Fig. 1 Structural comparison of the PC versus MV portal protein structures. Ribbon diagrams of P22 portal protein ring in PC (A) and MV (B) conformations. The portal oligomer is colored gray, with stalk, trigger loop, hammer loop, and barrel domain colored magenta, black, yellow, and red, respectively. Arrows indicate the coat-binding region present in the PC and MV portal protein structures.
Fig. 2 In vitro assembled PC and MV portals are exclusively dodecameric. (A and B) Top: Elution profiles of unlabeled PCs and PC portal (A) or MV portal (B). Bottom: Elution profiles of AF488-labeled PC portal (AF488 PC portal) or MV portal (AF488 MV portal) applied to a Superose 6 gel filtration column. The MW was determined on the basis of the calibration standards that are marked along the top x axis. The arrowheads on the bottom x axis indicate the location of the MW markers (D, dextran blue, 2000 kDa; T, thyroglobulin, 669 kDa). A.U., arbitrary units. Mass spectra of unlabeled and labeled PC portal (C) and MV portal (D) measured by CDMS. PC, MV, and AF488-labeled portal samples were buffer-exchanged using SEC into 100 mM ammonium acetate. The quadrupole mass filter was set to discard ions with m/z values below 4000 Da. The measured masses were binned using 5000-Da bins.
Fig. 3 Bacteriophage P22 in vitro assembly reaction. AF488 PC portal, AF488 MV portal, or AF488 PM were preincubated with SP in 20 mM Hepes (pH 7.5) and 70 mM potassium acetate (KAc) buffer for 4 hours at RT. The assembly reaction was initiated by the addition of CP monomers. After 4 hours, the assembly products were applied to a Superose 6 gel filtration column (GE Healthcare) to separate in vitro assembled PCs from unincorporated CP, SP, and labeled portal protein monomers or portal rings (PC portal or MV portal). The PC peak fractions were analyzed for AF488-labeled portal protein incorporation by SDS-PAGE.
Fig. 4 In vitro, both PC and MV portals are incorporated into PCs. Superose 6 elution profiles of the assembly reactions (Rx1) of CP and SP with AF488 PC portal (A) or AF488 MV portal (D) are shown along with the elution profiles from the mock reactions with empty PC shells (Rx2) and AF488-labeled PC portal, or MV portal alone (Rx3). The black arrowhead indicates the elution volume of in vitro assembled PC peak from the column. Three fractions from the PC peak from Rx1, Rx2, and Rx3 were subjected to SDS-PAGE. MW standard (MW, lane 1) and unlabeled PC marker (PC, lane 2) are also shown. The presence of labeled PC portal (B) or MV portal (E) in the PC peaks was visualized using a PharosFx Plus Molecular Imager (top), followed by silver staining (bottom). Electron micrographs of in vitro assembled P22 PC (Rx1) and empty PC shells (Rx2) in the presence of AF488 PC portal (C) or MV portal (F). Insets show magnified views of particles boxed in (C) and (F), respectively. In these particles, the immunogold-labeled portal protein is seen at a single capsid vertex. Scale bars, 100 nm. Note that, in the magnified view of (F), only the bead near the PC in the bottom right would have been counted. The other two beads between the PC were >15 μm from the PC.
Fig. 5 De novo oligomerized portal rings are assembly-competent. (A) Superose 6 elution profiles of the assembly reactions of CP and SP with AF488 PM (Rx1), along with the elution profiles from the mock reactions, where empty PC shells are incubated with AF488 PM (Rx2). (B) The three fractions corresponding to the SEC-purified PC peak from Rx1 and Rx2 were subjected to SDS-PAGE. The presence of AF488-labeled portal protein was visualized using a PharosFx Plus Molecular Imager at an excitation wavelength of 488 nm (top), followed by silver staining (bottom). MW standard (MW, lane 1) and unlabeled PC marker (PC, lane 2) are also shown. (C) Electron micrographs of in vitro assembled P22 PCs (Rx1) and empty PC shells (Rx2) in the presence of AF488 PM. Insets show magnified views of particles boxed in (C). Scale bars, 100 nm.
Fig. 6 SP acts as a “facilitator” of portal ring oligomerization. SP, CP, or ovalbumin was incubated with AF488 PM in 20 mM Hepes (pH 7.5) and 70 mM KAc buffer for 4 hours at RT. The reactions were applied over Superose 6 Increase column. The fractions harboring the PC, portal rings (PR), and PM peaks, 8 to 17.5 ml, were subjected to SDS-PAGE, and the presence of AF488-labeled portal rings or PMs in these fractions was visualized by PharosFx Plus Molecular Imager at an excitation wavelength of 488 nm.
