De novo Design of Proteins for Catalysis and Fluorescence Imaging

/Abstract Protein-small molecule interactions regulate protein function, signal transduction, and enzymatic catalysis in biological systems. Advances in protein engineering have enabled modifications of natural protein-small molecule interactions for diverse applications, including developing protein tags for fluorescence imaging and engineering enzymes for biocatalysis. However, designing de novo proteins to bind small molecules and catalyze reactions remains a significant challenge, often requiring extensive screening and experimental optimization. In my recent work, I developed a nature-inspired strategy leveraging weak protein affinities for non-primary substrates to create functional proteins. Using this approach, I designed Fluorescent ABLE (FABLE), a fluorophore-binding protein, and Kemp eliminase ABLE (KABLE), an enzyme for Kemp elimination. The initial Kemp eliminase design achieved an activity of 6600 M⁻¹s⁻¹ in just five attempts, surpassing previous computational designs by over an order of magnitude. Saturation mutagenesis produced a quadruple mutant with an activity of 600,000 M⁻¹s⁻¹, setting a new benchmark for base-catalyzed Kemp eliminase and outperforming mechanistically similar natural enzymes. These successes highlight the frontier of de novo protein design for specific ligand interactions. A deeper understanding of protein-small molecule interactions in de novo proteins is crucial for advancing both fundamental understanding of these interactions in Nature and practical applications in protein engineering, particularly in the development of protein tags for fluorescence imaging and the design of enzymes for biocatalysis. In this proposal, In this proposal, I aim to test my hypothesis that protein dynamics, particularly the enrichment of productive conformations, contribute to the enhanced rate of KABLE1.4 using a multidisciplinary approach that includes molecular dynamics, X-ray crystallography, and NMR (Aim 1). For fluorescence imaging, I will transform the proof-of-concept de novo protein (FABLE) into a set of ready-to- use protein tags by designing proteins that bind modern rhodamine fluorophores while equipping them with a predefined set of optimal features that no existing tool offers (Aim 2). Lastly, I propose engineering de novo proteins with covalent ligand interactions, allowing stable binding and a universal protein tag for various synthetic molecules without requiring sequence redesign (Aim 3). Harnessing de novo protein design will revolutionize the ability to engineer protein-small molecule interactions, driving transformative advancements in fluorescence imaging, enzyme catalysis, and broader applications requiring precise molecular recognition. Project Number: 1K99GM163003-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of General Medical Sciences (NIGMS) | Principal Investigator: Yuda Chen | Institution: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO, SAN FRANCISCO, CA | Award Amount: $124,767 | Activity Code: K99 | Study Section: Special Emphasis Panel[ZRG1 CDB-Z (80)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11282577