Genetically encoded fluorescent biosensors - fluorescent proteins attached to an additional protein sequence that make it sensitive to

small biomolecules or other physiological intracellular processes - fluorescent biosensors are introduced into cells, tissues or organisms to allow for detection

by fluorescence microscopy as a: -modulation of the fluorescent properties of a single fluorescent protein -translocation of the fluorescent protein -difference in FRET efficiency - many biosensors allow for long-term imaging and can be designed to specifically target cellular compartments or organelles - advantage of biosensors is that they permit investigation of a signaling pathway or

measurement of a biomolecule while largely preserving spatial and temporal cellular processes

https://www.addgene.org/fluorescent-proteins/biosensors/#GeneBiosensors

https://www.addgene.org/fluorescent-proteins/biosensors/#GeneBiosensors

Category Purpose

Arabinose Monitor intracellular levels of arabinose

ATP:ADP Monitor the ATP:ADP ratio during live-cell imaging

Calcium Large palette of genetically encoded Ca2+

indicators (GECOs)

Calcium Calcium indicator for neural activity imaging (GCaMP5G)

Calcium Calcium indicator based on C-terminal domain of Opsanus troponin C

Hydrogen Peroxide (H2O2) Measure changes in intracellular hydrogen peroxide concentration

Magnesium Fluorescent Mg2+

sensor

pH Image intracellular pH in live cells

Phosphate Monitor inorganic phosphate transport and metabolism

Zinc Visualize free zinc levels in live cells

https://www.addgene.org/fluorescent-proteins/biosensors/#GeneBiosensors

Genetically encoded fluorescent biosensors

Genetically encoded calcium indicators (GECIs) they enable chronic, noninvasive imaging of defined cells and compartments

Two important classes of genetically encoded Ca2+ indicators: 1. Förster resonance energy transfer (FRET)–based (D3cpVenus (D3cpV) (Palmer et al., 2006),

TN-XXL (Mank et al., 2008), YC3.60 (Nagai et al., 2004), cameleon type

2. single GFP type, such as GCaMPs (Tian et al., 2009) Cameleons - CaM-M13 is sandwiched between CFP and YFP. Binding of Ca2+ to calmodulin initiates an intramolecular interaction between CaM and M13 domains, causing the chimeric protein to become more compact and, thus, bringing together donor (CFP) and acceptor (YFP) fluorophores. Therefore, cameleons react to increases in the intracellular Ca2+ concentration with increases in their FRET response.

Figure 2. Visualizing Signal Events Using an Engineered Polypeptide Sequence (A) Sensing unit: a molecular switch, usually based on endogenous sequence of a kinase or its substrate. An on/off “switch” may occur endogenously, as in a conformational change or a binding event, or be engineered, as in a unimolecular adaptation of a binding event or a pseudoligand displacement probe. (B) The reporter unit generates optical readout, either by single-color fluorescence change or by a change in resonance energy transfer.

Major types of genetically encoded fluorescent indicators (GEFIs). A. The simplest GEFIs are based on intrinsic sensitivity of some of the FPs to small ions such as H+ or Cl−. Changes in concentration of the ions are reflected in spectral shifts. B. FRET-based sensors explore conformation changes in a linker flanked by two FPs comprising a FRET pair. Structural rearrangements of the domains comprising the linker lead to changes in FRET efficiency. C. Sensors based on circularly permuted proteins consist of cpFP fused to domain(s) undergoing conformation changes upon interaction with an environment.

Lukyanov and Belousov, Biochimica et Biophysica Acta 1840: 745–756 (2014)

circular permutation. The β-barrel of the GFP is very rigid and therefore unlikely to change conformation in response to forces applied to its termini. In a procedure named circular permutation the original N- and C-termini are fused by a short (6–10 a.a.) polypeptide linker and new termini are introduced at the side of the β-barrel near the chromophore. Circularly permuted FPs (cpFPs) are sensitive to conformational distortions induced by movements of the environment-sensing domains fused to their termini. Changes in structure or interactions of the sensing domains cause changes in the FP chromophore environment and spectral properties of the sensor.

