Buffer and salt dependent reassembly pathways of Myxococcus xanthus encapsulin

Protein cages that adopt multiple assembly states provide tractable systems for probing the mechanisms that govern icosahedral shell formation. The Myxococcus xanthus encapsulin shell protein assembles into either a T = 1 shell (60 subunits) or a T = 3 shell (180 subunits), making it an ideal model to investigate how solution conditions bias competing assembly pathways. Here, we examined the reassembly of M. xanthus encapsulin following complete urea-mediated disassembly, systematically varying buffer identity (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], Tris, or phosphate) and ionic strength (0-1M NaCl) at pH 7.5. Assembly products were analyzed by dynamic light scattering and size-exclusion chromatography, with selected fractions characterized by transmission electron microscopy. Buffer identity and salt concentration reproducibly determined the T = 1 versus T = 3 distribution: phosphate buffer strongly favored T = 1 under all conditions, Tris at low ionic strength promoted T = 3 formation, and increasing NaCl progressively shifted outcomes toward T = 1 or unassembled subunits depending on the buffer. Assembled populations remained fixed upon shell closure and did not interconvert upon buffer exchange, demonstrating kinetic trapping. Interface analysis of both assembly states reveals that T = 3 formation requires reversible sampling of multiple weak contacts following formation of one dominant interface, whereas T = 1 relies on repeated formation of a single hydrophobic interface. Together, these results show that M. xanthus encapsulin assembly proceeds through competing kinetic pathways, with the outcome determined by how solution conditions modulate the reversibility and selectivity of early subunit encounters.