Engineering GID4 for use as an N-terminal proline binder via directed evolution.

Area of Science:

• Biochemistry
• Molecular Biology
• Proteomics

Background:

• Advancements in nucleic acid sequencing have not been matched in proteomics.
• Protein sequencing requires selective N-terminal amino acid binders (NAABs).
• Currently, few engineered NAABs meet biotechnology reagent standards.

Purpose of the Study:

• To engineer novel NAABs for de novo protein sequencing.
• To improve binding affinity and reduce sequence context dependency for N-terminal proline (Nt-Pro) binders.
• To establish a viable strategy for engineering NAABs from native binding proteins.

Main Methods:

• Engineering of human protein GID4 using directed evolution.
• Yeast-surface display and fluorescence-activated cell sorting for variant selection.
• Characterization of engineered NAAB variants for Nt-Pro binding.

Main Results:

• Identified GID4 variants with enhanced binding response to Nt-Pro.
• A specific variant (A252V mutation) demonstrated reduced influence from adjacent amino acid residues.
• The engineered NAABs show promise for selective peptide identification.

Conclusions:

• The developed workflow is effective for engineering NAABs.
• Engineered GID4 variants offer improved Nt-Pro binding for protein sequencing applications.
• This approach facilitates the development of essential tools for de novo protein sequencing.