A dimer-monomer switch controls CHIP-dependent substrate ubiquitylation and processing.

The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.

Partitioning of an Enzyme-Polymer Surfactant Nanocomplex into Lipid-Rich Cellular Compartments Drives In Situ Hydrolysis of Organophosphates.

Most organophosphates (OPs) are hydrophobic, and after exposure, can sequester into lipophilic regions within the body, such as adipose tissue, resulting in long term chronic effects. Consequently, there is an urgent need for therapeutic agents that can decontaminate OPs in these hydrophobic regions. Accordingly, an enzyme-polymer surfactant nanocomplex is designed and tested comprising chemically supercharged phosphotriesterase (Agrobacterium radiobacter; arPTE) electrostatically conjugated to amphiphilic polymer surfactant chains ([cat.arPTE][S-]). Experimentally-derived structural data are combined with molecular dynamics (MD) simulations to provide atomic level detail on conformational ensembles of the nanocomplex using dielectric constants relevant to aqueous and lipidic microenvironments. These show the formation of a compact admicelle pseudophase surfactant corona under aqueous conditions, which reconfigures to yield an extended conformation at a low dielectric constant, providing insight into the mechanism underpinning cell membrane binding. Significantly, it demonstrated that [cat.arPTE][S-] spontaneously binds to human mesenchymal stem cell membranes (hMSCs), resulting in on-cell OP hydrolysis. Moreover, the nanoconstruct can endocytose and partition into the intracellular fatty vacuoles of adipocytes and hydrolyze sequestered OP.

Monitoring hippocampal glycine with the computationally designed optical sensor GlyFS.

Fluorescent sensors are an essential part of the experimental toolbox of the life sciences, where they are used ubiquitously to visualize intra- and extracellular signaling. In the brain, optical neurotransmitter sensors can shed light on temporal and spatial aspects of signal transmission by directly observing, for instance, neurotransmitter release and spread. Here we report the development and application of the first optical sensor for the amino acid glycine, which is both an inhibitory neurotransmitter and a co-agonist of the N-methyl-D-aspartate receptors (NMDARs) involved in synaptic plasticity. Computational design of a glycine-specific binding protein allowed us to produce the optical glycine FRET sensor (GlyFS), which can be used with single and two-photon excitation fluorescence microscopy. We took advantage of this newly developed sensor to test predictions about the uneven spatial distribution of glycine in extracellular space and to demonstrate that extracellular glycine levels are controlled by plasticity-inducing stimuli.

Sequential Electrostatic Assembly of a Polymer Surfactant Corona Increases Activity of the Phosphotriesterase arPTE.

We present a new methodology for the generation of discrete molecularly dispersed enzyme-polymer-surfactant bioconjugates. Significantly, we demonstrate that >3-fold increase in the catalytic efficiency of the diffusion-limited phosphotriesterase arPTE can be achieved through sequential electrostatic addition of cationic and anionic polymer surfactants, respectively. Here, the polymer surfactants assemble on the surface of the enzyme via ion exchange to yield a compact corona. The observed rate enhancement is consistent with a mechanism whereby the polymer-surfactant corona gives rise to a decrease in the dielectric constant in the vicinity of the active site of the enzyme, accelerating the rate-determining product diffusion step. The facile methodology has significant potential for increasing the efficiency of enzymes and could therefore have a substantially positive impact for industrial enzymology.

A novel route to copper(II) detection using 'click' chemistry-induced aggregation of gold nanoparticles.

A simple colorimetric method for the detection of copper ions in water is described. This method is based on the 'click' copper(I)-catalyzed azide-alkyne cycloaddition reaction and its use in promoting the aggregation of azide-tagged gold nanoparticles by a dialkyne cross-linker is described. Nanoparticle cross-linking, evidenced as a colour change, is used for the detection of copper ions. The lowest detected concentration by the naked eye was 1.8 µM, with the response linear with log(concentration) between 1.8-200 µM. The selectivity relative to other potentially interfering ions was evaluated.

A Rationally Designed Supercharged Protein-Enzyme Chimera Self-Assembles In Situ to Yield Bifunctional Composite Textiles.

Catalytically active materials for the enhancement of personalized protective equipment (PPE) could be advantageous to help alleviate threats posed by neurotoxic organophosphorus compounds (OPs). Accordingly, a chimeric protein comprised of a supercharged green fluorescent protein (scGFP) and phosphotriesterase from Agrobacterium radiobacter (arPTE) was designed to drive the polymer surfactant (S-)-mediated self-assembly of microclusters to produce robust, enzymatically active materials. The chimera scGFP-arPTE was structurally characterized via circular dichroism spectroscopy and synchrotron radiation small-angle X-ray scattering, and its biophysical properties were determined. Significantly, the chimera exhibited greater thermal stability than the native constituent proteins, as well as a higher catalytic turnover number (kcat). Furthermore, scGFP-arPTE was electrostatically complexed with monomeric S-, driving self-assembly into [scGFP-arPTE][S-] nanoclusters, which could be dehydrated and cross-linked to yield enzymatically active [scGFP-arPTE][S-] porous films with a high-order structure. Moreover, these clusters could self-assemble within cotton fibers to generate active composite textiles without the need for the pretreatment of the fabrics. Significantly, the resulting materials maintained the biophysical activities of both constituent proteins and displayed recyclable and persistent activity against the nerve agent simulant paraoxon.

