Fluorescent Proteins – Introduction and Photo Spectral Characteristics

April 10, 2012

The prospects of fluorescence microscopy changed dramatically with the discovery of fluorescent proteins in the 1950s. The starting point was the detection of the jellyfish Aequorea victoria green fluorescent protein (GFP) by Osamo Shimomura. Hundreds of GFP mutants later, the range of fluorescent proteins reaches from the blue to the red spectrum. The emergence of more fluorescent proteins coming from other species like Anthozoa shifted emission wavelengths even to the far-red region. This palette of possibilities offers scientists looking for a suitable fluorescing protein tag for their requirements a great choice. This article gives an overview of recent alternatives and provides a clearly arranged table with all relevant spectral characteristics.

Fig. 1: Molecular structure of Green Fluorescent Protein (GFP)

Fluorescent Proteins from A. victoria

Spectral properties of GFP or its variants lie in the amino acid structure forming the chromophore (Figure 1). This can be the three amino acids at positions 65–67 or residues close to this location (e.g. YFP). Besides the main mutations concerning the chromophore, research was also conducted on other site-directed mutagenesis to improve other factors like protein maturation and expression in heterologous cell systems (e.g. codon usage, protein folding at physiological temperature). Note that A. victoria is a relatively primitive marine organism with no body heat system.

Even if GFP is one of the most popular FPs because of its brightness and high photostability, it has two main disadvantages. These are a certain sensitivity to pH and a slight tendency to dimerize. Dimerization or oligomerization is a problem of many FPs. Their preference to agglutinate with each other can produce artifacts or misinterpretations concerning location and function of the fused protein. But scientists came up with some answers to that problem, too. Mutations at critical positions (F223R, L221K and A206K) where non-polar amino acids are replaced by hydrophilic ones show reduced dimerization. All the genetic changes which lead to an improvement of spectral as well as practical characteristics are summarized under the name of "enhanced" FPs.

In the case of wtGFP, enhancements lead to an EGFP (enhanced GFP) with a single excitation peak at 488 nm instead of the previous complex absorption spectrum at 395 nm and 475 nm. The first mutated version of wtGFP (the S65T mutant) developed by Roger Tsien et al. was five times brighter than the original and showed a shorter maturation  time. Together with better maturation efficiency at 37°C, based on another mutation (F64L), this plays an important role for people looking at living cells.

A very interesting GFP variant with one of the largest Stokes shifts is Sapphire. A mutation at a position close to the chromophore (T203I) leads to an alteration of the excitation maximum to 399 nm and the emission maximum to 511 nm. This is a Stokes shift of 112 nm. Emerald is another GFP modification with improved photostability and brightness and more efficient folding in mammalian cells.

Whereas all the green fluorescent proteins have a relatively high brightness, blue fluorescent proteins normally suffer from reduced emission intensity in microscopic applications. Nevertheless, they are used in optical assays because of other spectral properties. EBFP (Enhanced Blue Fluorescent Protein) was constructed by several rounds of mutating wtGFP. The first one (Y66H) skipped the emission peak from the green to the blue spectrum. More mutations followed, producing a protein with an excitation maximum at 380 nm and an emission maximum at 448 nm. These spectral properties make it a partner for EGFP in FRET microscopy. Recent blue fluorescent proteins with higher quantum yields and better photostability are Azurite, SBFP2 and EBFP2. A promising EBFP successor is a protein named Sirius which became popular because of its very high tolerance to pH (stable from pH 3–9) and its reputation for being the fluorescent protein with the shortest emission wavelength to date.

A second “blue” class of GFP variants is formed by cyan fluorescent proteins: CFPs. Substitution of tyrosine with tryptophan (Y66W) and further genetic alterations lead to a fluorochrome with improved brightness and photostability. This ECFP has a bimodal excitation and emission spectrum at 433/445 nm and 475/503 nm. Brightness is only about 40 % of that of EGFP. A prominent ECFP variant is Cerulean, which has a higher extinction coefficient and quantum yield. It is 1.5 times brighter than ECFP and is used as a FRET partner with YFP.

