Highly Sensitive and Selective Biosensor for a Disaccharide Based on an AraC-Like Transcriptional Regulator Transduced with Bioluminescence Resonance Energy Transfer.

Sensitive and selective quantification of individual sugars in complex media is technically challenging and usually requires HPLC separation. Accurate measurement without the need for separation would be highly desirable. The measurement of trace levels of lactose in lactose-reduced milk exemplifies the problem, with the added challenge that trace lactose must be measured in the presence of ≈140 mM glucose and galactose, the products of lactase digestion of lactose. Biosensing is an alternative to HPLC, but current biosensing methods, based on coupled-enzyme assays, tend to have poor sensitivity and complex biochemistry and can be time-consuming. We explored a fundamentally different approach, based on identifying a lactose-specific binding protein compatible with photonic transduction. We identified the BgaR transcriptional regulator of Clostridium perfringens, which is highly selective for lactose, as a suitable ligand binding domain and combined it with a bioluminescence energy resonance transfer transduction system. This BRET-based biosensor showed a 27% decrease in the BRET ratio in the presence of saturating (1 mM) lactose. Using a 5 min assay, the half maximal effective concentration (EC50) for lactose in phosphate-buffered saline (PBS) was 12 µM. The biosensor was 200 times more sensitive to lactose than to glucose or galactose. Sensitivity and selectivity were not significantly affected by the presence of 10% (v/v) dialyzed milk. The biosensor is suitable for direct determination of residual lactose in lactase-treated milk, with a limit of detection of 0.2 µM, 100 times below the most stringent lactose-free standard and without the need to remove fat or protein from the sample.

Visualization of Protein Interactions in Living Cells Using Bimolecular Luminescence Complementation (BiLC).

The number of intracellular protein-protein interactions (PPIs) far exceeds the total number of proteins encoded by the genome. Dynamic cellular PPI networks respond to external stimuli and endogenous metabolism in order to maintain homeostasis. Many PPIs are directly involved in disease pathogenesis and/or resistance to therapeutics; they therefore represent potential drug targets. A technology generally termed 'bimolecular complementation' relies on the physical splitting of a molecular reporter (such as a fluorescent or luminescent protein) and fusion of the resulting two fragments to a pair of interacting proteins. When these proteins interact, they effectively reconstitute the activity of the molecular reporter (typically leading to increased fluorescence or luminescence). This unit describes the selection and development of bimolecular luminescence complementation (BiLC) assays for reporting intracellular PPIs, and provides examples in which BiLC was used to identify small molecules that can modulate PPIs. © 2017 by John Wiley & Sons, Inc.

Genetically Encoded Molecular Tension Probe for Tracing Protein-Protein Interactions in Mammalian Cells.

Optical imaging of protein-protein interactions (PPIs) facilitates comprehensive elucidation of intracellular molecular events. We demonstrate an optical measure for visualizing molecular tension triggered by any PPI in mammalian cells. Twenty-three kinds of candidate designs were fabricated, in which a full-length artificial luciferase (ALuc) was sandwiched between two model proteins of interest, e.g., FKBP and FRB. One of the designs greatly enhanced the bioluminescence in response to varying concentrations of rapamycin. It is confirmed with negative controls that the elevated bioluminescence is solely motivated from the molecular tension. The probe design was further modified toward eliminating the C-terminal end of ALuc and was found to improve signal-to-background ratios, named "a combinational probe". The utilities were elucidated with detailed substrate selectivity, bioluminescence imaging of live cells, and different PPI models. This study expands capabilities of luciferases as a tool for analyses of molecular dynamics and cell signaling in living subjects.

Mechanical control of Renilla luciferase.

We report experiments where the activity of the enzyme luciferase from Renilla reniformis is controlled through a DNA spring attached to the enzyme. In the wake of previous work on kinases, these results establish that mechanical stress applied through the DNA springs is indeed a general method for the artificial control of enzymes, and for the quantitative study of mechano-chemical coupling in these molecules. We also show proof of concept of the luciferase construct as a sensitive molecular probe, detecting a specific DNA target sequence in an easy, one-step, homogeneous assay, as well as SNP detection without melting curve analysis.

Crystal structure of coelenterazine-binding protein from Renilla muelleri at 1.7 A: why it is not a calcium-regulated photoprotein.

Bioluminescence in the sea pansy Renilla involves two distinct proteins, a Ca2+-triggered coelenterazine-binding protein (CBP), and Renilla luciferase. CBP contains one tightly bound coelenterazine molecule, which becomes available for reaction with luciferase and O2 only subsequent to Ca2+ binding. CBP belongs to the EF-hand superfamily of Ca2+-binding proteins and contains three "EF-hand" Ca2+-binding sites. The overall spatial structure of recombinant selenomethionine-labeled CBP determined at 1.7 A, is found to approximate the protein scaffold characteristic of the class of Ca2+-regulated photoproteins. Photoproteins however, catalyze molecular oxygen addition to coelenterazine producing a 2-hydroperoxycoelenterazine intermediate, which is stabilized within the binding cavity in the absence of Ca2+. Addition of Ca2+ triggers the bioluminescence reaction. However in CBP this first step of oxygen addition is not allowed. The different amino acid environments and hydrogen bond interactions within the binding cavity, are proposed to account for the different properties of the two classes of proteins.

A new method of defense response analysis using a transient expression system in rice protoplasts.

We established a new plant defense response assay using a transient expression system in rice protoplasts. The assay system sensitively detected defense induction by flagellin, which had previously been assigned to a specific elicitor. Our assay system provides a rapid and efficient way to dissect rice defense mechanisms.