMs modulate eNP function.
(A) Differential HDX-MS between eNP-2 and eNP-3. The color gradient represented differential HDX shown at the bottom is generated by subtracting the amount of deuterium uptake of eNP-2 from that of eNP-3 at the 60 second time point. Regions colored white are not detected by HDX. HDX-MS measurements were performed in duplicate.
(B) Fluorescence polarization binding curves of eNP proteins for eVP35 NPBP. The measured KD values are 10.6 ± 2.4 nM (eNP WT,
), 1.9 ± 0.2 nM (eNP-2, ■), 5.4 ± 0.3 nM (eNP-2 K373A/K374A/K382A/K383A,
), 12.8 ± 6.8 nM (eNP-3,
), 9.7 ± 2.5 nM (eNP-3 K373A/K374A/K382A/K383A,
), and 1.2 ± 1.2 nM (eNP-4,
). Each measurement was performed at least twice independently in duplicate.
(C) Fluorescence polarization binding curves of eNP proteins for 20 nt ssRNA. The measured KD values are 0.013 ± 0.003 μM (eNP WT,
), 0.068 ± 0.020 μM (eNP-2, ■), > 10 μM (eNP-2 K373A/K374A/K382A/K383A,
), 0.015 ± 0.005 μM (eNP-3,
), 0.306 ± 0.006 μM (eNP-3 K373A/K374A/K382A/K383A,
), and 3.17 ± 0.28 μM (eNP-4,
). Each measurement was performed at least twice independently in duplicate.
(D) Differential HDX-MS between eNP-2 and eNP-2/ssRNA. The color gradient represented differential HDX shown at the bottom is generated by subtracting the amount of deuterium uptake of eNP-2 from that of eNP-2/ssRNA complex at the 60 second time point. Regions colored white are not detected by HDX. HDX-MS measurements were performed in duplicate. (E) Differential HDX-MS between eNP-3 and eNP-3/ssRNA. The color gradient represented differential HDX shown at the bottom is generated by subtracting the amount of deuterium uptake of eNP-2 complex from that of eNP-3/ssRNA complex at the 60 second time point. Regions colored white are not detected by HDX. HDX-MS measurements were performed in duplicate. (F) Differential HDX-MS between eNP-2/ssRNA and eNP-3/ssRNA. The color gradient represented differential HDX shown at the bottom is generated by subtracting the amount of deuterium uptake of eNP-2/ssRNA complex from that of eNP-3/ssRNA complex at the 60 second time point. Regions colored white are not detected by HDX. HDX-MS measurements were performed in duplicate.
See also Figure S2.