Helen Dacres

Greatly enhanced detection of a volatile ligand at femtomolar levels using bioluminescence resonance energy transfer (BRET)

Our goal is to develop a general transduction system for G-protein coupled receptors (GPCRs). GPCRs are present in most eukaryote cells and transduce diverse extracellular signals. GPCRs comprise not only the largest class of integral membrane receptors but also the largest class of targets for therapeutic drugs. In all cases studied, binding of ligand to a GPCR leads to a sub-nanometer intramolecular rearrangement. Here, we report the creation of a novel chimaeric BRET-based biosensor by insertion of sequences encoding a bioluminescent donor and a fluorescent acceptor protein into the primary sequence of a GPCR. The BRET(2)-ODR-10 biosensor was expressed in membranes of Saccharomyces cerevisiae. Assays conducted on isolated membranes indicated an EC(50) in the femtomolar range for diacetyl. The response was ligand-specific and was abolished by a single point mutation in the receptor sequence. Novel BRET-GPCR biosensors of this type have potential application in many fields including explosive detection, quality control of food and beverage production, clinical diagnosis and drug discovery.

Direct comparison of fluorescence- and bioluminescence-based resonance energy transfer methods for real-time monitoring of thrombin-catalysed proteolytic cleavage.

In this study, a representative FRET system (CFP donor and YFP acceptor) is compared with the BRET(2) system (Renilla luciferase donor, green fluorescent protein(2) (GFP(2)) acceptor and coelenterazine 400a substrate). Cleavage of a thrombin-protease-sensitive peptide sequence inserted between the donor and acceptor proteins was detected by the RET signal. Complete cleavage by thrombin changed the BRET(2) signal by a factor of 28.9+/-0.2 (R.S.D. (relative standard deviation), n=3) and the FRET signal by a factor of 3.2+/-0.1 (R.S.D., n=3). The BRET(2) technique was 50 times more sensitive than the FRET technique for monitoring thrombin concentrations. Detection limits (blank signal+3sigma(b), where sigma(b)=the standard deviation (S.D.) of the blank signal) were calculated to be 3.05 and 0.22nM thrombin for FRET and BRET(2), respectively. This direct comparison suggests that the BRET(2) technique is more suitable than FRET for use in proximity assays such as protease cleavage assays or protein-protein interaction assays.

Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage.

Bioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations of BRET are BRET(1) with Renilla luciferase (RLuc) and coelenterazine h (CLZ, lambda(em) approximately 475 nm) and BRET(2) with the substrate coelenterazine 400a (CLZ400A substrate, lambda(em)=395 nm) as the respective donors. For BRET(1) the acceptor is yellow fluorescent protein (YFP) (lambda(em) approximately 535 nm), a mutant of green fluorescent protein (GFP), and for BRET(2) it is GFP(2) (lambda(em) approximately 515 nm). It is not clear from previous studies which of these systems has superior signal-to-background characteristics. Here we directly compared BRET(1) and BRET(2) by placing two different protease-specific cleavage sequences between the donor and acceptor domains. The intact proteins simulate protein-protein association. Proteolytic cleavage of the peptide linker simulates protein dissociation and can be detected as a change in the BRET ratios. Complete cleavage of its target sequence by thrombin changed the BRET(2) ratio by a factor of 28.9+/-0.2 (relative standard deviation [RSD], n=3) and changed the BRET(1) ratio by a factor of 3.05+/-0.07. Complete cleavage of a caspase-3 target sequence resulted in the BRET ratio changes by factors of 15.45+/-0.08 for BRET(2) and 2.00+/-0.04 for BRET(1). The BRET(2) assay for thrombin was 2.9 times more sensitive compared with the BRET(1) version. Calculated detection limits (blank signal+3sigma(b), where sigma(b)=standard deviation [SD] of blank signal) were 53 pM (0.002 U) thrombin with BRET(1) and 15 pM (0.0005 U) thrombin with BRET(2). The results presented here suggest that BRET(2) is a more suitable system than BRET(1) for studying protein-protein interactions and as a potential sensor for monitoring protease activity.

Effect of enhanced Renilla luciferase and fluorescent protein variants on the Förster distance of Bioluminescence resonance energy transfer (BRET)

Bioluminescence resonance energy transfer (BRET) is an important tool for monitoring macromolecular interactions and is useful as a transduction technique for biosensor development. Förster distance (R(0)), the intermolecular separation characterized by 50% of the maximum possible energy transfer, is a critical BRET parameter. R(0) provides a means of linking measured changes in BRET ratio to a physical dimension scale and allows estimation of the range of distances that can be measured by any donor-acceptor pair. The sensitivity of BRET assays has recently been improved by introduction of new BRET components, RLuc2, RLuc8 and Venus with improved quantum yields, stability and brightness. We determined R(0) for BRET(1) systems incorporating novel RLuc variants RLuc2 or RLuc8, in combination with Venus, as 5.68 or 5.55 nm respectively. These values were approximately 25% higher than the R(0) of the original BRET(1) system. R(0) for BRET(2) systems combining green fluorescent proteins (GFP(2)) with RLuc2 or RLuc8 variants was 7.67 or 8.15 nm, i.e. only 2-9% greater than the original BRET(2) system despite being ~30-fold brighter.

Micromolar biosensing of nitric oxide using myoglobin immobilized in a synthetic silk film.

