Single fluorescent protein-based biosensors

This type of biosensors relies on a single fluorescent protein, where ligand-binding modulates the optical properties of the chromophore, typically the protonation state3. These sensors are most useful when ligand-binding results in a spectral shift, thus generating a signal that can be ratioed at two different wavelengths.

Examples: indicators for...

...pH

In general, the fluorescence of AFPs is quenched reversibly by moderate acidification5. This intrinsic pH sensitivity varies between different mutants and can be exploited to measure the ambient pH.

Both intensity-modulated and ratiometric pH-sensitive variants of GFP have been engineered and fused to a vesicle membrane protein to monitor vesicle exocytosis and recycling5. These "synapto-pHluorins"36 report synaptic neurotransmitter secretion by detecting the abrupt pH change that occurs when the acidic interior of the vesicle (pH ~5) is exposed to the outside of the cell (pH ~7) on fusion to the plasma membrane. Several rounds of mutagenesis generated two classes of pH-sensitive fluorescent proteins ("pHluorins"), termed "ratiometric" and "ecliptic"36. Ratiometric pHluorin contains mutations S202H, E132D, S147E, N149L, N164I, K166Q, I167V, R168H and L220F. The protein displays a reversible excitation ratio change between pH 7.5 and 5.5, with a response time of <20 ms. Ecliptic pHluorin, by contrast, gradually loses fluorescence as pH is lowered, until at pH values of <6.0 the excitation peak at 475 nm vanishes; in an environment of pH <6.0, the protein is therefore invisible (eclipsed) under 475 nm excitation. These changes are reversible within <20 ms after returning to neutral pH. Ecliptic pHluorin carries the substitutions S147D, N149Q, T161I, S202F, Q204T and A206T.

Some YFPs have particularly high pKas, in the range of 7 to 8, which makes them very useful for monitoring the pH of the cytosol and the mitochondrial matrix37. These same YFPs are quenched by halide ions (see below).

A major drawback for use in vivo of many of the proposed biosensors is that the analyte concentration often affects only the intensity of fluorescence38. Therefore, it is difficult to determine whether observed changes in fluorescence are due to changes in analyte concentration or GFP-based indicator concentration, and this is further complicated by inevitable photobleaching (i.e. the irreversible destruction, by any one of a number of different mechanisms, of a fluorophore that is under illumination5). Consequently, an independent means of measuring indicator concentration is required in most cellular applications.

Ratiometric probes, which have multiple excitation or emission maxima that show opposing changes in fluorescence excitation or emission in response to changes in analyte concentration, are potentially much more useful. Ratiometric measurements can reduce or eliminate distortions of data caused by photobleaching, indicator concentration, variable cell thickness, illumination stability, excitation path length, and nonuniform indicator distribution within cells or between groups of cells38.

[From Ref. 38] Fluorescence emission spectrum of deGFP1 for excitation at 400 nm.

Wild-type GFP can in principle provide a basis on which to construct indicators suitable for either emission or excitation ratiometric measurements38. The excitation spectrum of wild type is characterized by two major absorption bands (lmax= 397 nm and lmax= 475 nm) attributed to an internal ground-state equilibrium between the neutral and anionic forms of the chromophore, respectively6,13. The excited state of the neutral chromophore would be expected to fluoresce in the blue and the anionic chromophore in the green; however, in wild type the blue fluorescence is very weak and predominantly green fluorescence is observed upon excitation at either absorption maximum. This is because fast internal proton transfer from the excited state of the neutral chromophore produces the anion and results in green fluorescence8.

Although in wild-type GFP the ratio of the two absorption bands does not depend strongly on pH or ionic strength in the physiological range, pH-sensitive GFP variants that are ratiometric by excitation have been reported38. These variants retain the two absorption bands characteristic of wild type, but the ratio of the neutral and anionic populations depends strongly on pH. While such characteristics are very promising, two-wavelength excitation measurements show several limitations38.

