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The outer shell of the papillomavirus particle is comprised of 72 pentamers of …


Biology Articles » Virology » Structure-based engineering of papillomavirus major capsid L1: controlling particle assembly » Figures

Figures
- Structure-based engineering of papillomavirus major capsid L1: controlling particle assembly

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Figure 1. Structural elements of L1 important for papillomavirus particle assembly. A) The top view of an L1 pentamer, showing the lateral projections from each of the five monomers (each in a discreet color). B) A close-up view of the lateral projections two monomers, showing the detailed positions of h2, h3, h4, and h5 at the C-terminus of L1. C) The inter-pentameric helix-helix interactions between two pentamers in the T = 1 particle. The h4 of one pentamer (in yellow) interacts with h2 and h3 of another pentamer (in pink).

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Figure 2. Structure-based internal deletions of HPV16 L1. A) Sequence alignment of HPV 16 and HPV18 L1, showing the conserved regions of h2, h3, and h4. B) Diagram showing the design of the four internal deletions of HPV16 L1. D1 has a deletion within h4 and D2 removes h4 and a few residues outside the boundaries of h4 on both ends. C) Solubility and stability of the four constructs of HPV16 L1. Lanes 2–4: D1-L1 mutant; lanes 5–7: D2-L1 mutant; lanes 8–10: D3-L1 mutant; and lanes 11–12: D4-L1 mutant. Lanes 2, 5, 8, and 11 show the proteins eluted from the resin before thrombin digestion; lanes 3, 6, and 9 show the proteins eluted from the column after thrombin digestion; and lanes 4, 7, 10, and 12 show the proteins in the resin after thrombin digestion and elution. D) Size-exclusion chromatography analysis of the D1-L1 eluted from the column after thrombin treatment, showing the typical elution profile on Superdex-200. The major peak has an apparent molecular weight of ~235 kD, the expected size for a pentameric L1.

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Figure 3. EM examination of particle assembly of L1 mutant proteins. A) The purified proteins from four different L1 constructs. Lane 1: full-length HPV16 L1; lane 2: D1-L1 deletion mutant of HPV16 L1; lane 3: D2-L1 deletion mutant of HPV16 L1; lane 4: D2-L1 deletion mutant of HPV18 L1. B-E) EM images of the purified proteins treated under assembly condition. The proteins were in incubated in the assembly buffer at 25°C for 30 minutes. Uranyl acetate was then used to treat the protein samples on a carbon grid for EM examination. The scale of the images is indicated by the bar above panel B. The images shown are: (B) full-length HPV16 L1; (C): D1-L1 mutant of HPV16 L1; (D): D2-L1 mutant of HPV16 L1; (E): D2-L1 mutant of HPV18 L1.

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Figure 4. Examination of particle assembly of L1 mutants by size-exclusion chromatography. The proteins were incubated in assembly buffer at 25°C for 30 minutes before analysis on the Superdex-200 column (16/60, Pharmacia) at a flow rate at 1 mL/min. The positions of the molecular standards are indicated by arrows. An HPV16 L1 construct, ΔN10+ΔC30, shown previously to assemble into T = 1 particles, was included as a control (blue line).

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