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1.
Fig. 5

Fig. 5. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

(A). Overlay of size-exclusion chromatograms of WT gB ectodomain (gB730) and representative mutants. gB trimer elutes as a broad peak near the void volume. The elution volumes of the void and the molecular weight standards are shown (arrows).

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.
2.
Fig. 1

Fig. 1. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

Schematic view of the sequence of the full-length HSV-1 gB and constructs designed to stabilize the prefusion conformation. SS – signal sequence, MP – membrane-proximal region, TM – transmembrane region, Cyto – cytoplasmic domain are shown as rectangles of different shades of gray. GCNt (sequence: QIEDKIEEILSKIYHIENEIARIKKLIGE) and foldon (sequence: GYIPEAPRDGQAYVRKDGEWVLLSTF) are shown as black rectangles. Linkers GSGS, GSGTGS, or GGSGGTGGSG are shown as lines.

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.
3.
Fig. 3

Fig. 3. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

Most constructs are secreted from insect cells and are not misfolded. Proteins secreted into the supernatant (A) or remaining inside the cells (B) were detected by Western blot with anti-gB polyclonal antibody R68. Under nonreducing denaturing conditions, gB migrates as a monomer (M). Positions of molecular weight standards are labeled. Lanes are labeled at the top. gB730-GCNt-cyto lane corresponds to construct gB730-GCNt-cyto(821–904).

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.
4.
Fig. 6

Fig. 6. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

(A). All of the tested constructs adopted the postfusion conformation. gB molecules were visualized by negative-stain EM, and representative point mutants (A–C, H–I), “trimerization” constructs (D–G), and a truncation mutant (J) are shown. gB730-GCNt corresponds to construct gB730-GGSGGTGGSG-GCNt-Xa-His6, gB730-GCNt-cyto corresponds to construct gB730- GCNt-cyto(821-904), gB730-cyto corresponds to construct gB730-GSGTGS-cyto(796-904), and gB730-foldon corresponds to construct gB730-GSGS-foldon. Electron micrographs with multiple particles as well as close-up views of individual particles are shown. All large panels and all close-up panels are shown at the same magnification levels, respectively. (K) The crystal structure of gB730 is shown in surface representation, for comparison. Crown and base ends are labeled.

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.
5.
Fig. 4

Fig. 4. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

Most constructs react with anti-gB monoclonal antibody DL16, specific for the postfusion form. (A) Mutants designed to stabilize the prefusion conformation. (B) Mutants designed to destabilize the postfusion conformation. Proteins secreted into the supernatant were detected by a Western blot with anti-gB monoclonal antibodies DL16 (top panels) or C226 (bottom panels). Under nonreducing mildly denaturing conditions, gB migrates as a trimer. T – trimer, M – monomer. Positions of molecular weight standards are labeled. In A, lanes are numbered and corresponding constructs are listed underneath the blots. “L6” stands for linker sequence GSGTGS; “L10” stands for linker sequence GGSGGTGGSG. “Cyto” corresponds to the entire gB cytoplasmic domain. In B, lanes are labeled at the top.

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.
6.
Fig. 8

Fig. 8. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

Mutations in the gB Quad mutant do not affect the overall conformation of the gB ectodomain and cause only small local changes. (A) Crystal structure of the gB Quad mutant ectodomain (yellow) overlaid onto the structure of the WT gB (cyan). Only a single protomer (chain A) is shown. (B) A close-up view of the mutated region. Mutated residues are shown as sticks and labeled. (C) In gB Quad mutant, the side chain of F503 relocates to fill the void formed by R505G mutation. (D) A close-up view of the hydrogen bonds between the N-terminus of the central -helix αC in domain dIII (magenta) and domains dII and dV in the WT gB and gB Quad. Mutations eliminated all but one hydrogen bond. Residues involved in hydrogen bonds are shown as sticks and labeled. Hydrogen bonds are shown as dashed lines.

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.
7.
Fig. 7

Fig. 7. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

Several mutations affect the stability of the postfusion trimer. (A) Thermal denaturation stability of gB730, L700G/L711G, and gB730-GCNt-cyto(821-904) as monitored by measuring change in the CD signal at 218 nm with increasing temperature. Data are shown as fraction unfolded vs. temperature. (B) Thermal denaturation stability of gB730, L700G/L711G, and gB730-GCNt-cyto(821-904) as determined by Thermofluor assay. Data are shown as the first derivative of absorbance at 610 nm, in absorbance units per C, vs. temperature. (C) Oligomeric state of gB730 and several purified mutant proteins as analyzed by SDS-PAGE under mildly denaturing conditions (0.1% SDS, no boiling). Similar protein amounts were loaded into each lane. T – trimer, M – monomer. Positions of molecular weight standards are labeled.

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.
8.
Fig. 2

Fig. 2. From: Extensive mutagenesis of the HSV-1 gB ectodomain reveals remarkable stability of its postfusion form.

Regions targeted for mutagenesis. (A–C) Central coiled coil region as a target for mutagenesis. (A) The N terminus of the central alpha helix that undergoes refolding in VSV G is shown in magenta in the crystal structures of the prefusion and postfusion forms of the trimeric ectodomain 13; 18. The analogous region in the crystal structure of the postfusion form of HSV-1 gB ectodomain 12 is also shown in magenta. Domains in VSV G are labeled using gB domain numbering scheme, for clarity. Insets show close-up views of this region within single protomers. (B) A close-up view of the hydrogen bonds between the N-terminus of the central -helix αC in domain dIII (magenta) and domains dII and dV. Residues involved in hydrogen bonds are shown as sticks and labeled. Hydrogen bonds are shown as dashed lines. (C) Three symmetry-related C helices within the central coiled coil are shown in a top-to-bottom view. Residues L506, T509, and I513 at the coiled-coil interface are shown as sticks in all three protomers but labeled only in one protomer. (D–F) Domain dV as a target for mutagenesis. (D) A C-terminal helix in the ectodomain of paramyxovirus F protein, analogous to domain dV in HSV-1 gB, is shown in orange in the prefusion structure of PIV5 F and the postfusion structure of hPIV3 F 21; 32. Trimers are shown side by side with single protomers. (E) Domain dV in HSV-1 gB is shown in orange in the postfusion structure. The prefusion model of HSV-1 gB, including the hypothetical location of domain V, is shown schematically. Trimers are shown side by side with single protomers. dV truncations would occur within the orange stalk. (F) Truncation of dV (top) was expected to destabilize the postfusion form of gB with a minimal effect on the stability of the prefusion form of gB while the replacement of the dV with GCNt was expected to substitute the native helical stalk with an engineered one (bottom).

Elvira Vitu, et al. J Mol Biol. 2013 June 12;425(11):2056-2071.

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