Biology subjects

Castell, O.

Publications and source records attributed to Castell, O..

4 recordsLinked to original sources

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↗

bioRxiv2023

Manipulation of encapsulated artificial phospholipid membranes using sub-micellar lysolipid concentrations.

Droplet Interface Bilayers (DIBs) constitute a commonly used model of artificial membranes for molecular biology studies with applications in synthetic biology research. However, these model membranes have limited accessibility due to their requirement to be surrounded by an oil environment. Here, we demonstrate in-situ bilayer manipulation of submillimeter, free-standing, encapsulated droplet interface bilayers (eDIBs) in hydrogel capsules formed using dual-material, 3D-printed microfluidic devices. These microfluidic devices required no post-fabrication assembly, nor surface treatment to achieve the high-order emulsification, required for the formation of robust eDIBs. The eDIB capsules were exposed to various concentrations of lysophosphatidylcholine (LPC), in order to investigate the interaction of lysolipids with three-dimensional, encapsulated droplet bilayer networks. Micellar LPC concentrations trigger the bursting of the eDIB droplets, while at concentrations below the critical micelle concentration (CMC), the encapsulated aqueous inner droplet networks endure structural changes, precisely affecting the DIB contact angles and bilayer area. Manipulation of these enclosed, 3D-orchestrated membrane mimics facilitates the exploration of readily accessible compartmentalized artificial cellular machinery. Collectively, the multi-compartmentalized capsules and the lysolipid-mediated membrane modulation, introduce a chemical approach to control the properties and mechanics of artificial cellular membranes, toward responsive soft material developments.

synthetic biology↗

bioRxiv2022

Building programmable multicompartment artificial cells incorporating remotely activated protein channels using microfluidics and acoustic levitation

Intracellular compartments are functional units that support the metabolic processes within living cells, through spatiotemporal regulation of chemical reactions and biological processes. Consequently, as a step forward in the bottom-up creation of artificial cells, building analogous intracellular architectures is essential for the expansion of cell-mimicking functionality. Herein, we report the development of a droplet laboratory platform to engineer customised complex emulsion droplets as a multicompartment artificial cell chassis, using multiphase microfluidics and acoustic levitation. Such levitated constructs provide free-standing, dynamic, definable droplet networks for the encapsulation and organisation of chemical species. Equally, they can be remotely operated with pneumatic, heating, and magnetic elements for post-processing, including the incorporation of membrane proteins; alpha-hemolysin; and large-conductance mechanosensitive channel (MscL) and their activation. The assembly of droplet networks is three-dimensionally patterned with fluidic inputs configurations determining droplet contents and connectivity. Whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. In addition, a mechanosensitive channel, MscL, can be repeatedly activated and deactivated in the levitated artificial cell by the application of acoustic and magnetic fields to modulate membrane tension on demand. This offers possibilities beyond one-time chemically mediated activation to provide repeated, non-contact control of membrane protein function. Collectively, this will expand our capability to program and operate increasingly sophisticated artificial cells as life-like materials.

synthetic biology↗

bioRxiv2019

The effect of proximity on the function and energy transfer capability of fluorescent protein pairs

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Forster resonance energy transfer (FRET). Many FPs have a tendency to oligomerise, which is likely to be promoted through attachment to associating proteins through increases in local FP concentration. We show here that on association of FP pairs, the inherent function of the FPs can alter. Artificial dimers were constructed using a bioorthogonal Click chemistry approach that combined a commonly used green fluorescent protein (superfolder GFP) with itself, a yellow FP (Venus) or a red FP (mCherry). In each case dimerisation changes the inherent fluorescent properties, including FRET capability. The GFP homodimer demonstrated synergistic behaviour with the dimer being brighter than the sum of the two monomers. The structure of the GFP homodimer revealed that a water-rich interface is formed between the two monomers, with the chromophores being in close proximity with favourable transition dipole alignments. Dimerisation of GFP with Venus results in a complex displaying [~]86% FRET efficiency, which is significantly below the near 100% efficiency predicted. When GFP is complexed with mCherry, FRET and mCherry fluorescence itself is essentially lost. Thus, the simple assumptions used when monitoring interactions between proteins via FP FRET may not always hold true, especially under conditions whereby the protein-protein interactions promote FP interaction. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/838888v1_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@1863e41org.highwire.dtl.DTLVardef@61255dorg.highwire.dtl.DTLVardef@1dc188forg.highwire.dtl.DTLVardef@d066ad_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