Illustrate your essay with specific examples.
The following fluorescence excitation and emission patterns (solid and dashed lines respectively) are of the six major classes of GFP mutants and the wild type. The actual GFPs depicted are: a) wild type (Class 1); b) Emerald (Class 2); c) H9-40 (Class 3); d) Topaz (Class 4); e) W1B (Class 5) and f) P4-3 (Class 6).
Actual wavelengths of excitation (λexc) and emission (λem) are given in the following
a. Discuss why are the fluorescent proteins from Class 2 and Class 6 those most widely used in FRET systems. (7)
If the GFP from class 6 is used as the donor, whilst the GFP of class 2 is used as the acceptor. The emission wavelength of the donor (447 nm) overlaps fairly well with the excitation wavelength of the acceptor (489 nm). It is these two classes of GFP whose emission excitation spectra overlap the best.
A good answer is likely to describe how other pairs would not fit with this scheme.
b. The highest value for R0 (distance at which FRET is 50% efficient) between two GFP is 60 angstrom between H9-40 and Topaz, however this FRET pair has not been very successful. Discuss why this could be? (6)
The emission spectra of Topaz and of H9-40 are too close to each other to allow for good discrimination.
c. How would a FRET system work? What would be the function of the Class 2 GFP and the Class 6 GFP? (4)
FRET – Fluorescence Resonance Energy Transfer, it is a phenomenon that occurs when two fluorophores are in close proximity and the emission spectrum of one fluorophore, the donor overlaps the excitation spectrum of the second fluorophore, the acceptor.
Under these conditions excitation of the donor can produce emission from the acceptor at the expense of the emission from the donor. Anything that changes the distance or the configuration of the two fluorophores will change the efficiency of FRET.
The class 6 GFP would be the donor (since the excitation/emission spectra is lowest) and the class 2 GFP would be the acceptor.
d. Describe a FRET system that uses GFP as an active indicator. Discuss the advantages and disadvantages of such a system. (8)
There are many uses of FRET. These include, but are not limited to: enzymatic action, calcium sensitivity or protein interactions. Advantages of these systems may include high resolution, use in a wide variety of organisms, can be tissue specific. Disadvantages may include gene transfection required, GFPs must remain functional, rare or trace interactions may be hard to detect.
a) Discuss why are the fluorescent proteins from Class 2 and Class 6 those most widely used in FRET systems. (7)
The parameters affecting FRET efficiency are:
- Distance between the donor and the acceptor
- Spectral overlap of the donor emission spectrum and the acceptor absorption spectrum.
- Relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment
The two most widely used FRET systems are the Class 6 (BFP) – Class 2 (Phenolate) system and the Class 5 (CFP) – Class 4 (YFP) system. Class 2 and Class 6 proteins are used because:
- Emerald (Class 2 GFP) is the best choice for live-cell imaging of FRET reporters in the green colour class (Kremers, Piston, Davidson). It has improved the protein folding, expression at 37°C and brightness. The emission wavelength of Class 6 proteins (447 nm) overlaps considerably with the excitation wavelength of Class 2 proteins (488 nm).
- Class 6 (BFPs) as donors and Class 2 (phenolate anion) proteins as receptors are viable FRET systems because of their distinct wavelengths(Kremers, Piston, Davidson). The R0 for the system is 40- 43 A°.
- A landmark experiment by Tsein and Heim using BFP and eGFP (Tsein and Heim, 1996; Tsein, 1997) led to a series of other experiments to determine mutants.
- Easy availability from commercial suppliers.
- This system does not have an extenuated tail in its emission spectra like the one present in the emission spectrum of CFP.
It has been reported that BFPs have the poor extinction coefficients, quantum yields, and photostabilities. The CFP- YFP FRET systems have hence been proposed as alternatives(Miyawaki et al, 1997; Pollok and Heim, 1999).
b) The highest value for R0 (distance at which FRET is 50% efficient) between two GFP is 60 angstrom between H9-40 and Topaz, however this FRET pair has not been very successful. Discuss why this could be? (6)
A very high value of Ro (Förster radius) indicates a greater range of distances for which the FRET value would be close to maximal.
