Engineering fluorescent proteins for next- generation bioimaging applications

Zitti, Athena 2025. Engineering fluorescent proteins for next- generation bioimaging applications. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Modern biology and biomedical research have been transformed by advances in optical techniques that enable the sensitive detection and spatially resolved characterization of biomolecules. Among these, fluorescence microscopy has emerged as a pivotal tool for visualizing cellular structures and dynamics, allowing researchers to observe biological systems with high specificity and temporal resolution. Having the ability to selectively label cellular components with fluorophores has revolutionized the fields of cell biology, neurobiology and medical diagnostics. Central to the success of fluorescent-based approaches has been the engineering of fluorescent proteins. From the discovery of the first fluorescent protein, the green fluorescent protein (GFP), to its subsequent optimization that led to a diverse family of genetically encoded fluorescent reporters and later the discovery and development of a repertoire of di>erent coloured proteins, there is a constant need for fluorescent proteins (FPs) with better features to fit di>erent analysis requirements. This thesis explores the engineering of fluorescent proteins (FPs) to enhance their utility in bioimaging and Raman-based spectroscopy. Focusing on mCherry, mNeptune, and superfolder GFP (sfGFP), the work employs site-directed mutagenesis and non-natural amino acid (nnAA) incorporation to modulate fluorescence properties and introduce vibrational signatures detectable by Raman spectroscopy. In Chapter 3, nnAAs—p-cyano-phenylalanine (pCNPhe) and p-ethynylphenylalanine (pCCPhe)—were incorporated into specific residues of mCherry and mNeptune to introduce C≡N and C≡C vibrational bonds. Structural modelling guided residue selection near the chromophore to maximize Raman coupling. Although protein expression was successful, many mutants lacked colour, suggesting disrupted chromophore maturation. Nonetheless, this work laid foundational strategies for genetically encoded Raman-active probes. Chapter 4 details the creation of a novel mCherry variant (mCherryM66C), where cysteine was introduced at position 66 of the chromophore. Spectral analysis showed a 21 nm blue shift in excitation and a 25 nm shift in emission, along with a modest increase in v quantum yield. mCherryM66C displayed dual pH-dependent transitions (pKa = 5.7 and 8.8), consistent with a three-state chromophore model involving sequential protonation. While Raman signal was reduced compared to wild-type mCherry, the variant’s pH sensitivity and environmental responsiveness suggest potential use as a biosensor. Chapter 5 investigates the sfGFP H148S mutant (YuzuFP), developed through molecular dynamics simulations to enhance chromophore hydrogen bonding. Replacing histidine with serine resulted in increased brightness (~1.5-fold), improved absorbance, and significantly enhanced photobleaching resistance (~3-fold). Despite minimal change in pKa, YuzuFP maintained fluorescence more e>ectively across a wide pH range, a>irming its stability and suitability for imaging applications. Collectively, this work demonstrates the e>ectiveness of combining computational design with biochemical engineering to create next-generation FPs with enhanced spectral properties and environmental sensitivity. These engineered variants hold promise for advanced imaging techniques, including multiplexed fluorescence and Raman-based biosensing.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	26 February 2026
Last Modified:	26 Feb 2026 12:54
URI:	https://orca.cardiff.ac.uk/id/eprint/185321

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