Protein misfolding and inclusion body formation studied in vivo with the phage P22 system. (a) Crystal structure of a subunit of the P22 tailspike homotrimer (20), showing the parallel β-coil structure and ventral and dorsal “fins.” Blue spheres represent positions of temperature-sensitive folding mutants (21), which in general occupy solvent-exposed sites in this native form (see text). Two red spheres represent two global suppressor mutations (23). Reproduced with permission from The FASEB Journal (22). (b) Pathways of folding and aggregation in vivo of the phage P22 tailspike protein bearing temperature-sensitive folding mutations. The single-chain folding intermediates are thermolabile (designated I), with the mutations, potentially affecting turn formation, favoring off-pathway intermediate formation and association over productive folding. The global suppressor mutations reverse such partitioning, favoring the productive early folding intermediate over the aggregation-producing one (vertical arrow), even at 40°C. Reproduced with permission from Science (23). TSF, temperature-sensitive folding. (c) Folding and aggregation in vivo of phage P22 capsid protein bearing temperature-sensitive folding mutations (24). Pathways are similar to those for the tailspike. Here, the chaperonin system, GroEL/GroES/ATP, can act at 39°C to bind the aggregation-prone I* intermediate (monomer) and facilitate its productive folding. Reproduced with permission from The Journal of Biological Chemistry (24).