Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging

Abstract

The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three “ejection proteins” (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins’ locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection.

Measurement of the accurate mass of a 50 MDa infectious virus

“…The lower end of these ranges is incompatible with the measured mass of the intact phage. The most recent estimate of 11, 12, and 32 copies of gp7, gp16, and gp20, respectively, is used in Table . Adding the masses of all the components together, we calculate the average total mass of the intact P22 virion as 51,613 kDa (Table ).…”

“…There are three main contributions to the width of the peak: the mass resolution (which is known), the mass Table 1. Components of phage P22 and their masses [28,32,39,42] The total phage mass was calculated by multiplying the mass of each component by its stoichiometry and summing all those masses together. For the genome, the average DNA mass for the headful mechanism was used.…”

Cryo-EM Elucidation of the Structure of Bacteriophage P22 Virions after Genome Release

“…Density first tentatively assigned to the ejection proteins (27) was later found to be at least largely attributed to the C-terminal ''barrel'' of the portal protein (34). Ejection proteins have been indirectly located near the center of the head by the bubblegram imaging method (35), but details of their location are not known.…”

How disordered is my protein and what is its disorder for? A guide through the “dark side” of the protein universe

“…60,61 Finally, the spectacular rise of cryo-EM in the last decade 62 presages of an increasing number of examples where protein flexibility will be documented by this powerful, fast-growing structural technique (for 2 such examples see refs. 63,64 ). While there are IDPs/IDPRs that are able to perform their function while remaining completely disordered (e.g.…”