In this work we investigate the use of coiled-coil silk proteins, produced in recombinant Escherichia coli, as a new material for immobilizing biosensors. Myoglobin was embedded in transparent honeybee silk protein films. Immobilized myoglobin maintained a high affinity for nitric oxide (KD NO=52 µM) and good sensitivity with a limit of detection of 5 µM. The immobilized myoglobin-silk protein film was stable and could be stored as a dry film at room temperature for at least 60 days. The effect of immobilization on the structure of myoglobin was fully investigated using UV/visible, Fourier Transform Infrared and Raman spectroscopy, which indicated a weakening in the strength of the iron-histidine bond. This study demonstrates that recombinant coiled-coil silk proteins provide a safe and environmentally friendly alternative to sol-gels for stabilizing heme proteins for use as optical biosensors.

Comparison of enhanced bioluminescence energy transfer donors for protease biosensors.

Bioluminescence energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. We directly compared two recently developed variants of Renilla luciferase (RLuc), RLuc2 and RLuc8, as BRET donors using an in vitro thrombin assay. The comparison was carried out by placing a thrombin-specific cleavage sequence between the donor luciferase and a green fluorescent protein (GFP(2)) acceptor. Substitution of native RLuc with the RLuc mutants, RLuc2 and 8, in a BRET(2) fusion protein increased the light output by a factor of ~10. Substitution of native RLuc with either of the RLuc mutants resulted in a decrease in BRET(2) ratio by a factor of ~2 when BRET(2) components were separated by the thrombin cleavage sequence. BRET(2) ratios changed by factors of 18.8±1.2 and 18.2±0.4 for GFP(2)-RG-RLuc2 and GFP(2)-RG-RLuc8 fusion proteins, respectively, on thrombin cleavage compared to 28.8±0.20 for GFP(2)-RG-RLuc. The detection limits for thrombin were 0.23 and 0.26 nM for RLuc2 and RLuc8 BRET(2) systems, respectively, and 15 pM for GFP(2)-RG-RLuc. However, overall, the mutant BRET systems remain more sensitive than FRET and brighter than standard BRET(2).

Real-time, continuous detection of maltose using bioluminescence resonance energy transfer (BRET) on a microfluidic system

We have previously shown that a genetically encoded bioluminescent resonance energy transfer (BRET) biosensor, comprising maltose binding protein (MBP) flanked by a green fluorescent protein (GFP(2)) at the N-terminus and a variant of Renilla luciferase (RLuc2) at the C-terminus, has superior sensitivity and limits of detection for maltose, compared with an equivalent fluorescent resonance energy transfer (FRET) biosensor. Here, we demonstrate that the same MBP biosensor can be combined with a microfluidic system for detection of maltose in water or beer. Using the BRET-based biosensor, maltose in water was detected on a microfluidic chip, either following a pre-incubation step or in real-time with similar sensitivity and dynamic range to those obtained using a commercial 96-well plate luminometer. The half-maximal effective concentrations (EC50) were 2.4×10(-7)M and 1.3×10(-7) M for maltose detected in pre-incubated and real-time reactions, respectively. To demonstrate real-time detection of maltose in a complex medium, we used it to estimate maltose concentration in a commercial beer sample in a real-time, continuous flow format. Our system demonstrates a promising approach to in-line monitoring for applications such as food and beverage processing.

Fluorescent nitric oxide detection using cobalt substituted myoglobin

We report two advances in optical biosensing of nitric oxide (NO). Firstly, we developed an improved biomolecular gated system for fluorescent transduction of heme–NO binding. Secondly, through a cobalt substitution, the detection limit for NO was decreased an order of magnitude lower than that of native myoglobin.

Comparison of static and microfluidic protease assays using modified bioluminescence resonance energy transfer chemistry.

Background Fluorescence and bioluminescence resonance energy transfer (F/BRET) are two forms of Förster resonance energy transfer, which can be used for optical transduction of biosensors. BRET has several advantages over fluorescence-based technologies because it does not require an external light source. There would be benefits in combining BRET transduction with microfluidics but the low luminance of BRET has made this challenging until now. Methodology We used a thrombin bioprobe based on a form of BRET (BRETH), which uses the BRET1 substrate, native coelenterazine, with the typical BRET2 donor and acceptor proteins linked by a thrombin target peptide. The microfluidic assay was carried out in a Y-shaped microfluidic network. The dependence of the BRETH ratio on the measurement location, flow rate and bioprobe concentration was quantified. Results were compared with the same bioprobe in a static microwell plate assay. Principal Findings The BRETH thrombin bioprobe has a lower limit of detection (LOD) than previously reported for the equivalent BRET1–based version but it is substantially brighter than the BRET2 version. The normalised BRETH ratio of the bioprobe changed 32% following complete cleavage by thrombin and 31% in the microfluidic format. The LOD for thrombin in the microfluidic format was 27 pM, compared with an LOD of 310 pM, using the same bioprobe in a static microwell assay, and two orders of magnitude lower than reported for other microfluidic chip-based protease assays. Conclusions These data demonstrate that BRET based microfluidic assays are feasible and that BRETH provides a useful test bed for optimising BRET-based microfluidics. This approach may be convenient for a wide range of applications requiring sensitive detection and/or quantification of chemical or biological analytes.

Conversion of nitrous oxide to nitrogen by cobalt-substituted myoglobin

Developing technology to decrease greenhouse gas emissions is one of the greatest challenges we face in the 21st century. Nitrous oxide (N2O) is an important greenhouse gas, which is estimated to contribute 6% of the overall global warming effect. Herein we report the use of cobalt substituted heme proteins to reduce N2O to nitrogen (N2). This catalysis was electrochemically driven using methyl viologen or benzyl viologen as electron transfer partners for cobalt myoglobin. Using bulk electrolysis we demonstrated the production of 15N2 from 15N2. This catalysis, however, was noted to be poor, most likely due to oxidative damage to the protein scaffold.