GFP topology. Green dots mark locations of the termini in viable circular permutants. Orange dots mark places where long insertions have been made. Green arrows mark beta strands that can be left out and added back to reconstitute fluorescence. Red lines are connections created in rGFP3, rewired GFP. Topological changes and truncations are the least tolerated in the N-terminal 6 beta strands.

Crone et al., State of the Art in Biosensors Book 2013

Palmer et al., Trends in Biotechnology 29: 144-152 (2011)

VanEngelenburg and Palmer Current Opinion in Chemical Biology 2008, 12:60–65

Different types of FP-based biosensor. (A) The fusion of an FP such as GFP (green) to a specific binding domain (gray) can be used to report on the production of certain signaling molecules (blue circle), for example through the redistribution of the probe – and thus fluorescence – from the cytosol (left) to the plasma membrane (right). (B) The Ca2+ sensor GCaMP consists of a molecular switch that contains calmodulin (CaM) and M13 inserted into a circularly permuted GFP (green), in which the native N-and C-termini of GFP are linked together, and new termini are generated from within the core b-barrel structure of GFP; the addition of Ca2+ causes CaM to bind to M13, which leads to increased GFP fluorescence. (C) FRET-based reporter for kinase activity. A kinase-specific substrate peptide and a phosphoamino-acid-binding domain (PAABD) are sandwiched between two FPs that can undergo FRET (e.g. CFP and YFP). Phosphorylation (P) of the substrate through the cognate kinase induces binding of the substrate peptide by the PAABD, resulting in a conformational rearrangement that produces a change in FRET.

Sample et al., Journal of Cell Science (2014) 127, 1151–1160 Mehta and Zhang Annu. Rev. Biochem. 2011. 80:375–401

Hamers et al., Protoplasma (2014) 251:333–347

FRET basic principle

FRET basic principle

- GCaMPs are composed of a circularly permuted (cp) GFP fused to the calmodulin (CaM)–binding region of chicken myosin light chain kinase (M13) at its N terminus and a vertebrate CaM at its C terminus. Binding of Ca2+ causes the M13 and CaM domains to interact and the interface between CaM and the fluorescent protein (FP) to reorganize, which leads to an increase in fluorescence due to water-mediated interactions between the chromophore and R377 of CaM. Indicators are dim in the absence of Ca2+ and bright when bound to Ca2+.

GCaMP3 is based on circularly permuted green fluorescent protein (cpGFP), calmodulin (CaM), and the Ca2+/CaM-binding “M13” peptide (M13pep).

Design of GCaMP5s. A, Schematic of the GCaMP3 structure with sites of engineering shown. B, Structural effects of the D381Y mutation (D380Y in GCaMP3 numbering). Chromophore environment at the cpGFP/CaM interface in GCaMP2 (top, PDB 3EVR)(Akerboom et al., 2009) and GCaMP5G (bottom, PDB 3SG4) structure reported here. Structures are shown as a diagram and sticks colored by domain (cpGFP, green; linker, white; CaM, cyan). Selected portions of the model around the GFP chromophore (CRO) are represented as sticks with ordered water molecules represented as red spheres.

Biosensors for Ca: Calcium indicator for neural activity imaging (GCaMP5G)

Akerboom et al. The Journal of Neuroscience, 32: 13819 –13840, 2012

Biosensors for Ca: Large palette of genetically encoded Ca2+ indicators (GECOs)

Developed a colony-based screen for Ca2+-dependent fluorescent changes. Ca2+ indicators targeted to the E. coli periplasm can be shifted toward the Ca2+-free or Ca2+-bound states by experimental manipulation of the environmental Ca2+ concentration.

Zhao et al., Science 333: 1888-1891, 2011

Schematic of the system for image-based screening of E. coli colonies. The GCaMP variant, as represented by GCaMP2 has a TorA periplasmic export tag.

Spectral profiles of GECOs. Fluorescence excitation (Ex) and emission spectra (Em) normalized to the Ca2+-free state. Normalized excitation spectra of Ca2+-free (dashed line) and Ca2+-bound (solid line) B-GECO1 (blue), G-GECO1 (green), and R-GECO1 (red). Absorbance (Abs) and emission spectra for Ca2+-free (dashed line) and Ca2+-bound (solid line) GEM-GECO1.