Fig. 7 De novo assembled portal rings analyzed by sucrose gradient and TEM. (A) Densitometry profile of de novo ring nucleation reaction (indicated on the left), where SP was incubated with unlabeled PMs (Rx1) in 20 mM Hepes (pH 7.5) and 70 mM KAc buffer for 4 hours at RT and separated over a linear 5 to 20% sucrose gradient. The profiles of the control reactions, where PM, SP, and preassembled dodecameric portal rings were individually incubated in the buffer for 4 hours at RT and separated, are also shown. The black arrow indicates a shift in the portal protein migration down the gradient into higher sucrose concentration toward the position of the preassembled dodecameric portal rings. The black dashed lines indicate the range of fractions where portal oligomers are observed. The green dashed line shows the center of the monomer peak. (B) TEMs of negatively stained particles present in fraction 14 (indicated by black arrowhead) from Rx1 (top) and Rx4 (bottom), taken at ×150,000 magnification. Scale bars, 100 nm. Red arrows indicate portal rings that are magnified ×4 and shown in the inset. Scale bars, 20 nm.
Fig. 8 SP interacts with PMs, not preassembled portal rings (PC portal or MV portal). Elution profile of SP, CP monomers, and ovalbumin from an IMAC bound with His-tagged PMs (A) or His-tagged PC portal (B) and through an NHS-activated agarose bead column conjugated to non–His-tagged MV portal (C). The elution profile of each protein was determined by monitoring intrinsic tryptophan fluorescence of each fraction using AMINCO-Bowman AB2 spectrofluorometer or Horiba FluoroMAX 4 spectrofluorometer at an excitation wavelength at 280 nm, the emission wavelength set to 340 nm, and the bandpasses set to 1 and 8 nm, respectively.
Fig. 9 Structural comparison of the coat-binding region of the PC and MV portal protein structures. Overlay of the PC and MV portal protomers (residues 10 to 599) generated using the program Chimera is presented. The helical barrel domain (residues 600 to 725) is not shown in the overlay. The PC portal protomer is colored gray, with the coat-binding region highlighted in cyan. The MV portal protomer is colored gold, with the coat-binding region highlighted in red. The stem and wing regions that interact with CP in the capsid do not undergo significant conformational transitions between the two portal structures (black box). The regions in the coat binding that undergoes marked conformation change between the two portal structures are highlighted in the gray dashed circle, whereas the black curved arrow points to the major conformational change that occurs in the trigger loop between the two structures.
Fig. 10 Proposed model of portal protein incorporation into PCs of bacteriophage P22. Model for the incorporation of portal rings during P22’s PC assembly. During initiation of PC assembly, the SP interacts with PMs, instead of interacting exclusively with the CP. The interaction between SP and PM leads to the oligomerization of portal protein monomers into dodecameric rings, which are subsequently incorporated into growing PC head.
- Table 1 Mass of the PC and MV portals determined from CDMS spectra.
Sample Theoretical mass of
12-mer (kDa)Measured mass of
12-mer (kDa)*PC portal 1005.6 1027 ± 0.009 MV portal 992.9 997.2 ± 0.004 *The measured masses were obtained by fitting a Gaussian to the measured peak and taking the center of the Gaussian. The mass difference observed between the PC and MV portals is due to the His tag bound to the PM. The uncertainties for the measured masses are ±1 SD for multiple measurements on different samples.
- Table 2 In vitro assembly reaction setup.
Reaction no. Components of assembly reaction* Concentration* Rx1 SP 9 μM AF488 PM or AF488 PC portal or
AF488 MV portal1.1 μM Coat monomers 6.4 μM Rx2 AF488 PM or AF488 PC portal or
AF488 MV portal1.1 μM Shells 3.2 μM Rx3 AF488 PM or AF488 PC portal or
AF488 MV portal1.1 μM *The components and the concentration of which each protein was added in the reaction are mentioned.
- Table 3 In vitro assembled P22 PCs containing gold particles.
PC type* Total number of PCs counted Number of PCs with gold particles % (A) (Pre: SP + AF488 PC portal) + CP 954 114 12.0 (B) Shells + AF488 PC portal 1340 7 0.5 (C) (Pre: SP + AF488 MV portal) + CP 1000 64 6.4 (D) Shells + AF488 MV portal 739 6 0.8 *PCs or empty PC shells assembled in vitro in the presence of AF488 PC portal or AF488 MV portal.
Supplementary Materials
Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/3/7/e1700423/DC1
fig. S1. Analysis of PC and MV portals by native protein gel electrophoresis.
fig. S2. Ovalbumin is not coassembled into PC.
fig. S3. Charge detection mass histogram of SP catalyzed de novo portal rings.
Additional Files
Supplementary Materials
This PDF file includes:
- fig. S1. Analysis of PC and MV portals by native protein gel electrophoresis.
- fig. S2. Ovalbumin is not coassembled into PC.
- fig. S3. Charge detection mass histogram of SP catalyzed de novo portal rings.
Files in this Data Supplement:
- fig. S1. Analysis of PC and MV portals by native protein gel electrophoresis.
