• Benjaman Alvarez posted an update 7 months, 3 weeks ago

    Ion of two.eight A. The crystal was in the space group H3 with eight polypeptides (designated as A to H) inside the asymmetric unit. The threefold crystallographic symmetry operated around the asymmetric unit content to produce a 24-mer cage. The packing with the Hpf-E2 subunits within the nanocage structure was identical to that in the Hpf-E1, with minor shifts in relative positions (see alignments of subunits below for facts). Due to the somewhat low resolution, only 11 water molecules and no other ions/small molecules were modeled (Table 1). The Hpf-E2 protein was purified from refolding of inclusion bodies. As a result, there were no endogenously bound metal ions, including Fe, identified in the crystal structure. The His side chains that coordinated the Fe in Hpf-E1 had a slight shift away from each other in Hpf-E2. Place in the inserted MtrE loops The packing in the 24-mer cage structure of both Hpf-E1 and Hpf-E2 was identical to that on the wild-type Hpf. Even so, the inserted N. gonorrhoeaeFEBS Open Bio 7 (2017) 1196207 2017 The Authors. Published by FEBS Press and John Wiley Sons Ltd.Structure-based design of nanoparticle immunogensL. Wang et al.Fig. 3. Protein purification and characterization. Panels (A), (B), and (C) are SDS/PAGE of Hpf-E2, Hpf, and Hpf-E1, respectively, stained by SimplyBlue (Invitrogen, Carlsbad, CA, USA). Lanes marked M are markers; T, total cell lysate; S, supernatant; P, pellets. Lanes marked Q elute 1 and two are from peaks marked 1 and 2 in (D). Lanes marked S6 are just after the Superose 6 gel filtration as shown in (E). Lanes in (B) marked (NH4)2SO4 S and P are the supernatant and pellet fractions of ammonium sulfate precipitation (65 saturation) on the cell lysate supernatant. In panel (C), the lane marked Q-ft is definitely the flow-through in the loading of a HiTrap Q Ari, 1994; Roy et al., 2007). SSB-ssDNA complexes can transition involving these modes column in 20 mM Tris pH 8.0 and 50 mM NaCl, and lane HIC may be the protein following hydrophobic interaction chromatography working with a butyl HP column. A minor contaminant 100 kDa on S6 was removed by HIC. (D) Chromatogram of a HiTrap Q column elution of Hpf-E2. (E) Gel filtration chromatograms. The standards are thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa), in the Gel Filtration HMW Calibration Kit (GE). The calculated molecular weight from the Hpf-E2 nanoparticle is 514 kDa, and that for the wild-type Hpf is 464 kDa. (F) An electron micrograph of purified Hpf-E2. The scale bar is 20 nm. The EM images of Hpf and Hpf-E1 are identical.peptides in each structures had been disordered. To ascertain the place and prospective conformations with the inserted N. gonorrhoeae peptides, we superposed the Hpf-E1 subunit structure using the computationally developed model (Fig. 4A). The Hpf-E1 structure was basically identical to the computational model. The majority of the subunits of Hpf-E1 had some weak electron density for part of the inserted peptide that recommended that the MtrE loop 1 peptide adopted many conformations (Fig. 5A). It can be most likely that the N. gonorrhoeae peptide could have conformations that did not fold into its native b-hairpin structure.