Dynamic tuneable G protein-coupled receptor monomer-dimer populations

G protein-coupled receptors (GPCRs) are the largest class of membrane receptors, playing a key role in the regulation of processes as varied as neurotransmission and immune response. Evidence for GPCR oligomerisation has been accumulating that challenges the idea that GPCRs function solely as monomeric receptors; however, GPCR oligomerisation remains controversial primarily due to the difficulties in comparing evidence from very different types of structural and dynamic data. Using a combination of single-molecule and ensemble FRET, double electron–electron resonance spectroscopy, and simulations, we show that dimerisation of the GPCR neurotensin receptor 1 is regulated by receptor density and is dynamically tuneable over the physiological range. We propose a “rolling dimer” interface model in which multiple dimer conformations co-exist and interconvert. These findings unite previous seemingly conflicting observations, provide a compelling mechanism for regulating receptor signalling, and act as a guide for future physiological studies

Bioluminescent detection of isothermal DNA amplification in microfluidic generated droplets and artificial cells

Microfluidic droplet generation affords precise, low volume, high throughput opportunities for molecular diagnostics. Isothermal DNA amplification with bioluminescent detection is a fast, low-cost, highly specific molecular diagnostic technique that is triggerable by temperature. Combining loop-mediated isothermal nucleic acid amplification (LAMP) and bioluminescent assay in real time (BART), with droplet microfluidics, should enable high-throughput, low copy, sequence-specific DNA detection by simple light emission. Stable, uniform LAMP–BART droplets are generated with low cost equipment. The composition and scale of these droplets are controllable and the bioluminescent output during DNA amplification can be imaged and quantified. Furthermore these droplets are readily incorporated into encapsulated droplet interface bilayers (eDIBs), or artificial cells, and the bioluminescence tracked in real time for accurate quantification off chip. Microfluidic LAMP–BART droplets with high stability and uniformity of scale coupled with high throughput and low cost generation are suited to digital DNA quantification at low template concentrations and volumes, where multiple measurement partitions are required. The triggerable reaction in the core of eDIBs can be used to study the interrelationship of the droplets with the environment and also used for more complex chemical processing via a self-contained network of droplets, paving the way for smart soft-matter diagnostics

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

Abstract: Intracellular compartments are functional units that support the metabolism 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 complex emulsion-based, multicompartment artificial cells, using microfluidics and acoustic levitation. Such levitated models provide free-standing, dynamic, definable droplet networks for the compartmentalisation 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 mechanosensitive channel of large-conductance. The assembly of droplet networks is three-dimensionally patterned with fluidic input configurations determining droplet contents and connectivity, whilst acoustic manipulation can be harnessed to reconfigure the droplet network in situ. The mechanosensitive channel 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 expands our growing capability to program and operate increasingly sophisticated artificial cells as life-like materials

Site-specific protein photochemical covalent attachment to carbon nanotube side walls and its electronic impact on single molecule function

Functional integration of proteins with carbon-based nanomaterials such as nanotubes holds great promise in emerging electronic and optoelectronic applications. Control over protein attachment poses a major challenge for consistent and useful device fabrication, especially when utilizing single/few molecule properties. Here, we exploit genetically encoded phenyl azide photochemistry to define the direct covalent attachment of four different proteins, including the fluorescent protein GFP and a β-lactamase binding protein (BBP), to carbon nanotube side walls. AFM showed that on attachment BBP could still recognize and bind additional protein components. Single molecule fluorescence revealed that on attachment to SWCNTs function was retained and there was feedback to GFP in terms of fluorescence intensity and improved resistance to photobleaching; GFP is fluorescent for much longer on attachment. The site of attachment proved important in terms of electronic impact on GFP function, with the attachment site furthest from the chromophore having the larger effect on fluorescence. Our approach provides a versatile and general method for generating intimate protein–CNT hybrid bioconjugates. It can be potentially applied to any protein of choice; the attachment position and thus interface characteristics with the CNT can easily be changed by simply placing the phenyl azide chemistry at different residues by gene mutagenesis. Thus, our approach will allow consistent construction and modulate functional coupling through changing the protein attachment position

Positive functional synergy of structurally integrated artificial protein dimers assembled by Click chemistry

Construction of artificial higher order protein complexes allows sampling of structural architectures and functional features not accessible by classical monomeric proteins. Here, we combine in silico modelling with expanded genetic code facilitated strain promoted azide-alkyne cycloaddition to construct artificial complexes that are structurally integrated protein dimers and demonstrate functional synergy. Using fluorescent proteins sfGFP and Venus as models, homodimers and heterodimers are constructed that switched ON once assembled and display enhanced spectral properties. Symmetrical crosslinks are found to be important for functional enhancement. The determined molecular structure of one artificial dimer shows that a new long-range polar network comprised mostly of organised water molecules links the two chromophores leading to activation and functional enhancement. Single molecule analysis reveals the dimer is more resistant to photobleaching spending longer times in the ON state. Thus, genetically encoded bioorthogonal chemistry can be used to generate truly integrated artificial protein complexes that enhance function

Single molecule fluorescence for membrane proteins

The cell membrane is a complex milieu of lipids and proteins. In order to understand the behaviour of individual molecules is it often desirable to examine them as purified components in in vitro systems. Here, we detail the creation and use of droplet interface bilayers (DIBs) which, when coupled to TIRF microscopy, can reveal spatiotemporal and kinetic information for individual membrane proteins. A number of steps are required including modification of the protein sequence to enable the incorporation of appropriate fluorescent labels, expression and purification of the membrane protein and subsequent labelling. Following creation of DIBs, proteins are spontaneously incorporated into the membrane where they can be imaged via conventional single molecule TIRF approaches. Using this strategy, in conjunction with step-wise photobleaching, FRET and / or single particle tracking, a host of parameters can be determined such as oligomerisation state and dynamic information. We discuss advantages and limitations of this system and offer guidance for successful implementation of these approaches

VarLOCK: sequencing-independent, rapid detection of SARS-CoV-2 variants of concern for point-of-care testing, qPCR pipelines and national wastewater surveillance

The COVID-19 pandemic demonstrated the need for rapid molecular diagnostics. Vaccination programs can provide protection and facilitate the opening of society, but newly emergent and existing viral variants capable of evading the immune system endanger their efficacy. Effective surveillance for Variants of Concern (VOC) is therefore important. Rapid and specific molecular diagnostics can provide speed and coverage advantages compared to genomic sequencing alone, benefitting the public health response and facilitating VOC containment. Here we expand the recently developed SARS-CoV-2 CRISPR-Cas detection technology (SHERLOCK) to provide rapid and sensitive discrimination of SARS-CoV-2 VOCs that can be used at point of care, implemented in the pipelines of small or large testing facilities, and even determine the proportion of VOCs in pooled population-level wastewater samples. This technology complements sequencing efforts to allow facile and rapid identification of individuals infected with VOCs to help break infection chains. We show the optimisation of our VarLOCK assays (Variant-specific SHERLOCK) for multiple specific mutations in the S gene of SARS-CoV-2 and validation with samples from the Cardiff University Testing Service. We also show the applicability of VarLOCK to national wastewater surveillance of SARS-CoV-2 variants and the rapid adaptability of the technique for new and emerging VOCs