Rational design of FRET sensor proteins based on mutually exclusive domain interactions

Abstract Proteins that switch between distinct conformational states are ideal to monitor and control molecular processes within the complexity of biological systems. Inspired by the modular architecture of natural signalling proteins, our group explores generic design strategies for the construction of FRET-based sensor proteins and other protein switches. In the present article, I show that designing FRET sensors based on mutually exclusive domain interactions provides a robust method to engineer sensors with predictable properties and an inherently large change in emission ratio. The modularity of this approach should make it easily transferable to other applications of protein switches in fields ranging from synthetic biology, optogenetics and molecular diagnostics

Sensor properties of the different MagFRET variants.

1<p>Mutations introduced in the first or second 12-residue metal binding loops of HsCen3 are indicated in bold and are underlined.</p>2<p>The dissociation constant (<i>K</i>_d) for each variant's Mg²⁺ and first Ca²⁺ binding event is indicated, together with the standard error (SE).</p>3<p>A binding event's dynamic range (D.R.) is defined as the difference in emission ratio between the unbound and fully metal bound form divided by the emission ratio in the unbound form, multiplied by 100%.</p

Metal binding properties of MagFRET-1.

<p>(A) Normalized fluorescence emission spectra of MagFRET-1 at 0 and at 16 mM Mg²⁺ after excitation at 420 nm. (B, C) Emission ratio (Citrine to Cerulean) of MagFRET-1 as a function of the Mg²⁺ (B) or Ca²⁺ (C) concentration. Solid lines indicate a fit to a single (B) or a double (C) binding event, yielding a <i>K</i>_d of 0.15±0.02 mM for Mg²⁺ and <i>K</i>_d's of 10±4 µM and ∼35 mM for Ca²⁺, respectively. (D) Emission ratios of MagFRET-1 in absence of metal, in the presence of 10 µM Ba²⁺, Ni²⁺, Cu²⁺, Zn²⁺ or Fe³⁺, and in the presence of the same metals and 1 mM Mg²⁺. Measurements were performed in triplicate, error bars indicate SEM. All measurements were performed in 150 mM Hepes (pH 7.1), 100 mM NaCl and 10% (v/v) glycerol with 0.2 µM sensor protein.</p

Design of the genetically encoded magnesium FRET sensor MagFRET.

<p>(A) Crystal structure (PDB code 2GGM) of HsCen2 in the calcium-bound, compact state. The typical helix-loop-helix structure can be observed, with EF-hands indicated by Roman numerals. The dotted lines indicate the N-terminal truncated part of the domain used in the sensor. In HsCen3, the high-affinity Mg²⁺/Ca²⁺ binding site is in loop I, and a much weaker Ca²⁺-binding site is found in loop II. (B) Schematic representation of MagFRET, where the N-terminal truncation of HsCen3 is flanked by Cerulean and Citrine. In absence of Mg²⁺, the HsCen3 domain is in a molten globule-like state, with little tertiary structure and a relatively large average distance between the fluorescent domains. Mg²⁺-binding induces a compact, well-defined tertiary structure, resulting in increased energy transfer between Cerulean and Citrine.</p

<i>In situ</i> characterization of MagFRET-1 in HEK293 cells.