A FRET system comprising of H9-40 and Topaz has not met with great success because the emission spectra of H9-40 (511 nm) and Topaz (527nm) are very close to each other thus preventing an easy distinction between the two emission spectra (Tsien, 1998). If the two emission spectra can not be distinguished from each other, the identification of the biochemical activity that ultimately leads to the emission from the acceptor fluorophore of the FRET system is difficult. Many FRET systems are employed to elucidate dynamic processes like protein-protein interactions. While one protein is labelled with the donor fluorophore, the other is labelled with the acceptor fluorophore. Upon association of the biomolecules the fluorophores undergo intermolecular FRET. When the two biomolecules are at a distance, the emission observed would be of an individual fluorophore. The emission spectra thus reveal association or dissociation. Since the emission spectra of H9-40 and Topaz overlap, this distinction is not possible.
c) How would a FRET system work? What would be the function of the Class 2 GFP and the Class 6 GFP? (4)
FRET is Förster/ Fluorescence Resonance Energy Transfer. The energy transfer is non-radiative in nature. Energy transfer occurs from a donor to an acceptor chromophore. The distance between the two fluorophores is 8-10nm or less and the energy transfer occurs through dipole-dipole coupling.
If a suitable acceptor is present, the donor fluorophore can transfer excited state energy directly to the acceptor without emitting a photon (violet arrow) i.e., through a virtual photon. This emission spectrum from the donor fluorophore has similar characteristics to those of the emission spectrum of the acceptor fluorophore. The FRET efficiency would be altered with change of the distance between the two fluorophores or their configurations.
One of the most used FRET systems is Class 2- Class 6 system where the former acts as acceptor and the latter as donor. The emission wavelength of Class 6 proteins (447 nm) overlaps considerably with the excitation wavelength of Class 2 proteins (488 nm). The efficiency of a FRET system is high. The emission wavelengths of the two species are widely separated at 509 for Class 6 proteins and 448 nm for Class 2 proteins.
It is hence possible to obtain clear results from the FRET system consisting of Class 2 and Class 6 proteins.
d) Describe a FRET system that uses GFP as an active indicator. Discuss the advantages and disadvantages of such a system. (8)
GFP was discovered as a companion to a chemiluminescent protein called aequorin by Shimomura et al. from Aequorea jellyfish (Shimomura et al, 1962).
Several FRET systems employing GFP as an active indicator for detection of:
- assays for protease action(Tsien and Heim,1996; Mitra, Silva and Youvan, 1996)
- Ca2+ sensors (Romoser, Hinkle and Persechini, 1997; Miyawaki et al, 1997)
- transcription factor dimerization (Periasamy, Kay and Day, 1997)
Development of Ca2+ sensors
These were developed by Miyawaki(Miyawaki et al, 1997) and Rosomer(Romoser, Hinkle and Persechini, 1997) independently. These could detect changes and respond dynamically.
Sensors developed by Rosomer et al :
Class 6 BFP and class 2 GFP mutants were used. FRET between the two entities was facilitated with a 26-residue spacer containing the calmodulin (CaM)-binding domain from avian smooth muscle myosin light chain kinase. The length of the space enabled the dimerization of the mutants. Addition of Ca2+-CaM disrupted FRET because of prevention of the dimerization of mutants.
Bacterially expressed recombinant protein was injected into individual HEK- 293 cells. The decrease in emission at 510nm was recorded upon increase in the cytosolic Ca2+ concentration. It was concluded that the response of the indicator in cells was limited by CaM availability and that the indicator is responsive to the Ca2+-CaM complex and not mere Ca2+ entities.
Sensors developed by Miyawaki et al
Class 5 Cyan-Fluorescent Protein (CFP) and Class 4 Yellow-Fluorescent Protein (YFP) were used. While the former was attached to the N-terminus of CaM, the latter was bound to the C-terminus of M13, the CaM-binding peptide from skeletal muscle myosin light chain kinase. The two complexes could either be fused via two glycines or left disjointed. FRET increased in this system due to attachment of CaM to M13 after the binding of Ca2+ to the former. The Indicators were introduced via transfection subsequent to the optimization of mammalian expression using mutant GFPs. The brightness of the indicators enabled the transfection. Several organelles were targeted by employing these indicators as the chimera were developed in situ.
- Can be targeted to specific tissues, cells, organelles, or proteins.
- Potential to be employed in measurement of many bioactive species other than Ca2+ subject to the availability of a conformationally sensitive receptor.
- Spatial and temporal resolutions are high.
- Mutagenesis can be readily employed for structural modifications.
- cDNA construction and/or sequence modification is economical and viable for distribution.
- Amenable for use with most organisms.
- Possess good optical properties like visible excitation, emission ratio and high photostability.
- The ability of cells to inherently synthesize GFPs was not exploited by the system developed by Rosomer et al.
- The biological activity of CaM or M13 might disrupt the process.
- Sensors developed by Rosomer et al can be used to detect only cytosolic signals.
- Presence of a significant background signal while illuminating at the donor’s excitation maximum and observing at the acceptor’s emission maximum(Tsein, 1998).