Zhao et al., Science 333: 1888-1891, 2011

Biosensors for Ca: Large palette of genetically encoded Ca2+ indicators (GECOs)

design of green fluorescent genetically encoded Ca2+ indicators for optical imaging as G-GECO1, G-GECO1.1, and G-GECO1.2. The G-GECOs share a Ca2+-dependent increase in fluorescence (2300 to 2600%) that is approximately double that of GCaMP3

Troponin C-based Ca2+ sensor proteins. (a) Schematic drawing illustrating the FRET-based mechanism of sensor function. (b) An outline of the structure of four TnC-based FCIPs (Genetically encoded fluorescent Ca2+ indicator proteins).

Biosensors for Ca: Calcium indicator based on C-terminal domain of Opsanus troponin C

Troponin C - Ca2+ binding protein from cardiac and skeletal muscle. It is a specialized Ca2+ binding protein with the only known function to regulate muscle contraction.

Garaschuk et al., Cell Calcium 42: 351–361, 2007

Biosensors for Ca: Calcium indicator based on C-terminal domain of Opsanus troponin C

Troponin C - Ca2+ binding protein from cardiac and skeletal muscle. It is a specialized Ca2+ binding protein with the only known function to regulate muscle contraction.

A two state model of pH dependent ground states of wtGFP. (A) Four molecules of water and side-chains of amino acids depicted in grey are involved in hydrogen-bonding with the chromophore. (B) Chromophore formation involves cyclization, imidazolinone ring system formation, dehydration and oxidation.

Biosensors for pH : Image intracellular pH in live cells

Benčina Sensors 2013, 13, 16736-16758

The pH of individual subcellular compartments in a prototypic eukaryotic cell. We show only pH values obtained using the genetically encoded pH-sensitive FPs as indicators.

Biosensors for pH : Image intracellular pH in live cells

Benčina Sensors 2013, 13, 16736-16758

(A) A set up of dual excitation sequential scanning confocal microscope for imaging ratiometric pHluorin probes. (B) Fluorescence excitation spectra of pHluorin in buffers with pH ranging from 5.2 to 8.5. (C) A hypothetical pHluorin dose reponse curve; a plot of fluorescence ratio versus pH. The ratios between the emission intensities at 405- and 476-nm are calculated. The physiological target pH range and physiologically relevant dynamic range are indicated. (D) Pseudocolored image (right) of permeabilized yeast cells in buffered solution with nigericin is calculated from images taken at 405- and 476-nm excitation (Scans 1 and 2). Different colors are assigned to defined pH values in accordance with the in situ calibration curve (blue, alkalinic; red, acidic). The in situ calibration curve is shown in panel D.

Biosensors for pH : Image intracellular pH in live cells

Benčina Sensors 2013, 13, 16736-16758

Han et al., Anal. Chem., Publication Date (Web): 21 Apr 2015

Basic principles for oxidant detection using (a) oxidant-sensitive dyes and (b) “non-redox” probes.

Winterbourn Biochimica et Biophysica Acta 1840: 730–738 (2014)

Biosensors for redox/ROS: Redox-sensitive cytosolic protein sensors

Biosensors for disaccherides: Monitor intracellular levels of arabinose in E. coli

In vivo analysis of metabolite accumulation in live bacterial cells using fluorescence resonance energy transfer sensors. Gram-negative bacterial cells are shown surrounded by two membranes expressing the fluorescence resonance energy transfer sensor, which consists of an episomally encoded recognition element, a periplasmic binding protein for arabinose or maltose, sandwiched by an N-terminal ECFP and a C-terminal EYFP. The sensor contains no targeting information and will thus be present in the cytosol of Escherichia coli. The cell in the top panel is cultivated in the absence of the ligand; therefore, endogenous ligand concentrations are low (all sensors are in the unbound state, with low fluorescence resonance energy transfer). The cell in the bottom panel is cultivated in the presence of the ligand; therefore, endogenous ligand concentrations are high (all sensors are in the bound state, with high fluorescence resonance energy transfer).

