Scanning calorimetry combined with cryo-electron microscopy affords a powerful approach to investigating hierarchical interactions in multi-protein complexes. Calorimetry can detect the temperatures at which certain interactions are disrupted and cryo-EM can reveal the accompanying structural changes. The procapsid of bacteriophage HK97 (Prohead I) is a 450 Å-diameter shell composed of 60 hexamers and 12 pentamers of gp5, organized with icosahedral symmetry. Gp5 consists of the N-terminal Δ-domain (11 kDa) and gp5 ∗ (31 kDa): gp5 ∗ forms the contiguous shell from which clusters of Δ-domains extend inwards. At neutral pH, Prohead I exhibits an endothermic transition at 53 °C with an enthalpy change of 14 kcal/mole (of gp5 monomer). We show that this transition is reversible. To capture its structural expression, we incubated Prohead I at 60 °C followed by rapid freezing and, by cryo-EM, observed a capsid species 10% larger than Prohead I. At 11 Å resolution, visible changes are confined to the gp5 hexamers. Their Δ-domain clusters have disappeared and are presumably disordered, either by unfolding or dispersal. The gp5 ∗ hexamer rings are thinned and flattened as they assume the conformation observed in Expansion Intermediate I, a transition state of the normal, proteolysis-induced, maturation pathway. We infer that, at ambient temperatures, the hexamer Δ-domains restrain their gp5 ∗ rings from switching to a lower free energy, EI-I-like, state; above 53°, this restraint is overcome. Pentamers, on the other hand, are more stably anchored and resist this thermal perturbation.
Journal of Structural Biology – Elsevier
Published: May 1, 2007
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