Figure 1. Biochemical characterization of oligomerization in wildtype and mutant eNP proteins.

(A) Domain organization of eNP WT and key constructs used in this study listed below. NTD, N-terminal domain; CTD, C-terminal domain. (B) Elution profile from SEC for eNP-2 in buffer containing 500 mM NaCl. Inset. Coomassie stained SDS-PAGE of corresponding peak fractions from SEC. p1, peak 1; p2, peak 2. (C) DLS data in PBS for: (i) eNP-2 p1 (R_h = 71.6 ± 2.0 nm), eNP-2 p2 (R_h = 33.7 ± 3.6 and 308.8 ± 18 nm), and (iii) eNP-2 p2 + eVP35 NPBP (R_h = 4.2 ± 0.1 nm). Two independent DLS experiments were performed in triplicate for each construct shown. (D) Elution profile from SEC-MALS of eNP-2 WT (black) and eNP-2 K373A/K374A/K382A/K383A (red). Mw = > 10⁴ kDa (^*) and 45 ± 1 kDa (^**) for the peaks indicated. The theoretical molecular mass for monomeric eNP-2 is 48 kDa. Two independent experiments were performed. (E) DLS data in PBS for eNP-2 K373A/K374A/K382A/K383A (R_h = 4.2 ± 0.2 nm). Two independent DLS experiments were performed in triplicate. (F) Differential HDX-MS highlighted on eNP-2 (PDB: 4YPI); regions not present in the structure are indicated by a dotted line. The color gradient represented differential HDX shown at the bottom is generated by subtracting the amount of deuterium uptake of eNP-2 K373A/K374A/K382A/K383A from that of eNP-2 at the 60 second time point. Regions colored white are not detected by HDX. HDX-MS measurements were performed in duplicate.

See also Figure S1.