<p>(A–D) Confocal fluorescence microscopy images showing HEK293 cells expressing MagFRET-1 (A, B) and MagFRET-1-NLS (C, D) showing Cerulean (A,C) or Citrine emission (B, D). (E, F) Investigation of MagFRET-1's <i>in situ</i> Ca²⁺ sensitivity. (E) Emission ratio over time of intact HEK293 cells expressing MagFRET-1 measured by widefield fluorescence microscopy. At t = 120 s, 50 µM of PAR-1 agonist peptide was added to activate Ca²⁺ signaling. (F) To confirm Ca²⁺ signaling took place in stimulated cells, the fluorescence intensity of intact HEK293 cells loaded with Ca²⁺-dye Oregon Green–BAPTA was followed. At t = 120 s, 50 µM of PAR-1 agonist peptide was added to activate Ca²⁺ signaling, and at t = 240 s, 20 µM A23187 was added. In E and F, each trace represents the response of an individual cell, with ratio (E) or intensity (F) normalized to the value at t = 0 s. (G, H) Response of MagFRET-1 expressed in permeabilized HEK293 cells to changes in [Mg²⁺]. MagFRET-1 emission ratio was followed over time as the concentration of MgCl₂ (G) or EDTA (H) was increased, as indicated on the panels. (I, J) Response of negative control construct Cerulean-linker-Citrine expressed in permeabilized HEK293 cells to changes in [Mg²⁺]. To maintain an isotonic solution, the increase in Cl⁻ concentration due to addition of MgCl₂ was compensated for by reducing the KCl concentration in the buffer. Prior to imaging, cells were permeabilized using 10 µg/mL digitonin. Traces in G to J represent averages of at least 9 cells, error bars indicate SEM, ratios were normalized to the emission ratio at t = 0.</p

Genetically encoded FRET sensors to monitor intracellular Zn2+ homeostasis

We developed genetically encoded fluorescence resonance energy transfer (FRET)-based sensors that display a large ratiometric change upon Zn2+ binding, have affinities that span the pico- to nanomolar range and can readily be targeted to subcellular organelles. Using this sensor toolbox we found that cytosolic Zn2+ was buffered at 0.4 nM in pancreatic ß cells, and we found substantially higher Zn2+ concentrations in insulin-containing secretory vesicles

Dynamic imaging of cytosolic zinc in Arabidopsis roots combining FRET sensors and RootChip technology

Zinc plays a central role in all living cells as a cofactor for enzymes and as a structural element enabling the adequate folding of proteins. In eukaryotic cells, metals are highly compartmentalized and chelated. Although essential to characterize the mechanisms of Zn(2+) homeostasis, the measurement of free metal concentrations in living cells has proved challenging and the dynamics are difficult to determine. Our work combines the use of genetically encoded Förster resonance energy transfer (FRET) sensors and a novel microfluidic technology, the RootChip, to monitor the dynamics of cytosolic Zn(2+) concentrations in Arabidopsis root cells. Our experiments provide estimates of cytosolic free Zn(2+) concentrations in Arabidopsis root cells grown under sufficient (0.4 nM) and excess (2 nM) Zn(2+) supply. In addition, monitoring the dynamics of cytosolic [Zn(2+) ] in response to external supply suggests the involvement of high- and low-affinity uptake systems as well as release from internal stores. In this study, we demonstrate that the combination of genetically encoded FRET sensors and microfluidics provides an attractive tool to monitor the dynamics of cellular metal ion concentrations over a wide concentration range in root cells

Evanescent field biosensor using polymer slab waveguide-based cartridges for the optical detection of nanoparticles

We present a polymer optical waveguide integration technology for the detection of nanoparticles in an evanescent field (EF)-based biosensor. Polymer waveguides together with their light coupling structures are designed to be integrated in a novel cartridge concept which will eventually lead to cost-effective, rapid, and easy-to-use point-of-care testing (POCT). The selected slab waveguides generate a homogeneous and well-defined EF, illuminating magnetic nanoparticles that are used as optical contrast labels and are measured using dark-field microscopy. The nanoparticles quantitatively bind to the sensor surface in the presence of target molecules, mediated by antibodies. Compatibility of the waveguide materials with these biomolecules is therefore a strong requirement. When designing a biosensor for POCT, it is also necessary to consider fabrication and manipulation tolerances, targeting the compatibility with mass-production technologies. In this context, polymer optics offer unique advantages, and in combination with the optical contrast labels a very high sensitivity can be achieved. The sensing concept was assessed by comparing various commercially available polymer materials (LightLink, Ormocer, Epocore/Epoclad) in terms of waveguide design and fabrication, optical performance (detection of nanoparticles), and biological compatibility and performance as a sensor surface in an immuno-assay for cardiac troponin I