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

Droplet-based screening of phosphate transfer catalysis reveals how epistasis shapes MAP kinase interactions with substrates.

The combination of ultrahigh-throughput screening and sequencing informs on function and intragenic epistasis within combinatorial protein mutant libraries. Establishing a droplet-based, in vitro compartmentalised approach for robust expression and screening of protein kinase cascades (>107 variants/day) allowed us to dissect the intrinsic molecular features of the MKK-ERK signalling pathway, without interference from endogenous cellular components. In a six-residue combinatorial library of the MKK1 docking domain, we identified 29,563 sequence permutations that allow MKK1 to efficiently phosphorylate and activate its downstream target kinase ERK2. A flexibly placed hydrophobic sequence motif emerges which is defined by higher order epistatic interactions between six residues, suggesting synergy that enables high connectivity in the sequence landscape. Through positive epistasis, MKK1 maintains function during mutagenesis, establishing the importance of co-dependent residues in mammalian protein kinase-substrate interactions, and creating a scenario for the evolution of diverse human signalling networks.RCUK | Biotechnology and Biological Sciences Research Council (BBSRC) - BB/M011194/1 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 721613 EC | Horizon 2020 Framework Programme (EU Framework Programme for Research and Innovation H2020) - 659029 EC | EU Framework Programme for Research and Innovation H2020 | H2020 Priority Excellent Science | H2020 European Research Council (H2020 Excellent Science - European Research Council) - 69566

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

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

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

Robust Red FRET Sensors Using Self-Associating Fluorescent Domains

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn²⁺ sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn²⁺ FRET sensors to image Zn²⁺ over a broad concentration range in the same cellular compartment

<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

Quantifying stickiness: thermodynamic characterization of intramolecular domain interactions to guide the design of förster resonance energy transfer sensors

The introduction of weak, hydrophobic interactions between fluorescent protein domains (FPs) can substantially increase the dynamic range (DR) of Forster resonance energy transfer (FRET)-based sensor systems. Here we report a comprehensive thermodynamic characterization of the stability of a range of self-associating FRET pairs. A new method is introduced that allows direct quantification of the stability of weak FP interactions by monitoring intramolecular complex formation as a function of urea concentration. The commonly used S208F mutation stabilized intramolecular FP complex formation by 2.0 kCal/mol when studied in an enhanced cyan FP (ECFP)-linker-enhanced yellow FP (EYFP) fusion protein, whereas a significantly weaker interaction was observed for the homologous Cerulean/Citrine FRET pair (Delta G(o-c)(0) = 0.62 kCal/mol). The latter effect could be attributed to two mutations in Cerulean (Y145A and H148D) that destabilize complex formation with Citrine. Systematic analysis of the contribution of residues 125 and 127 at the dimerization interface in mOrange-linker-mCherry fusion proteins yielded a toolbox of new mOrange-mCherry combinations that allowed tuning of their intramolecular interaction from very weak (Delta G(o-c)(0) = -0.39 kCal/mol) to relatively stable (Delta G(o-c)(0) = 2.2 kCal/mol). The effects of these mutations were also studied by monitoring homodimerization of mCherry variants using fluorescence anisotropy. These mutations affected intramolecular and intermolecular domain interactions similarly, although FP interactions were found to be stronger in the latter. The knowledge thus obtained allowed successful construction of a red-shifted variant of the bile acid FRET sensor BAS-1 by replacement of the self-associating Cerulean-Citrine pair by mOrange-mCherry variants with a similar intramolecular affinity. Our findings thus allow a better understanding of the subtle but important role of intramolecular domain interactions in current FRET sensors and help guide the construction of new sensors using modular design strategie

Improved RAD51 binders through motif shuffling based on the modularity of BRC repeats.

Exchanges of protein sequence modules support leaps in function unavailable through point mutations during evolution. Here we study the role of the two RAD51-interacting modules within the eight binding BRC repeats of BRCA2. We created 64 chimeric repeats by shuffling these modules and measured their binding to RAD51. We found that certain shuffled module combinations were stronger binders than any of the module combinations in the natural repeats. Surprisingly, the contribution from the two modules was poorly correlated with affinities of natural repeats, with a weak BRC8 repeat containing the most effective N-terminal module. The binding of the strongest chimera, BRC8-2, to RAD51 was improved by -2.4 kCal/mol compared to the strongest natural repeat, BRC4. A crystal structure of RAD51:BRC8-2 complex shows an improved interface fit and an extended β-hairpin in this repeat. BRC8-2 was shown to function in human cells, preventing the formation of nuclear RAD51 foci after ionizing radiation.BBSRC (BB/K013629/1), Cancer Research UK (C7905/A25715), EPSRC (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z), MRC (grants MR/K015850/1 and MR/K02292X/1), ERC, H2020 Marie-Curi