Three-Dimensional Printable Enzymatically Active Plastics.

Here, we describe a facile route to the synthesis of enzymatically active highly fabricable plastics, where the enzyme is an intrinsic component of the material. This is facilitated by the formation of an electrostatically stabilized enzyme-polymer surfactant nanoconstruct, which, after lyophilization and melting, affords stable macromolecular dispersions in a wide range of organic solvents. A selection of plastics can then be co-dissolved in the dispersions, which provides a route to bespoke 3D enzyme plastic nanocomposite structures using a wide range of fabrication techniques, including melt electrowriting, casting, and piston-driven 3D printing. The resulting constructs comprising active phosphotriesterase (arPTE) readily detoxify organophosphates with persistent activity over repeated cycles and for long time periods. Moreover, we show that the protein guest molecules, such as arPTE or sfGFP, increase the compressive Young's modulus of the plastics and that the identity of the biomolecule influences the nanomorphology and mechanical properties of the resulting materials. Overall, we demonstrate that these biologically active nanocomposite plastics are compatible with state-of-the-art 3D fabrication techniques and that the methodology could be readily applied to produce robust and on-demand smart nanomaterial structures.

Method for Developing Optical Sensors Using a Synthetic Dye-Fluorescent Protein FRET Pair and Computational Modeling and Assessment.

Biosensors that exploit Förster resonance energy transfer (FRET) can be used to visualize biological and physiological processes and are capable of providing detailed information in both spatial and temporal dimensions. In a FRET-based biosensor, substrate binding is associated with a change in the relative positions of two fluorophores, leading to a change in FRET efficiency that may be observed in the fluorescence spectrum. As a result, their design requires a ligand-binding protein that exhibits a conformational change upon binding. However, not all ligand-binding proteins produce responsive sensors upon conjugation to fluorescent proteins or dyes, and identifying the optimum locations for the fluorophores often involves labor-intensive iterative design or high-throughput screening. Combining the genetic fusion of a fluorescent protein to the ligand-binding protein with site-specific covalent attachment of a fluorescent dye can allow fine control over the positions of the two fluorophores, allowing the construction of very sensitive sensors. This relies upon the accurate prediction of the locations of the two fluorophores in bound and unbound states. In this chapter, we describe a method for computational identification of dye-attachment sites that allows the use of cysteine modification to attach synthetic dyes that can be paired with a fluorescent protein for the purposes of creating FRET sensors.

Construction of a robust and sensitive arginine biosensor through ancestral protein reconstruction.

Biosensors for signaling molecules allow the study of physiological processes by bringing together the fields of protein engineering, fluorescence imaging, and cell biology. Construction of genetically encoded biosensors generally relies on the availability of a binding "core" that is both specific and stable, which can then be combined with fluorescent molecules to create a sensor. However, binding proteins with the desired properties are often not available in nature and substantial improvement to sensors can be required, particularly with regard to their durability. Ancestral protein reconstruction is a powerful protein-engineering tool able to generate highly stable and functional proteins. In this work, we sought to establish the utility of ancestral protein reconstruction to biosensor development, beginning with the construction of an l-arginine biosensor. l-arginine, as the immediate precursor to nitric oxide, is an important molecule in many physiological contexts including brain function. Using a combination of ancestral reconstruction and circular permutation, we constructed a Förster resonance energy transfer (FRET) biosensor for l-arginine (cpFLIPR). cpFLIPR displays high sensitivity and specificity, with a Kd of ∼14 µM and a maximal dynamic range of 35%. Importantly, cpFLIPR was highly robust, enabling accurate l-arginine measurement at physiological temperatures. We established that cpFLIPR is compatible with two-photon excitation fluorescence microscopy and report l-arginine concentrations in brain tissue.

Protein engineering with unnatural amino acids.

Protein engineering has become an extensively used tool in many fields, allowing us to probe protein function, characterize proteins using a range of biophysical techniques, chemically modify proteins and improve protein function for medical and industrial applications. It is now possible to site-specifically incorporate unnatural, or non-canonical, amino acids (uAAs) into proteins, which has had a major impact on protein engineering. In this review, we discuss the recent technical developments in the field and how uAA-protein engineering is becoming an increasingly valuable molecular tool, with the unique chemical functionalities of some uAAs allowing a range of otherwise impossible experiments to be performed. Finally, the impediments that have resulted in a relatively small number of recent studies in which uAA-protein engineering has been used to improve protein function are discussed, alongside some of the recent technical developments that may serve to overcome these obstacles.