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

## TL;DR

This paper introduces a new method combining protein design and evolutionary data to identify key amino acids in red-light sensing proteins.

## Contribution

The FSA approach integrates evolutionary conservation and ProteinMPNN to assign functional roles to amino acids in proteins.

## Key findings

- The FSA method identified new allosteric regulation residues in phytochromes.
- Substituted amino acids affected spectral and thermal properties of phytochromes.
- The method is applicable to other proteins beyond phytochromes.

## Abstract

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.

•Comparison of natural and ProteinMPNN sequences for structure-function assignment•Discovery of allosteric network residues in the second shell cofactor environment•Correlation of conformational dynamics with thermal reversion supported by HDX-MS•Identification of tuning residues relevant for the whole phytochrome family

Comparison of natural and ProteinMPNN sequences for structure-function assignment

Discovery of allosteric network residues in the second shell cofactor environment

Correlation of conformational dynamics with thermal reversion supported by HDX-MS

Identification of tuning residues relevant for the whole phytochrome family

In this work, Eder et al. demonstrate that the structure-based design model ProteinMPNN can be used together with natural amino acid conservation to uncover functionally important residues in proteins. In a light sensing phytochrome, previously unknown functional residues were identified and their role was assessed by in vitro experiments.