(629b) Developing N-Terminal Amino Acid Binder Proteins for Next-Generation Protein Sequencing

The emerging next-generation protein sequencing technologies show promise to revolutionize the field of proteomics as next-generation DNA and RNA sequencing did for the fields of genomics and transcriptomics. One promising approach involves recognition of N-terminal amino acids of surface-immobilized polypeptides by N-terminal amino acid binder (NAAB) proteins. The fluorescent tags on the NAAB regents will enable fluoro-sequencing of the polypeptides. Currently, however, the amino acids that can be recognized by published NAAB reagents are limited to arginine and several hydrophobic residues. More residues need to be identifiable to enable sequencing without a priori knowledge of the target protein. In this work, we leverage the naturally occurring proteins to engineer new NAAB reagents that preferentially binds to proline or glycine. We characterize the variants’ affinity and specificity to the target amino acid, thermostability, and compare them to the wild-type. The engineered proline- and glycine-binding proteins from this work can be added to the repertoire of NAAB reagents that can be used to develop next-generation sequencing platform and bring one step closer to making protein sequencing without a priori knowledge of the target protein a reality.