Capturing intrinsic protein dynamics for explaining beneficial substitutions from protein engineering campaigns

Directed evolution and knowledge-based design have been proven to be successful strategies for engineering proteins to desired applications. Traditional engineering strategies have focused on analyzing "hot spots" such as the substrate binding pocket to identify key positions. Yet, with a deeper understanding of protein structure-function relationships, many protein residues could significantly contribute to protein functionality, as they have the potential to influence one another and consequently alter protein conformation through the interconnected network formed by amino acids. Hence, it is essential to highlight the intrinsic dynamics in guiding protein engineering. This involves two main aspects: firstly, focusing on the exploration of protein conformation dynamics in order to understand how substitutions affect global and local conformations. Secondly, analyzing the intricate networks of amino acids in order to capture the amino acid interaction network and understand the impact of distal mutagenesis on the protein mutability landscape. Understanding intrinsic dynamics can deepen the understanding of both global and local enzyme properties, and help identify critical hotspots that researchers often overlooked in traditional protein engineering campaigns but are essential for property improvements. Additionally, we also discuss the current computational and experimental approaches in capturing protein conformations and amino acid networks.