Monomer-dimer control and crystal engineering in TASPs

Area of Science:

• Protein engineering
• Biocrystallography
• Synthetic biology

Background:

• Template-assembled synthetic proteins (caviteins) exhibit monomer-dimer equilibrium in solution.
• Previous work demonstrated modulating this equilibrium through sequence design, including histidine metal chelation and disulfide bonds.
• The forces governing dimeric cavitein crystal nucleation and stabilization remain largely uncharacterized.

Purpose of the Study:

• To investigate the molecular interactions responsible for dimeric cavitein crystal formation and stabilization.
• To probe the role of specific amino acid residues in cavitein crystallization.
• To elucidate the structural basis for histidine-mediated bias towards dimeric forms.

Main Methods:

• Design and synthesis of glutamine variants of cavitein.
• Crystallization of cavitein variants.
• X-ray crystallography to determine the structure of a histidine-modified cavitein.

Main Results:

• A critical glutamate hydrogen-bonding interaction was identified as essential for crystal nucleation and stabilization.
• A crystal structure of a cavitein variant (Q4-E3H), designed for dimeric stability via histidine metal coordination, was obtained.
• The crystal structure revealed a histidine cluster interaction, providing a likely explanation for the observed dimeric preference in solution.

Conclusions:

• Glutamate hydrogen bonding is a key determinant for dimeric cavitein crystal formation and stability.
• Histidine-mediated metal coordination can bias caviteins towards a dimeric state, as evidenced by structural analysis.
• Understanding these interactions advances the design principles for controlling protein self-assembly and stability.