D. Dafydd Jones

Cardiff University | CU · School of Biosciences

Carbon nanotube field effect transistors (CNT-FET) hold great promise as next generation miniaturised biosensors. One bottleneck is modelling how proteins, with their distinctive electrostatic surfaces, interact with the CNT-FET to modulate conductance. Using advanced sampling molecular dynamics combined with non-canonical amino acid chemistry, we...

The photochemically active sites of the proteins sfGFP66azF and Venus66azF, members of the green fluorescent protein (GFP) family, contain a non-canonical amino acid residue p-azidophenylalanine (azF) instead of Tyr66. The light-induced decomposition of azF at these sites leads to the formation of reactive arylnitrene (nF) intermediates followed by...

The RNA dependent RNA polymerase (RdRp) of the SARS-CoV-2 virus is error prone, with errors being corrected by the exonuclease (NSP14) proofreading mechanism. However, the mutagenesis and subsequent evolutionary trajectory of the virus is mediated by the delicate interplay of replicase fidelity and environmental pressures. Here, we have shown that...

Objectives Carbon nanotube field-effect biosensors (bioCNTFETs) are a promising platform to detect biomolecular analytes because they are highly sensitive, specific, low-cost, portable, and integrable into arrays for multiplexed sensing [1]. Their sensitivity originates from carbon nanotubes having electrical properties that are strongly dependent...

Carbon nanotube field effect transistor (CNT-FET) setups hold great promise for constructing next generation miniaturised biosensors whereby a biomolecular event gates conductance. The main issue is predicting how proteins, with their innate mosaic and distinctive electrostatic surfaces, interact with and thus modulate conductance of the CNT-FET. T...

Glycosylation is the most prevalent protein post‐translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet, the full impact of glycosylation on the protein structure–function relationship, especially in enzymes, is still limited. Here, we show that glycosylation rigidifies the important commercial enzyme h...

From enzymes to hormones, proteins are the most versatile macromolecules that serve a vital function throughout all biological systems. In nature, organisms are restricted to a 20 amino acid repertoire, which in turn limits the chemistry. Nature evolves and adapts but currently does so under a “chemistry-limited” circumstance. This in turn has limi...

Nanocarbon‐based field‐effect transistor (NC‐FET) biosensors are at the forefront of future diagnostic technology. By integrating biological molecules with electrically conducting carbon‐based platforms, high sensitivity real‐time multiplexed sensing is possible. Combined with their small footprint, portability, ease of use, and label‐free sensing...

Glycosylation is the most prevalent protein post-translational modification, with a quarter of glycosylated proteins having enzymatic properties. Yet the full impact of glycosylation on the protein structure-function relationship, especially in enzymes, is still limited. Here we show glycosylation rigidifies the important commercial enzyme horserad...

Fluorescent proteins have revolutionized cell biology and cell imaging through their use as genetically encoded tags. Structural biology has been pivotal in understanding how their unique fluorescent properties manifest through the formation of the chromophore and how the spectral properties are tuned through interaction networks. This knowledge ha...

The nanoscale organization of functional (bio)molecules on solid substrates with nanoscale spatial resolution and single-molecule control—in both position and orientation—is of great interest for the development of next-generation (bio)molecular devices and assays. Herein, we report the fabrication of nanoarrays of individual proteins (and dyes) vi...

Cold active esterases have gained great interest in several industries. The recently determined structure of a family IV cold active esterase (EstN7) from Bacillus cohnii strain N1 was used to expand its substrate range and to probe its commercially valuable substrates. Database mining suggested that triacetin was a potential commercially valuable...

Integrating photoactive proteins with synthetic nanomaterials holds great promise in developing optoelectronic devices whereby light, captured by a antenna protein, is converted to a modulated electrical response. The protein–nanomaterial interface is critical to defining optoelectronic properties; successful integration of bionanohybrids requires...

Here we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surface that is thought to play a role in cold-adaption by retaining solvation under changed water solvent entropy at lower temperatures. The conformation of the f...

