Local disorder is associated with enhanced catalysis in an engineered photoswitch

Area of Science:

• Biochemistry
• Optogenetics
• Enzyme Engineering

Background:

• The LOV2 domain from *A. sativa* is a key component in optogenetic switches.
• Previous work created light-regulated Dihydrofolate Reductase (DHFR) using LOV2, with mutations tuning light response.
• Mechanisms of light-induced allostery in DHFR and mutation effects were unclear.

Purpose of the Study:

• To elucidate the mechanism of light-induced allosteric activation of DHFR.
• To understand how mutations modulate the allosteric response to light.
• To improve rational design of light-regulated enzymes.

Main Methods:

• Eyring analysis to quantify enzyme kinetics and thermodynamics.
• Circular Dichroism (CD) spectroscopy to assess protein thermal stability.
• Characterization of DHFR/LOV2 fusion protein and its mutants.

Main Results:

• DHFR/LOV2 fusion protein showed marginal stability at physiological temperatures.
• LOV2 photoactivation destabilized the fusion and reduced catalytic transition free energy.
• Light activation involved an enthalpic benefit offset by an entropic penalty.
• Mutations modulated this tradeoff, affecting enthalpy or entropy, but not both.
• An activity-stability tradeoff was observed for many mutations.

Conclusions:

• Light allosterically activates DHFR by altering the balance of enthalpy and entropy in the transition state.
• Mutations fine-tune light regulation by exploiting an activity-stability tradeoff.
• Understanding these mechanisms enables rational design of novel optogenetic tools.