Creating novel proteins by combining design and selection.

Area of Science:

• Protein engineering
• Computational biology
• Biochemistry

Background:

• Protein-peptide interactions are crucial in biological processes.
• Remodeling these interfaces is challenging but important for therapeutic and diagnostic applications.

Purpose of the Study:

• To demonstrate a combined computational design and experimental selection strategy for remodeling protein-peptide binding interfaces.
• To engineer a novel TPR1 module variant with enhanced binding affinity for the PTIEEVD peptide.

Main Methods:

• Utilized Rosetta software for computational interrogation of TPR1 sequences compatible with PTIEEVD peptide binding.
• Screened a library of TPR1 variants using a split GFP fluorescent assay to identify functional binders.
• Compared computational predictions with experimental selection results.

Main Results:

• Identified specific TPR1 sequences predicted to bind the PTIEEVD peptide using Rosetta.
• Experimental screening validated some predicted binding interactions.
• A 'consensus' TPR1 variant, derived from screening results, demonstrated successful binding to the PTIEEVD peptide.

Conclusions:

• The combination of computational design and experimental selection offers a powerful and synergistic approach to protein interface engineering.
• This strategy is effective for remodeling protein-peptide recognition interfaces.
• The engineered TPR1 variant shows potential for applications requiring specific protein-peptide interactions.