Fluorescent indicators that are ratiometric by emission are therefore very desirable. An example of such indicators is given by deGPFs (which result from substitution of wild-type residue 65 with threonine and residues 148 and/or 203 with cysteine)38: these variants display dual emission and provide improved ratiometric pH indicators. In fact, they respond to pH changes by opposing changes in blue and green emission and have biologically relevant pKas, ranging from 6.8 to 8.0. Emission switches from a green form (lmax about 515 nm) to a blue form (lmax about 460 nm) with acidifying pH. Crystal structure analyses of one of these GFP variants (deGFP1) at high and low pH were performed to elucidate the basis for the dual emission characteristics38. At low pH, the structure does not contain a hydrogen bond network that would support rapid transfer of a proton from the excited state of the neutral chromophore to a suitable acceptor; hence blue emission is observed. At high pH, backbone rearrangements induced by changes in the associated hydrogen bond network permit excited state proton transfer from the excited state of the neutral chromophore to the bulk solvent via Ser147 and bound water molecules, resulting in green emission from the anionic chromophore38.

...halides5,39

The fluorescence emission of YFPs has been shown to respond rapidly and reversibly to changes in the concentration of some small anions such as halides; this allows for the use of YFPs as genetically encodable Cl- sensors that may be targeted to specific organelles in living cells.

The halide-binding site is found near van der Waals contact with the chromophore imidazolinone oxygen atom, in a small buried cavity adjacent to Arg96, which provides electrostatic stabilization. The halide ion is hydrogen bonded to the phenol group of T203Y, consistent with a mutational analysis that indicates that T203Y is indispensible for tight binding.

Halide ions bind selectively in the order F- > I- > Cl- > Br- (with a stronger level of interaction at low pH values) and raise the chromophore pKa values, since delocalization of the phenolate negative charge over the chromophore skeleton is suppressed. The absorbance spectrum of YFP is a function of halide concentration, with conversion of the chromophore anion (lmax= 514 nm) to the neutral form (lmax= 392 nm) upon addition of halide. Only the anion is fluorescent in the YFPs, leading to suppression of fluorescence as halide concentration is increased.

...Ca2+

Conformationally responsive elements, which range from short peptide motifs to full-length proteins, can be inserted into AFPs5. The best-characterized example is the insertion of calmodulin (CaM) in place of Tyr145 of YFP, which results in Ca2+ sensors (known as camgaroos) that increase fluorescence sevenfold on binding of Ca2+ 5,24. Camgaroos have proved to be useful for imaging Ca2+ inside mitochondria40.

An important topological variation of this is to insert an AFP inside a conformationally responsive protein or pair of protein domains5. In a circularly permuted AFP (cpAFP), the original amino and carboxyl termini are joined by a flexible linker, and new amino and carboxyl termini are introduced at one of several possible locations near the chromophore. Such permutation increases the flexibility and optical responsiveness to stresses that are applied on the new termini5. Insertion of circularly permuted GFP (cpGFP) between CaM and M13 (a 26-residue peptide derived from the CaM-binding region of the skeletal muscle myosin light-chain kinase27, which binds calmodulin in a Ca2+-dependent fashion) yields Ca2+ indicators that are known as GCaMP28 or pericams27 (the latter contain cpYFP). The inclusion of M13 increases the apparent Ca2+ affinity of the CaM by allowing the formation of ternary complexes, so that these molecules are more sensitive than camgaroos to small elevations in physiological levels of Ca2+ 5. In fact, the Ca2+-bound CaM and M13 peptide form a stable and compact complex; the fluorescence properties of the chimeric protein change according to the Ca2+-dependent interaction between CaM and M13.

About pericams, the behavior of the spectral changes has been varied by mutations which could affect the proton network of the cpYFPs27. Some pericam variants shift their excitation wavelengths on binding of Ca2+, as opposed to just increasing fluorescence, which thereby enables ratiometric observation5,27.

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