Buffer and salt dependent reassembly pathways of Myxococcus xanthus encapsulin

Area of Science:

• Biochemistry and structural biology
• Protein assembly and self-organization
• Biophysics of macromolecular complexes

Background:

• Protein cages, like Myxococcus xanthus encapsulin, can form distinct icosahedral shells (T=1 or T=3).
• Understanding the factors influencing these assembly pathways is crucial for controlling protein structure formation.
• Investigating how solution conditions bias assembly is key to deciphering self-assembly mechanisms.

Purpose of the Study:

• To investigate how buffer identity and ionic strength affect the reassembly of Myxococcus xanthus encapsulin.
• To determine the influence of solution conditions on the formation of T=1 versus T=3 encapsulin shells.
• To elucidate the kinetic pathways and interface dynamics governing encapsulin assembly.

Main Methods:

• Urea-mediated disassembly of Myxococcus xanthus encapsulin.
• Systematic variation of buffer identity (HEPES, Tris, phosphate) and ionic strength (0-1M NaCl) at pH 7.5.
• Analysis of assembly products using dynamic light scattering, size-exclusion chromatography, and transmission electron microscopy.

Main Results:

• Buffer identity and salt concentration reproducibly determined the T=1 versus T=3 shell distribution.
• Phosphate buffer favored T=1, Tris at low ionic strength favored T=3, and increasing NaCl shifted outcomes.
• Assembly products were kinetically trapped and did not interconvert upon buffer exchange.

Conclusions:

• Myxococcus xanthus encapsulin assembly proceeds through competing kinetic pathways.
• Solution conditions modulate the reversibility and selectivity of early subunit encounters, dictating the final assembly state.
• The formation of T=3 shells requires reversible sampling of weak contacts, while T=1 relies on hydrophobic interface formation.