Kaper et al., Biotechnology for Biofuels 2008, 1:11

Detection of intracellular arabinose with FLIPara-200n. Detection of intracellular arabinose in Escherichia coli BL21-Gold(DE3) cells expressing FLIPara-250n. The arrow indicates arabinose addition

Accumulation of intracellular maltose detected with FLIPmal sensors. Cytosolic maltose accumulation measured in 30-second intervals after injection of buffer or 50 mM maltose to microtiter plate wells containing Escherichia coli BL21-Gold(DE3) cells expressing FLIPmal-40μΔ1-enhanced yellow fluorescent protein. The arrow indicates the time point of maltose addition

Expression of Nanosensors in Arabidopsis Wild Type and Silencing Mutants.(A) Number of mature, soil-grown transformants showing significant eYFP fluorescence as determined using an epifluorescence stereomicroscope.(B) Representative fluorescence images of leaves from the different transformants.

Biosensors for disaccherides: Monitor intracellular levels of glucose in A. thaliana

Bacterial Periplasmic Binding Protein superfamily (PBPs) recognize hundreds of substrates with high affinity (attomolar to low micromolar) and specificity. PBPs have been shown to undergo a significant conformational change upon ligand binding; fusion of an individual sugar, amino acid, or phosphate binding PBP with a pair of GFP variants produced nanosensors for maltose, ribose, glucose, Glu, and phosphate.

Construct Maps for FLIPglu-Δ13.(A) FLIPglu-Δ13 cassette containing linearly fused eCFP-mglB-eYFP genes. The size of each gene, restriction sites, and transcription start and stop are indicated.(B) pPZP 312 binary vector T-DNA containing a FRET glucose nanosensor. L, left border; MglB, E. coli periplasmic glucose binding protein; PMAS, MAS promoter; P35S, CaMV 35S promoter; R, right border; TRbcs, Rbcs terminator; T35S, CAMV 35S terminator. Arrows indicate the direction of transcription. The restriction enzymes used for cloning are indicated.

Deuschle et al., Plant Cell 18: 2314–2325, 2006

Glucose-Induced FRET Changes in the Cytosol of Leaf Epidermal Cells.The FRET sensor FLIPglu-600μΔ13 with an affinity of 600 μM for glucose in stably transformed rdr6-11 Arabidopsis plants responds in the epidermis to perfusion with 50 mM glucose. Quantitative data were derived by pixel-by-pixel integration of the ratiometric images. The scale at right gives fluorescence intensity in arbitrary units (A.U.) for the individual eCFP (480/30) and eYFP (535/40) emission channels; the scale at left gives the ratio of eYFP intensity divided by eCFP intensity. For each phase (plus/minus glucose), one ratiometric image with a pavement and a guard cell is shown.

Biosensors for disaccherides: Monitor intracellular levels of glucose in A. thaliana

Deuschle et al., Plant Cell 18: 2314–2325, 2006

Sensor Sequence Kd (M) a ΔRmax a, b Hill coefficient

Dynamic

rangec

FLIPPi-260n Wild-type 2.6 × 10−7

−0.13 1.03 25–1200 nM

FLIPPi-770n S161A 7.7 × 10−7

−1.07 1.03 80–5600 nM

FLIPPi-4μ T163A 3.9 × 10−6

−1.34 1.02 0.4–25 μM

FLIPPi-5μ S52A 5.1 × 10−6

−1.33 1.03 0.5–34 μM

FLIPPi-200μ G162A 2.1 × 10−4

−1.13 1.00 25–1600 μM

FLIPPi-30m T22A 3.3 × 10−2

−1.03 1.03 3–170 mM

FLIPPi affinity mutants

FLIPPi-WT construct (above); FLIPPi-260n construct (below; nine amino acids were deleted from the C-terminus of the eCFP, one from the N-terminus of Venus, and four from each binding protein-fluorophore linker).

