Genetically encoded fluorescent biosensors

The aim of fluorescent biosensors for live-cell imaging is to convert a molecular event into an optical signal that can be detected microscopically. Fluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Biosensors based on fluorescent proteins have many advantages over synthetic dyes including construction by genetic manipulation, spontaneously acquired fluorescence and simple delivery and intracellular targeting. A potential drawback of FP - based biosensors are their bulkiness, rate-limiting chromophore formation and photobleaching. The strategy is premised on the idea that a cellular signal can induce a change in the FP thus altering fluorescence.

37 1. Introduction

1.4.2.1 Single FP biosensors

The design of single fluorophore biosensors aims at reversibly destabilizing a bright chromophore state. Single FPs exhibit natural pH sensitivity as the deprotonation of the chromophore correlates with high fluorescence quantum yield and an absorption shift from 405 to 488 nm.

This direct correlation was exploited for the design of pH-sensors called pHluorins. The engineering of the redox-sensor roGFP required mutagenesis that renders chromophore protonation dependent on the redox status of the FP was used (Dooley et al. 2004). roGFP contains two surface-exposed cysteines that form disulfide bonds which promote chromophore protonation. Ratios from excitation at 405 and 488 nm indicate the extent of roGFP protonation and oxidation.

In order to render biophysical properties dependent on an analyte molecule, e.g. H2O2 or Calcium, a detecting protein is fused to an FP endowing the resulting biosensor with a specific functionality. Detector proteins can be inserted into specific sites of the FP barrel while preserving its fluorescent properties (G S Baird et al. 2000). Circularly permuted FPs (cpFPs) have been opened at these positions and rejoined at the original N- and C-termini thereby destabilizing the deprotonated state of the fluorophore (Nakai et al., 2001). Chimeric proteins GCaMP and pericam contain a cpGFP fused to calmodulin (CaM) and its target peptide, M13. Their spectral properties change reversibly with Ca2+ concentration probably due to the interaction between CaM and M13, leading to an alteration of the environment surrounding the chromophore.

1.4.2.2 FRET biosensors

A donor chromophore in its excited state can transfer energy to an acceptor chromophore in a mechanism called Förster (or Fluorescence) Resonance Energy Transfer (FRET). This non-radiative transfer can be described by a dipole-dipole coupling of the chromophores. Effective energy transfer depends on the distance of the two chromophores (usually less than 10 nm) and on the Förster Radius R0. R0 describes the distance between the donor and acceptor at which 50% of the excited state energy is transferred from the donor to the

38 1. Introduction

acceptor. It is dependent on the quantum yield of the donor in the absence of the acceptor (QD), the refractive index (n) of the medium, the relative orientation of the transition dipoles (j2), and the spectral overlap integral (J). The most sensitive range of the distance between the fluorophores R is 0.7–1.4 R0, corresponding to 90–10% FRET efficiency. R0 is usually between 40 Å to 70 Å, hence, protein conformational change in this range is ideal for the largest dynamic range in FRET biosensors. The FRET signal of a given FRET pair is also modulated by the orientation of the chromophores. For a FRET pair that is not restricted in its movement, the orientation factor is defined by an averaged value of 2/3.

Figure 11 FRET principle. (A) FRET equations EFRET=FRET-efficiency, R0=Förster radius [nm],

r=distance [nm], R06=Förster distance [cm6], n=refractive index of the medium, QD=quantum yield of

donor in the absence of acceptor, J(λ)=overlap integral [M-1 cm3], FD(λ)=normalized fluorescence

intensity of the donor, εA(λ)= extinction coefficient of the acceptor at λ *M-1 cm-1], λ= wavelength

[cm]. (B) Simplified Jablonski energy diagram for FRET. Donor excitation (blue) acceptor emission (green). (C) FRET efficiency decreases with the distance of the fluorophores and depends on the Förster radius R0 of the respective FRET pair. (D) FRET rate depends on the orientation ot the

transtition dipole moments of donor and acceptor relative to the vector joining their centers. (Maurel Damien, S. Jähnichen)

39 1. Introduction

Dependency of FRET efficiency on orientation and distance of the donor/acceptor couple can be exploited for the design of biosensors if a detector protein is incorporated. FRET biosensors have been engineered to detect a broad range of molecular events such as protein-binding interactions, conformational changes, catalytic functions and concentration of biomolecules including signaling molecules, cellular metabolites and nucleic acids.

Different FRET biosensor strategies can be distinguished (Figure 12). Bipartite FRET biosensors detect intermolecular interactions and comprise split detector domains that are separately fused to donor or acceptor FP. Intramolecular FRET sensors undergo structural changes in response to the analyte as the FRET pair is covalently linked via a detector domain. FRET protease biosensors consist of a protease cleavage site separating the donor from the acceptor.

Figure 12 FRET biosensor strategies. (A) FRET between a separate sensory domain and substrate fused to CFP and YFP is induced upon binding. (B) FRET biosensor featuring a single sensory domain and effector ligand that induces a conformational change. (C) Proximity-based FRET sensor based on intramolecular substrate-sensory domain interaction. (D) The FRET pair is separated by a protease cleavage site. CFPs (cyan), YFPs (yellow), sensory domains (flesh-colored), effector ligand (spheres or ellipses) (T.B. Gines, M. W. Davidson, Zeiss tutorial)

Spectral microscopy and confocal microscopy are applied to detect FRET efficiency Energy transfer results in decreased quantum yield and lifetime of donor fluorescence, the increase

40 1. Introduction

of acceptor fluorescence brightness, and a loss of emission light polarization. These parameters can be measured by stimulated emission (SE) and fluorescence lifetime imaging (FLIM), respectively.

Desirable spectroscopic properties of the FRET pair include sufficient separation in excitation spectra for selective stimulation of the donor GFP, an overlap between the emission spectrum of the donor and the excitation spectrum of the acceptor to obtain efficient energy transfer and reasonable separation in emission spectra between donor and acceptor FPs. Regarding cellular sensor performance, parameters such as bleaching, tissue autofluorescence and pH sensitivity have to be taken into account. Currently, CFP donor and YFP acceptor are the most commonly used FPs to form a FRET pair.