A GFP mutation not directly altering one of the three central amino acids in the chromophore resulted in the rise of yellow fluorescent proteins. YFPs have a common threonine at position 203 which is exchanged by a tyrosine (T203Y). This amino acid is part of the β-barrel and lies in close proximity to the chromophore. Compared to GFP, excitation and emission properties have been shifted to longer wavelengths with excitation and emission maxima at 514 nm and 527 nm (EYFP). One characteristic of EYFP is its pH sensitivity. At pH 6.5 EYFP has only about 50 % of its fluorescence, which is not always a disadvantage. When it comes to pH measurement (e.g. of vesicles, endosomes etc.), EYFP can be used as an indicator. Interestingly, a further mutation (Q69M) developed a better acid stability and dramatically improved brightness (75 % brighter than EGFP). This protein, which still has a poor photostability compared to EGFP, was called Citrine. Another YFP mutant (F46L) showed a drastically faster maturation speed and also an improved pH resistance.  This protein was named Venus and is a frequent FRET acceptor with Cerulean.

Fluorescent Proteins from Anthozoa

As one can see from the first section, most FPs, coming from the jellyfish A. victoria emit light in the blue to the yellow spectrum. Red fluorescing proteins are missing. The Russian scientist Sergey A. Lukyanov closed the gap when he discovered FPs in Anthozoa. Red FPs have a big advantage over others because there is much less autofluorescence in the cells in the red spectrum. Furthermore they are excited by longer wavelengths, which is good for living cells. Light of shorter wavelengths does far more damage to specimens. A further general advantage of coral proteins over jellyfish proteins is their efficient maturation at 37°C. Whereas A. victoria GFP and derivates had to be genetically modified to fold in the right way, anthozoan proteins mature without the necessity of molecular engineering. This might be due to warmer water temperatures of their biotope.

Fig. 2: Molecular structure of DsRed

The first and still one of the most used FPs discovered in Anthozoa is DsRed. The name is derived from the sea anemone Discosoma striata. DsRed has an excitation maximum at 558 nm and an emission peak at 583 nm. However, the first euphoria stagnated when structural information was published. DsRed maturates much more slowly than jellyfish FPs and has an intermediate chromophore stage. This stage emits light in the green spectrum and causes overlap with other FPs. As already mentioned in the GFP section, DsRed also has another problem. The red fluorescing sea anemone protein is an obligate tetramer with the predisposition to form oligomers. This can lead to misinterpretations concerning location and function of the fused protein. In general Anthozoa FPs have a similar structure to that of Aequorea FPs. The chromophore is hidden in a β-barrel structure with the size of 4 nm x 3 nm (height x diameter). The difference lies in the more elliptical appearance of the Anthozoa β-barrel (Figure 2).

In parallel to GFP "evolution", researchers started to modify the original DsRed to overcome its structural deficiencies. The second DsRed generation – DsRed2 – has a reduced oligomer formation tendency and a faster maturation speed, minimizing the green light emitting intermediate stage. Further mutagenesis led to a red FP which had totally lost its tetramer status but also forfeited some of its quantum yield (25 % of DsRed2). This work of Tsien et al. was the first monomeric red fluorescent protein and therefore called mRFP1.

This mRFP1 was then the starting point for the creation of a group of six monomeric FPs collectively called "mFruit". Their individual names are derived from their emission color: mHoneydew, mBanana, mOrange, mTangerine, mStrawberry and mCherry. mCherry is the most useful of these FPs, emitting light in the range of 610 nm with a brightness of 50 % that of EGFP.