Biosensors for phosphate : Monitor inorganic phosphate transport and metabolism

Gu et al., FEBS Letters 580 (2006) 5885–5893

Genetically-encoded fluorescence resonance energy transfer (FRET) sensors for phosphate (Pi) (FLIPPi) were engineered by fusing a predicted Synechococcus phosphate-binding protein (PiBP) to eCFP and Venus. Purified fluorescent indicator protein for inorganic phosphate (FLIPPi), in which the fluorophores are attached to the same PiBP lobe, shows Pi-dependent increases in FRET efficiency.

Specificity and pH sensitivity of FLIPPi-5μ. (a) Selectivity of FLIPPi-5μ for phosphate over nitrate and sulfate. Solid lines were plotted by curve fitting. (b) Phosphate binding of FLIPPi-5μ was tested in 20 mM Tris buffers adjusted to pH 6.8, 7.0, 7.2, 7.4 and 7.6, then titrated with phosphate solutions of the same respective pH. Statistical analysis (ANOVA) showed no significant difference regarding the maximal ratio change between pH 6.8 and 7.4.

Biosensors for phosphate : Monitor inorganic phosphate transport and metabolism

Gu et al., FEBS Letters 580 (2006) 5885–5893

Principle of genetically encoded FRET sensors for the intracellular imaging of transition metal (TM) ions. (i) Plasmid DNA encoding for a FRET sensor can be transfected into the nucleus of cells, whereby (ii) ribosomes and mRNA are responsible for the controlled non-invasive expression of the FRET sensor. Genetically encoded FRET sensors can accurately monitor fluctuations in the influx and efflux of TM ions in real time. (iii) By incorporating various localization tags, the FRET sensors can be targeted to the cell membrane or to specific organelles, such as the mitochondria and the Golgi apparatus, where the local compartmentalized TM ion concentrations can be significantly higher compared to free cytosolic concentrations.

Biosensors for metals and ions: Visualize free zinc levels in live cells

Vinkenborg et al., Current Opinion in Chemical Biology 2010, 14:231–237

The eCALWY sensor family: genetically encoded Zn2+ sensors based on conformational switching. (a) In the absence of Zn2+, hydrophobic mutations on the surface of the fluorescent domains promote complex formation of Cerulean and Citrine. Binding of Zn2+ to ATOX1 and WD4 disrupts this complex, generating a large decrease in energy transfer. (b) Sensor occupancy in pancreatic β-cells (INS-1(832/13)) as a function of the sensor Kd. Datapoints show the occupancy of the different eCALWY variants under resting conditions. The dashed lines depict the expected responses assuming free zinc concentrations of 0.05, 0.1, 0.2 (0.4, solid line), 0.8, 1.6, and 3.2 nM, respectively. (c) False-colored spinning disc confocal microscopy images of INS-1(832/13) cells expressing eCALWY-4 in resting cells (left image), after addition of 50 μM of the membrane permeable zinc chelator TPEN (middle image) and after addition of 5 μM pyrithione (pyr) together with 100 μM Zn2+ (right image).

Biosensors for metals and ions: Visualize free zinc levels in live cells

Vinkenborg et al., Current Opinion in Chemical Biology 2010, 14:231–237

Figure 1. The Pipeline for Kinase Activity Reporter Development. (A) Design. The majority of two-fluorescent-protein (FP) reporter blueprints position the fluorescent protein pair around a molecular switch, either endogenous or engineered. (B) Development is typically an iterative process. (C) Initial characterization of a reporter usually involves measuring its response to a known stimulus of the signal of interest; here, “a” represents an earlier version of a sensor with lower dynamic range, whereas “b” represents an optimized version. (D) Optimization of a reporter may involve changing the fluorescent proteins, linker, or length/identity of the specific protein components that recognize the signal. After such optimization, new versions of a probe are compared to earlier versions. (E) Application of a probe in cell lines (typically to investigate kinase activity and spatiotemporal regulation, or crosstalk with other pathways), primary cells (to explore kinase involvement in cell type-specific behaviors), and live animals (to explore the involvement of a kinase in specific processes or pathologies in vivo) can be both an end goal of probe development and a spur to further optimization.

Oldach and Zhang Chemistry & Biology 21, February 20, 2014