The ability to detect proteins through gating conductance by their unique surface electrostatic signature holds great potential for improving biosensing sensitivity and precision. Two challenges are: (1) defining the electrostatic surface of the incoming ligand protein presented to the conductive surface; (2) bridging the Debye gap to generate a me...

The formation of protein complexes is central to biology, with oligomeric proteins more prevalent than monomers. The coupling of functionally and even structurally distinct protein units can lead to new functional properties not accessible by monomeric proteins alone. While such complexes are driven by evolutionally needs in biology, the ability to...

Nanoscale protein-based sensing devices designed to present proteins in defined orientations allowed the control of local electrostatic surface presented within the Debye length, and thus modulation of the conductance gating effect upon sensing protein targets. Abstract The ability to detect proteins through gating conductance by their unique surf...

Here, we report the controlled assembly of SWCNT–GFP hybrids employing DNA as a linker. Two distinct, enriched SWCNTs chiralities, (6,5), (7,6), and an unsorted SWCNT solution, were selectively functionalized with DNA and hybridized to a complementary GFPDNA conjugate. Atomic force microscopy images confirmed that GFP attachment occurred predominan...

A central challenge in nanobiotechnology is the bottom-up assembly of platforms capable of monitoring and exploiting biomolecular interactions with nanoscale control; this in turn can allow the development of novel bioelectronics interfaces. In this regard, electrical detection methodologies, using nanomaterials, are one of the most promising candi...

Fluorescent proteins (FPs) have revolutionised the life sciences, but the mechanism of chromophore maturation is still not fully understood. Here we show that incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; a crystal structure indicates the presen...

The Hbl toxin is a three-component haemolytic complex produced by Bacillus cereus sensu lato strains and implicated as a cause of diarrhoea in B. cereus food poisoning. While the structure of the HblB component of this toxin is known, the structures of the other components are unresolved. Here, we describe the expression of the recombinant HblL1 co...

This tutorial provides an introduction to the powerful molecular graphics program known as PyMOL. This program is now used routinely to generate pretty but useful and informative pictures of protein structures present in many journal publications and in lectures. But PyMOL is more than that – it is a very useful tool to interact with, inspect and a...

Cold-active esterases hold great potential for undertaking useful biotransformations at low temperatures. Here, we determined the structure of a cold active family IV esterase (EstN7) cloned from Bacillus cohnii strain N1, which has an apparent melting temperature of 26°C. EstN7 is a dimer with a classical α/β hydrolase fold. It has an acidic surfa...

Cold active esterases represent an important class of enzymes capable of undertaking useful chemical transformations at low temperatures. EstN7 from Bacillus cohnii represents a true psychrophilic esterase with a temperature optimum below 20°C. We have recently determined the structure of EstN7 and have used this knowledge to understand substrate s...

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in proximity via distance dependent processes such as Förster resonance energy transfer (FRET). The impact of FP association is assessed by predicting dimerization sites in silico and stabilizing the dimers by bio‐orthogonal covalent linkage...

Fluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O 2 located a...

In this article, we report the number of cyclin B1 proteins tagged with enhanced green fluorescent protein (eGFP) in fixed U‐2 OS cells across the cell cycle. We use a quantitative analysis of epifluorescence to determine the number of eGFP molecules in a nondestructive way, and integrated over the cell we find 10⁴ to 10⁵ molecules. Based on the me...

Metalloporphyrins play important roles in areas ranging from biology to nanoscience. Using computational design, we converted metalloporphyrin specificity of cytochrome b562 from iron to fluorogenic zinc. The new variant had...

Protein oligomers are more common in nature than monomers, with dimers being the most prevalent final structural state observed in known structures. From a biological perspective, this makes sense as it conserves vital molecular resources that may be wasted simply by generating larger single polypeptide units, and allows new features such as cooper...

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...

Fluorescent proteins (FPs) are commonly used in pairs to monitor dynamic biomolecular events through changes in their proximity via distance dependent processes such as Förster 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 loc...