The brightest FP so far is a follower of the mFruit faction and has the name tdTomato. As far as it was genetically changed, dTomato was an obligate dimer. But dimerization was avoided by putting the two dimerization partners in one molecule. Two dTomato units were coupled by a 12 amino acid linker, creating the tandem dimer FP tdTomato with an emission maximum at 581 nm and a photostability in the highest region.

The emission spectrum was shifted further into the far-red area (630 nm – 700 nm) when another mFruit successor was created. mPlum is the mFruit member with the deepest red emission of any of the mFruit proteins at 649 nm.

The amount of scientifically used green fluorescing anthozoan proteins is very small, which is not very surprising in view of the availability of the well-known and user-friendly A. victoria GFP. It is obvious that people did not see any necessity to establish a new green fluorescent protein. Nevertheless there are some, like a bright fluorescent protein from the stony coral Galaxeidae, Azami Green. Its sequence homology with EGFP is interestingly less than 6 %.

An anthozoan FP which has had a high impact on deep tissue imaging is Katushka. Applying mutagenesis on RFPs coming from E. quadricolor, Katushka was identified as a dimeric protein with an emission maximum at 635 nm and the highest brightness levels of all deep-red fluorescing proteins. The monomeric form of Katushka is called mKate and was supplied with a higher brightness later on, resulting in mKate2.

In summary, all the fluorescent proteins which are used today for microscopic applications were derived from primitive marine creatures. Table 1 comprises the most important ones together with their relevant spectral characteristics like excitation and emission maxima, photostability, quantum yield and brightness.

Outlook

What turned out to be a very interesting story was the discovery of a FP expressed by a vertebrate. Amphioxus, a small fish-like marine chordate, produces AmphiGFP at its anterior side. The sequence analysis of that FP predicts a typical β-barrel structure and seems to be related to CopGFP of the copepod Pontellina plumata (Crustacea). This finding showed that the phenomenon of fluorescence is not restricted to primitive invertebrates, but can also be found in animals of higher evolutionary stages. Furthermore this discovery shows the ongoing process of fluorescent protein finding, manipulation and enhancement which is still a topical issue of researchers. This fact corroborates the importance and high impact of fluorescent proteins in recent and future life science research.

Spectral Characteristics of Fluorescent Proteins

Table 1: Fluorescent Proteins, Source: http://flowcyt.salk.edu/fluo.html

Table 1: Fluorescent Proteins, Source: http://flowcyt.salk.edu/fluo.html
Ex: Peak excitation wavelength (nm)
Em: Peak emission wavelength (nm)
MW: Molecular weight
QY: Quantum yield
BR: Brightness; Extinction coefficient * Quantum yield/1,000
PS: Photostability; time to 50 % brightness (sec)

Fluorescent Proteins

 

Ex (nm)

Em (nm)

MW

QY

BR

PS

Source

Other

Y66H

360

442

           

Y66F

360

508

           

EBFP

380

440

 

0.18

9

 

Addgene

Monomer

EBFP2

383

448

 

0.56

18

 

Addgene

Monomer

Azurite

383

447

 

0.55

14

   

Monomer

GFPuv

385

508

           

T-Sapphire

399

511

 

0.60

26

25

 

Weak dimer

TagBFP

402

457

26 k

0.63

33

++

Evrogen

Monomer

Cerulean

433

475

 

0.62

27

36

 

Weak dimer

mCFP

433

475

 

0.40

13

64

 

Monomer

ECFP

434

477

 

0.15

3

     

CyPet

435

477

 

0.51

18

59

 

Weak dimer

Y66W

436

485

           

dKeima-Red

440

616

 

0.31

8

 

MBL

Dimer

mKeima-Red

440

620

 

0.24

3

 

MBL

Monomer

TagCFP

458

480

 

0.57

29

 

Evrogen

Dimer

AmCyan1

458

489

 

0.75

29

 

Clontech

Tetramer

mTFP1 (Teal)

462

492

 

0.85

54

   

Dimer

S65A

471

504

           

Midoriishi-Cyan

472

495

 

