Integrating protein sequence design and evolutionary sequence conservation to uncover spectral tuning sites in red-light photoreceptors.

Protein structure and function are defined by non-covalent interactions of the polypeptide backbone and amino acid side chains providing specific chemical environments. Understanding how these interactions impact stability and/or functional aspects of proteins is critical to understand fundamental mechanisms of life itself. However, assigning functional or structural roles to individual amino acids is challenging even if structural models are available. This study introduces the function-structure-adaptability (FSA) approach, a semi-automated pipeline leveraging evolutionary sequence conservation and ProteinMPNN to assign amino acid-level roles in proteins. Here, we show that the pipeline can identify previously undescribed functional allosteric regulation residues in a specific family of target proteins-red light-responsive phytochromes. Identified sites were targeted by amino acid substitution to explore their role in phytochromes spectral characteristics and thermal reversion properties. These results expand our understanding of the intricate regulation mechanisms in phytochromes. Furthermore, the FSA approach can be readily employed for other target proteins.