Photoactivatable, photoconvertable, and photoswitchable Fluorescent Proteins

June 20, 2012

The terms "photoactivatable, photoconvertable, and photoswitchable Fluorescent Proteins" combine a group of genetically altered fluorescent proteins with very special attributes: They can be activated from a non fluorescent state, they can change their emission spectrum or they are even able to be switched "on and off" for a lot of times. This behavior is due to some distinct photo physical attributes. At the single molecule level many fluorescent proteins show an intrinsic blinking activity which can be influenced by external illumination. Optical highlighter proteins are mutated fluorescence proteins with pronounced emissive and non-emissive respectively new emissive states, what is expressed in the attributes photoactivatable, photoconvertible or photoswitchable. Another interesting type of fluorescent proteins are Fluorescent Timers which are able to change their color over time. The following article will give an overview of a selection of these "extraordinary" fluorescent proteins.

Photoactivatable proteins

Photoactivatable proteins can be "switched on" from a low fluorescent state to a higher fluorescent state. This switch is happening during less than a second by applying a short light pulse in the violet/blue spectrum and allows detection of dynamic cellular processes. Directed photoactivation of molecules in a region of interest (ROI) can be used to monitor the movement of these activated proteins inside the cell. Whereas in other pulse-chase experimental setups like FRAP one cannot distinguish between proteins which are re-entering the ROI and newly synthesized molecules, photoactivation is one way to bypass this problem.

The first photoactivatable protein was a wtGFP variant and had been created by Lippincott-Schwarz and Patterson. It comprised a single point mutation (T203H), which lead to a very low absorbance in the area from 450 to 550 nm. Being photoactivated with the help of violet light, PA-GFP (photoactivatable green fluorescent protein) switches its absorption maximum from 400 to 504 nm. Therefore its fluorescence increases ca. 100-fold when excited with a wavelength of 488 nm, what is reflected in a sharp contrast between activated and non-activated proteins [1].

The underlying process during the activation of PA-GFP seems to be a light-induced decarboxylation of the glutamic acid side chain in residue 222. This loss of a carbon dioxide alters the chromophore configuration from a neutral to an anionic state [2].

Another candidate which should be mentioned here is Phamret (photoactivation-mediated resonance energy transfer), which is a special tandem dimeric fluorescent protein, photoactivated by FRET. Phamret is a fusion protein, consisting of one PA-GFP coupled covalently to its FRET partner ECFP. Exposure to 458 nm wavelength leads to emission of ECFP at 480 nm. Adjacent photoactivation of PA-GFP is performed with a 405 nm laser beam. After that, repeated exposure to 458 nm leads to FRET between the excited ECFP and PA-GFP, resulting in a green fluorescence. So the "inactive" and "active" form can be excited by the same laser wavelength. A negative characteristic could be the size of the protein which is consisting of two fluorescent proteins, possibly evoking sterical problems.

It is noteworthy that photoactivatable proteins often display a reduced brightness compared to EGFP and show a reduced photostability.

Fig. 1: Photoactivatable FPs can be switched "on" from a non fluorescent state in a fluorescent state by irradiation with light in the blue/violet spectrum. Photoconvertable FPs are able to change their fluorescence emission spectrum from one maximum to another. This is also triggered by exposure to light with a certain wavelength. Photoswitchable FPs can be switched "on and off" with the help of light pulses of two different wavelengths. This loop can be executed for up to several hundred times. Fluorescent Timers are altering their emission spectrum by time. With their help it is possible to correlate age and cellular position of a protein e.g.

Photoconvertible proteins

In contrast to photoactivatable proteins, photoconvertible proteins emit fluorescence already in their non-converted state. Therefore it is easier to define your ROI. Photoconversion was discovered in the stony open brain coral Trachyphyllia geoffroyi. Due to their emission attributes, which are shifting from green to red after photoconversion with UV light, the fluorescent protein was given the name Kaede like the leaves of Japanese maple trees [4]. In autumn they are turning their color from green to red. If Kaede is photoconverted with wavelengths between 380 and 400 nm, its emission maximum is shifting from 518 nm to 582 nm. This is displayed in a dramatically altered ratio between green and red fluorescence by the factor 2,000. This conversion is irreversible. Another limiting factor is the tetrameric nature of Kaede, what makes it rather unattractive for live cell imaging studies.

The underlying process of photoconversion is again a light induced process. In this case a histidine residue inside the chromophore (His61-Tyr63-Gly64) is cleaved by irradiation and finally leads to a formation of a highly conjugated dual imidazole ring system. This process is connected to a fluorescence shift to red wavelengths [2].

A red to green convertible FP is coming from the coral Dendronephtytha sp. Its name and the red activatable attribute are mirrored in the expression Dendra [5]. The commercial development Dendra2 is the first monomeric red-to-green photoconvertible protein.

Another widely used optical highlighter is tdEosFP [6] and was primarily isolated from Labophyllia hemprichii, a stony coral. The tandem dimer can convert from green to red fluorescence after near-ultraviolet exposure and is gladly used for superresolution microscopy, what is also true for the monomeric versions mEos and mEos2.

A photoconvertible protein with very similar characteristics to Keade was found in a third stony coral: Favia favus. After near-ultraviolet irradiation tetrameric KikGR changes its fluorescence from green to red, but both in a much brighter way than Kaede. Its commercial variant is called Kikume and can be photoconverted by multiphoton excitation with 750 nm wavelength. Like this it can be used in thick tissue specimens. Mutagenesis of KikGR lead to the monomeric form mKikGR. Together with mEos2 and Dendra2 it is an often used optical highlighter for superresolution imaging.