Metalloporphyrins play important roles in areas ranging from biology to nanoscience. Biology uses a narrow set of metal centres comprising mainly of iron and magnesium. Here, we convert metalloporphyrin specificity of cytochrome b 562 from iron (haem) to fluorogenic zinc protoporphyrin IX (ZnPP). Through a computationally guided iterative design pr...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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 i...

Enhancing the substrate specificity of a psychrophilic esterase EstN7 via protein engineering

p>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 geneticall...

We combined in silico modelling with fully genetically encoded strain promoted azide-alkyne cycloaddition, to construct bespoke protein dimers. Using fluorescent proteins GFP and Venus as models, homo and heterodimers were constructed that switched ON once assembled and displayed enhanced spectral properties. The determined molecular structure reve...

There is now crucial medical importance placed on understanding the role of early-stage, sub-visible protein aggregation, particularly in neurodegenerative disease. While there are strategies for detecting such aggregates in vitro, there is no ap-proach at present that can detect these toxic species associated with cells and specific subcellular co...

We have created modified protein variants by introducing a non-canonical amino acid p-azido-L-phenylalanine (azF) into defined positions for photochemically-induced covalent attachment to graphene. Attachment of GFP, TEM and cyt b562 proteins was verified through a combination of atomic force and scanning tunnelling microscopy, resistance measureme...

The single-molecule properties of metalloproteins have provided an intensely active research area in recent years. This brief review covers some of the techniques used to prepare, measure and analyse the electron transfer properties of metalloproteins, concentrating on scanning tunnelling microscopy-based techniques and advances in attachment of pr...

We report the site-specific coupling of single proteins to individual carbon nanotubes (CNTs) in solution and with single-molecule control. Using an orthogonal Click reaction, Green Fluorescent Protein (GFP) was engineered to contain a genetically encoded azide group and then bound to CNT ends in different configurations: in close proximity or at l...

The bright bioluminescence catalysed by Photinus pyralis firefly luciferase (Fluc) enables a vast array of life science research such as bioimaging in live animals and sensitive in vitro diagnostics. The effectiveness of such applications is improved using engineered enzymes that to date have been constructed using amino acid substitutions. We desc...

We demonstrate an approach that allows attachment of single stranded DNA (ssDNA) to a defined residue in a protein of interest (POI) so as to provide optimal and well-defined multi-component assemblies. Using an expanded genetic code system, azido-phenylalanine (azF) was incorporated at defined residue positions in each POI; copper-free click chemi...

Through the genetic incorporation of a single phenyl azide group into superfolder GFP (sfGFP) at residue 148 we provide a molecular description of how this highly versatile chemical handle can be used to positively switch protein function in vitro and in vivo via either photochemistry or bioconjugation. Replacement of H148 with p-azido-l-phenylalan...

Correction for ‘Functional modulation and directed assembly of an enzyme through designed non-natural post-translation modification’ by Andrew M. Hartley et al., Chem. Sci., 2015, 6, 3712–3717.

The photochemical properties of phenyl azide have been exploited to modulate the function of a red autofluorescent protein, mCherry. Using genetic code reprogramming, phenyl azide chemistry has been introduced at functionally strategic positions in mCherry leading to deactivation, activation or enhancement upon UV irradiation.

Expanded genetic code approaches are a powerful means to add new and useful chemistry to proteins at defined residues positions. One such use is the introduction of non-biological reactive chemical handles for site-specific biocompatible orthogonal conjugation of proteins. Due to our currently limited information on the impact of non-canonical amin...

Post-translational modification (PTM) modulates and supplements protein functionality. In nature this high precision event requires specific motifs and/or associated modification machinery. To overcome the inherent complexity that hinders PTM’s wider use, we have utilized a non-native biocompatible Click chemistry approach to site-specifically modi...

