Simulation-guided engineering of split GFPs with efficient β-strand photodissociation

Area of Science:

• Biochemistry
• Biophysics
• Molecular Biology

Background:

• Split green fluorescent proteins (GFPs) are essential tools for studying protein-protein interactions via complementation.
• Current methods for split-GFP dissociation are often irreversible or inefficient.
• Photodissociation of split GFPs has low quantum efficiency, limiting its application.

Purpose of the Study:

• To rationally design and engineer split GFPs with enhanced light-induced dissociation rates.
• To overcome limitations of traditional protein engineering approaches for split-GFP optimization.
• To demonstrate the utility of computational methods in accelerating protein engineering.

Main Methods:

• Combined classical and enhanced sampling molecular dynamics simulations.
• Utilized Quantum Mechanics/Molecular Mechanics (QM/MM) calculations to model key dissociation states.
• Employed computational modeling to guide rational amino acid substitutions.

Main Results:

• Identified non-intuitive amino acid changes that significantly increase photodissociation rates.
• Achieved up to a 20-fold increase in photodissociation speed for engineered split GFPs.
• Validated the effectiveness of computational approaches in protein engineering.

Conclusions:

• Computational modeling can effectively guide the engineering of split GFPs for improved photodissociation.
• Rational design using advanced simulations accelerates the development of functional protein tools.
• This approach holds potential for increasing success rates in complex protein engineering projects.