Photoswitchable dyes

Whereas photoactivation is an irreversible conversion from a non fluorescent state to a fluorescent state, photoswitchable proteins are able to shuttle between both conditions. With the help of light pulses of different wavelengths these fluorescent proteins can be switched on and off for several hundred times without photobleaching. This phenomenon, to switch between fluorescent and dark states, is called photochromism and shows up already at the single molecule level of wtGFP, although to a very low extend.

One well known photoswitchable protein, which is used in super-resolution microscopy, was derived from a stony coral: Dronpa is a monomer and has one absorption maximum at 503 nm due to its anionic, deprotonated chromophore, and a minor absoption maximum at 390 nm due to its neutral, protonated chromophore. Whereas the anionic form has an emission maximum at 518 nm, the neutral form depicts a non fluorescent state. Furthermore there is a cis-trans isomerization connected with the protonation of the chromophore. In the neutral state of the chromophore the Tyr66 side chain is in a trans conformation (s. Figure 2). In its anionic state Tyr66 has a cis conformation. Upon irradiation with a 405 nm laser pulse Dronpa is forced into its fluorescent cis conformation. A 488 nm laser pulse then switches the Dronpa conformation to its non fluorescent trans state. This cycle can be repeated several hundred times.

Figure 2: The photoswitchable protein Dronpa used in super-resolution microscopy

A tetrameric photoswitchable protein with emission maxima in the red region is Kindling FP (KFP1).

Latest efforts to create new optical highlighters came up with a protein which is combining both, photoconversion and photoswitching. IrisFP is a wtEosFP derivate with an on and off switchable state in its green fluorescent as well as in its red fluorescent conformation. In other words, upon irradiation with a high intensity 405 nm laser beam IrisFP is transferred from its green to a red emitting status. Green IrisFP can be switched off with the help of 488 nm laser light. Low intensity laser light at 405 nm switches it on again. On the other hand red IrisFP is switched off with 532 nm laser light and can be switched on again with a 440 nm laser. The adjacent construction of the monomeric form mIrisFP opened the door for further applications [3].

Fluorescent timers

Fluorescent proteins which are changing their emission spectrum by time are called fluorescent timers. This behavior is not the result of pH change, ionic strength or protein concentration but happens independently from these biochemical parameters. In other words the age of the relevant protein can be estimated by its color. With these characteristics it is possible to measure time – and time depending happenings – inside a living cell.

The first fluorescent timer was described by the lab of Sergey A. Lukyanov in the year 2000. This DsRed mutant named FT (for fluorescent timer) alters its emission spectrum from red to green wavelengths during 18 hours. Due to the fact that FT was a tetramer Vladislav Verkhusha saw the necessity to create a monomeric one. Mutagenesis of mCherry lead to 3 more fluorescent timers with different transformation times. These proteins are turning from blue to red in fast, medium or slow time scales but still too slow to measure cellular processes happening during minutes (s. Table 1).

Nevertheless, with the help of fluorescent timers it is possible to watch the behavior of proteins in living cells, both in spatial and temporal resolution. For example differentiation and cellular polarization processes can be observed. Moreover protein transport events or viral assembly can be tracked or gene activity can be monitored. This list of applications surely could be extended and shows the potential use of Fluorescent Timers in the future.

Table 1: Selected Highlighter Proteins and Fluorescent Timers

 Protein

Exmax(nm)

Emmax(nm)

QY

Brightness

pKa

Structure

Miscellaneous

 Photoactivatable
 proteins

 

 

 

 

 

 

Condition of activation

PA-GFP

400
504

515
517

0.13
0.79

2.7
14

 

Monomer

violet

Phamret

458
458

475
517

0.40
0.79

13
14

 

Monomer

violet

 Photoconvertible
 proteins

 

 

 

 

 

 

Condition of conversion

Dendra2

490
553

507
573

0.50
0.55

22
19

6.6
6.9

Monomer

Initial state
violet

Kaede

508
572

518
580

0.88
0.33

87
20

 

Tetramer

Initial state
violet

mEos2

506
573

519
584

0.84
0.55

47
30

5.6
6.4

Monomer

Initial state
violet

mKikGR

505
580

515
591

0.69
0.63

34
18

ND
ND

Monomer

Initial state
violet

 Photoswitchable
 proteins

 

 

 

 

 

 

Condition of activation (quenching)

Dronpa

503

518

0.85

80.8

5.0

Monomer

violet (blue)

Kindling FP (KFP1)

580

600

0.07

4.1

ND

Tetramer

green (450 nm)

 Photoconvertible/
 Photoswitchable
 proteins

 

 

 

 

 

 

 

IrisFP

488
551

516
580

0.43
0.47

22
17

 

Tetramer

 

mIrisFP

486
546

516
578

0.54
0.59

25
19

5.4
7.6

Monomer

violet (dark to green)
cyan (red to dark)
violet (green to red)

 Fluorescent
 timers

 

 

 

 

 

 

Transition time (h)

Slow-FT

402
583

465
604

0.35
0.05

12
4

2.6
4.6

Monomer

9.8
28

Medium-FT

401
579

464
600

0.41
0.08

18
6

2.7
4.7

Monomer

1.2
3.9

Fast-FT

403
583

466
606

0.30
0.09

15
7

2.8
4.1

Monomer

0.25
7.1

mK-Go

500
548

509
561

ND
ND

ND
ND

6.0
4.8

Monomer

10

Exmax: Excitation maximum, Emmax: Emission maximum, QY: Quantum Yield, Brightness is calculated by the product of Quantum Yield and molar extinction coefficient divided by 1,000

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