A serine protease (N.protease), from Nocardiopsis sp., was cloned and expressed in E.coli and investigated for its potential kinetic stability. Protein expression using two vectors, pET-22b (+) and pET-39b (+) was compared based on proper folding and soluble expression of the protein. pET-39b (+) was found to be a better vector for soluble expressi...

Genetic code reprogramming allows proteins to sample new chemistry through the defined and targeted introduction of non-natural amino acids (nAAs). Many useful nAAs are derivatives of the natural aromatic amino acid tyrosine, with the para OH group replaced with useful but often bulkier substituents. Extending residue sampling by directed evolution...

Single-amino-acid deletions are a common part of the natural evolutionary landscape but are rarely sampled during protein engineering owing to limited and prejudiced molecular understanding of mutations that shorten the protein backbone. Single-amino-acid deletion variants of enhanced green fluorescent protein (EGFP) have been identified by directe...

This chapter introduces a set of transposon-based methods that were developed to sample trinucleotide deletion, trinucleotide replacement, and domain insertion. Each approach has a common initial step that utilizes an engineered version of the Mu transposon called MuDel. The inherent low sequence specificity of MuDel results in its random insertion...

Altering a protein's backbone through amino acid deletion is a common evolutionary mutational mechanism, but is generally ignored during protein engineering primarily because its effect on the folding-structure-function relationship is difficult to predict. Using directed evolution, enhanced green fluorescent protein (EGFP) was observed to tolerate...

Introducing new physicochemical properties into proteins through genetically encoded Uaa (unnatural amino acid) incorporation can lead to the generation of proteins with novel properties not normally accessible with the 20 natural amino acids. Phenyl azide chemistry represents one such useful addition to the protein repertoire. Classically used in...

Expanding the genetic code opens new avenues to modulate protein function in real time. By genetically incorporating photoreactive phenyl azide, the fluorescent properties of green fluorescent protein (GFP) can be modulated by light. Depending on the residue in GFP programmed to incorporate the phenyl azide, different effects on function and photoc...

A genetically encoded precursor to an aryl nitrene, para-azidophenylalanine, was introduced site specifically into proteins to deduce if distinct environments were capable of caging a reactive organic intermediate. Following photolysis of mutant T4 lysozyme or green fluorescent proteins, EPR spectra showed, respectively, the presence of a triplet n...

The electron transfer properties of proteins are normally measured as molecularly averaged ensembles. Through these and related measurements, proteins are widely regarded as macroscopically insulating materials. Using scanning tunnelling microscopy (STM), we present new measurements of the conductance through single-molecules of the electron transf...

Enhanced Green Fluorescent Protein (EGFP) is one of the most widely used engineered variants of the original wild-type Green Fluorescent Protein. Here, we report the high resolution (1.35 Å) structure of EGFP crystallised in its untagged sequence form that reveals the combined impact of the F64L and S65T, that give rise to improved folding and spec...

Rationale behind modelling of E222 as a double conformer. Modelling of residue E222 as either the single conformer A (A), the single conformer B (B) or as a double conformer as observed in PDB entry 2Y0G [S1] (C) does not fully satisfy the electron density difference map. Modelling of the double conformer as seen here (D) satisfies the electron den...

Structural comparison of E222 double conformers in the present structure (4EUL) and 2Y0G. (A) Overlay of all four conformers from 4EUL and 2Y0G. Conformer A and B from 4EUL are coloured green and yellow, respectively. Conformer A and B from 2Y0G [S1] are coloured orange and blue, respectively. The significant difference in placement of the side cha...

Residues with multiple conformers in EGFP. Electron density difference maps and residues in EGFP with multiple conformers are shown as sticks and coloured green, yellow or grey for conformer A, B or C respectively. (TIFF)

Size exclusion chromatography of EGFP. Samples of EGFP were applied to a Superdex™ 75 gel filtration column and the elution monitored at 488 nm. Protein concentrations of 10 µM (solid black line), 25 µM (long dashed line), 50 µM (medium dashed line) or 100 µM (short dashed line) were applied to the column. A small decrease in peak elution volume (∼...