Overview

Our project focuses on the design of a novel transcriptional regulator protein, the development of a protein engineering workflow that can drive future design efforts, and advancing molecular dynamics simulations for protein-DNA and protein-ligand interactions. Additionally, we have deepened our understanding of the modular nature of the TetR family of proteins.

We created a new protein design workflow that targets an underexplored area of protein engineering: designing a protein based on a ligand, rather than the other way around. This approach opens up possibilities for designing systems that detect difficult-to-identify ligands. The in silico design allowed us to generate multiple protein models efficiently, reducing both economic and time costs.

Moreover, the development and mutation of this protein enabled us to further investigate the modularity of this protein family and to elucidate the mechanisms of binding and separation for DNA and ligands. This discovery paves the way for the creation of new transcriptional regulators responsive to different ligands, marking a valuable technological advancement.

We hope these contributions will aid future rational protein design efforts and expand the possibilities for developing novel transcriptional regulation proteins based on the TetR family. Looking ahead, the integration of neural network technologies could enhance the development of these proteins by leveraging existing models.

Design of a novel transcriptional regulator protein

The novel design of ESTER (Estrogen Sensitive Transcriptional Engineered Regulator) enables precise transcriptional control of target genes in response to the presence of 17α-ethynylestradiol (EE2). This innovation opens new avenues for estrogen-focused systems, such as developing genetically engineered microorganisms for water purification and detecting EE2 in various environmental sources.

Protein engineering workflow

We developed a protein engineering workflow focused on designing proteins based on specific ligands. This process requires an initial template for targeted mutations and a deep understanding of various protein engineering software. Our approach follows rational design principles, grounded in the knowledge of biological activity and protein conformations.

Molecular dynamics simulations for protein interactions

For the in silico validation of protein function, we performed molecular dynamics simulations using GROMACS. This allowed us to evaluate the protein’s functionality under various conditions and provided valuable insights into its DNA separation mechanism and conformational changes within its subunits. This powerful technique enhances our understanding of the molecular mechanisms involved, offering a foundation for future protein design and in silico validation efforts.

Modular nature of the TetR family of proteins

Building on the development of ESTER, we analyzed the modular nature of TetR proteins, demonstrating that the DNA-binding domain can be exchanged for another binding domain based on protein alignment with a template. It is important to note that this modularity is dependent on the dimerization domain and the mechanism in which the template protein induces a conformational change in this DNA binding domain.