Dynamics differentiate between active and inactive inteins.

Area of Science:

• Biochemistry
• Molecular Biology
• Protein Engineering

Background:

• Inteins are protein elements that catalyze their own excision and ligation of flanking protein sequences (exteins).
• Engineered mini-inteins, lacking the homing endonuclease domain, exhibit reduced splicing activity compared to full-length counterparts.
• A V67L mutation was identified to potentially restore native splicing activity in these engineered mini-inteins.

Purpose of the Study:

• To investigate the impact of the V67L mutation on the stability and conformational dynamics of engineered mini-inteins.
• To elucidate the factors governing the active state of mini-inteins, including linker length and protein flexibility.
• To understand the relationship between protein dynamics and intein catalytic function.

Main Methods:

• Computational molecular dynamics simulations to model mini-intein structures and dynamics.
• Experimental analysis of intein splicing activity and kinetics.
• Examination of protein flexibility and allosteric interactions within the mini-intein system.

Main Results:

• The V67L mutation was found to stabilize mini-intein constructs by reducing global fluctuations.
• Linker length significantly influences intein dynamics, with shorter linkers potentially impacting flexibility.
• Active intein structures exhibit higher flexibility in both the linker and the intein core.
• Allosteric interactions between the linker and a specific turn region (residues 35-41) were identified.

Conclusions:

• While the V67L mutation enhances global stability, cooperative protein dynamics are more critical for intein function than high stability alone.
• Intein catalysis is characterized by non-linear Arrhenius kinetics, underscoring the role of conformational dynamics.
• Engineered allosteric interactions that modulate conformational dynamics could be used to control intein splicing or cleaving activity.