Caspase-Based Fusion Protein Technology: Substrate Cleavability Described by Computational Modeling and Simulation.

Area of Science:

• Biochemistry
• Structural Biology
• Protein Engineering

Background:

• Caspase-based fusion protein technology (CASPON) enables universal cleavage of fusion tags to restore native N-termini.
• CASPON enzyme optimization has yielded promiscuity against various N-terminal peptides.
• However, cleavage efficiency can be unexpectedly low for larger proteins.

Purpose of the Study:

• To develop an efficient computational method for predicting CASPON cleavage efficiency.
• To rationalize cleavage outcomes based on structural interactions between CASPON and N-terminal peptides.

Main Methods:

• Utilizing molecular dynamics simulations to generate diverse N-terminal peptide conformations.
• Analyzing the structural representation of intrinsically disordered N-terminal peptides.
• Employing a fitting procedure to model interactions with the CASPON enzyme.

Main Results:

• The number of favorable N-terminal conformations strongly correlates with experimentally observed cleavage efficiency.
• This agreement supports a conformational selection model for CASPON activity.
• The method provides a computationally inexpensive way to assess cleavability.

Conclusions:

• A novel computational approach accurately predicts CASPON cleavage efficiency.
• Understanding N-terminal peptide conformations is key to optimizing protein purification.
• This method allows for a priori assessment of CASPON cleavability, streamlining protein engineering workflows.