0.9

25

 

MBL

Dimer

Wild Type GFP

396,475

508

26 k

0.77

16

     

S65C

479

507

           

TurboGFP

482

502

26 k

0.53

37

 

Evrogen

Dimer

TagGFP

482

505

27 k

0.59

34

++

Evrogen

Monomer

TagGFP2

483

506

27 k

0.6

34

++

Evrogen

Monomer

AcGFP1

484

510

27 k

0.82

27

 

Clontech

 

S65L

484

510

           

Emerald

487

509

 

0.68

39

0.69

Invitrogen

Weak dimer

S65T

488

511

           

EGFP

488

507

26 k

0.60

34

174

Addgene

Weak dimer

Azami-Green

492

505

 

0.74

41

 

MBL

Tetramer (monomeric available)

ZsGreen1

493

505

105 k

0.91

40

 

Clontech

Tetramer

Dronpa-Green

503

518

 

0.85

81

 

MBL

Photoswitchable

TagYFP

508

524

27 k

0.62

47

 

Evrogen

Monomer

EYFP

514

527

26 k

0.61

51

60

 

Weak dimer

Topaz

514

527

 

0.60

57

   

Monomer

Venus

515

528

 

0.57

53

15

 

Weak dimer

mCitrine

516

529

 

0.76

59

49

 

Monomer

>YPet

517

530

 

0.77

80

49

 

Weak dimer

TurboYFP

525

538

26 k

0.53

56

 

Evrogen

Dimer

PhiYFP

525

537

26.8 k

0.40

52

++

Evrogen

Weak dimer

PhiYFP-m

525

537

26.8 k

0.39

48

++

Evrogen

Monomer

ZsYellow1

529

539

 

0.65

13

 

Clontech

Tetramer

mBanana

540

553

 

0.70

4

 

Clontech

Monomer

Kusabira-Orange

548

559

 

0.60

31

 

MBL

Monomer

mOrange

548

562

 

0.69

49

9

 

Monomer

mOrange2

549

565

 

0.60

35

 

Clontech

Monomer

mKO

548

559

 

0.60

31

122

 

Monomer

TurboRFP

553

574

26 k

0.67

62

 

Evrogen

Dimer

tdTomato

554

581

 

0.69

95

98

Clontech

Tandem dimer

DsRed-Express2

554

591

 

0.42

15

 

Clontech

 

TagRFP

555

584

27 k

0.48

48

 

Evrogen

Monomer

DsRed monomer

557

592

~28 k

0.1

3.5

16

Clontech

Monomer

DsRed2 ("RFP")

563

582

~110 k

0.55

24

 

Clontech

 

mStrawberry

574

596

 

0.29

26

15

Clontech

Monomer

TurboFP602

574

602

26 k

0.35

26

 

Evrogen

Dimer

AsRed2

576

592

~110 k

0.21

13

 

Clontech

Tetramer

mRFP1

584

607

~30 k

0.25

   

Tsien lab

Monomer

J-Red

584

610

 

0.20

8.8

13

 

Dimer

mCherry

587

610

 

0.22

16

96

Clontech

Monomer

HcRed1

588

618

~52 k

0.03

0.6

 

Clontech

Dimer

mKate2

588

633

26 k

0.40

25

+

Evrogen

Monomer

Katushka (TurboFP635)

588

635

26 k

0.34

22

++

Evrogen

Dimer

mKate (TagFP635)

588

635

 

0.30

15

 

Evrogen

Monomer

TurboFP635

588

635

26 k

0.34

22

 

Evrogen

Dimer

mPlum

590

649

 

0.10

4.1

53

Clontech

 

mRaspberry

598

625

 

0.15

13

 

Clontech

Monomer; faster photobleach than mPlum

mNeptune

600

650

 

0.20

13

 

Tsien Lab

Monomer

E2-Crimson

611

646

 

0.23

29

 

Clontech

 

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