Biology subjects

Vitsupakorn, D.

Publications and source records attributed to Vitsupakorn, D..

3 recordsLinked to original sources

bioRxiv2025

Structure, function and dynamics of mCoral, a pH responsive engineered variant of the mCherry fluorescent protein with improved hydrogen peroxide tolerance.

The red fluorescent protein mCherry is one of the most utilised fluorescent proteins in biology. Here, we have changed the chromophore chemistry by converting the thioether group of M66 to a thiol group through mutation to cysteine. The new variant termed mCoral due to its orange fluorescence hue has similar brightness to mCherry but has improved resistance to hydrogen peroxide. The variant is also responsive to pH with a low and high pKa forms that have distinct spectral properties, which DFT analysis suggests is due to protonation state changes in the newly introduced thiol group as well as the phenol group. The structure of mCoral reveals that the M66C mutation creates a space within the {beta}-barrel structure that is filled by a water molecule, which makes new polar interactions including with backbone carbonyl group of F65. Molecular dynamic simulations suggests that this additional water molecule, together with local solvation around the chromophore, could play a role in promoting planarity of the full conjugated system comprising the chromophore; the mCoral chromophore makes slightly more H-bonds with water than mCherry. The main water exit point for mCherry is also narrower in mCoral potentially explaining the increased resistance to hydrogen peroxide. Overall, a small structural change to mCherry has resulted in a new fluorescent protein with potentially useful characteristics and an insight into the role of dynamics and water in defining structure-function relationship in red fluorescent proteins.

biochemistry↗

bioRxiv2025

Chromophore charge-state switching through copper-dependent homodimerisation of an engineered green fluorescent protein.

Here, we have linked one of the most common protein-protein interaction events, homodimerisation, to an essential trace metal, copper, through engineering green fluorescent protein. Mutation of H148 to cysteine promotes the neutral chromophore in the monomer that excites predominantly at [~]400 nm. Homodimerisation via a copper-dependent disulphide bridge, switches the chromophore to the charged phenolate that excites at [~]490 nm. The result is [~]30 fold change in the fluorescence emission ratio. Homo-dimerisation kinetics are further improved by optimising the sfGFP homodimer interface, generating the variant termed GFP-diS2. Structures of the monomeric and dimeric GFP-diS2 suggests charge switching is through peptide bond flipping and the formation of a buried organised water networks around the chromophore that span the interface region. Fusion to a leucine zipper protein dimerisation element greatly increased GFP-diS2 association rate making it a more effective copper sensor in vitro and in vivo with Cu(I) instigating the signal change quicker and at lower ion concentrations than Cu(II). Thus, GFP-diS2 provides the framework for generating a sensitive genetically encoded copper sensor and will eventually be adapted to monitor one of the most important protein-protein interactions in biology, homo-oligomerisation.

biochemistry↗

bioRxiv2025

Molecular dynamics guided engineering of Aequorea victoria Green Fluorescent Protein chromophore interactions generates a brighter variant with improved photobleaching resistance

Fluorescent proteins (FPs) are a crucial tool for cell imaging, but with developments in fluorescence microscopy and researcher requirements there is still a need to develop brighter versions that remain fluorescent for longer. Using short time-scale molecular dynamics-based modelling to predict changes in local chromophore interaction networks and solvation, we constructed an Aequorea victoria GFP (avGFP) variant called YuzuFP that is 1.5 times brighter than the starting superfolding variant (sfGFP) with a near 3-fold increased resistance to photobleaching in situ. YuzuFP contained a single mutation that replaces the chromophore interacting residue H148 with a serine. Longer time scale molecular dynamics revealed the likely mechanism of action is S148 makes more persistent polar interactions with the chromophore phenol group and increases the residency time of an important water molecule. As demonstrated by live cell imaging, YuzuFP not only offers a timely upgrade as a useful green-yellow avGFP for cell imaging applications over longer timescales, but it also provides a basic scaffold for future avGFP engineering efforts.

biochemistry↗