Green Fluorescent Proteins and Color Variants

1
Green Fluorescent Proteins and Color Variants

• Structure and Applications

Michael Kirberger Department of Chemistry,
Georgia State University, Atlanta, GA, 30303
2
Summary of Topics

1. Overview of GFP
2. Fluorescent Chromophore
3. Importance of GFP
4. Summary of Variants
5. Variants Mutated for Faster Maturation
6. Fluorescent Protein Applications

3
Introduction to GFP

• Green Fluorescent Protein first found to
  fluoresce under UV light in 1955.
• Aequorea Victoria
• Bioluminescent jellyfish
• In A. Victoria, bioluminescence involves aequorin
  (luciferase), coelenterazine (luciferin), GFP.

Davenport, D. and J.A.C. Nicol, Proc. R. Soc.
London, Ser. B, 1955. 144 p. 399-411. Zimmer,
M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81. http//animaldiversity.ummz.umich.edu/s
ite/resources/Grzimek_inverts/Hydrozoa/Aequorea_vi
ctoria_medusa.jpg/view.html
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GFP B-Barrel

• Wt-GFP
• 238 residues
• 11 ?-sheet barrel-like structure,
• monomeric tertiary structure diameter of 24 Å,
  and a height of 42 Å
• Chromophore (65-67).

http//www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP3.
htm
5
GFP Fluorescence in A. Victoria

• Aequorin first binds three Ca2 ions then
  oxidizes coelenterazine.
• The resulting complex, Ca3-apo-aequorin-coelentera
  mide, emits a blue light in vivo.
• However, this blue light (470 nm) is not emitted
  externally, rather it is transferred to a
  chromophore in GFP, where the energy is reemitted
  as green light (508 nm).

Tsien, R.Y., The green fluorescent protein. Annu
Rev Biochem, 1998. 67 p. 509-44. Morin, J.G. and
J.W. Hastings, Energy transfer in a
bioluminescent system. J Cell Physiol, 1971.
77(3) p. 313-8. Ward, W.W. and M.J. Cormier, An
energy transfer protein in coelenterate
bioluminescence. Characterization of the Renilla
green-fluorescent protein. J Biol Chem, 1979.
254(3) p. 781-8. McDowell, J. Luciferase
Mechanism. cited Available from
http//www.ebi.ac.uk/interpro/potm/2006_6/Page2.ht
m.
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GFP Chromophore
Tyr-66
Gly-67
Ser-65
Cyclicized Chromophore of 65Ser-Tyr-Gly67 from
1GFL.pdb
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Possible Mechanism for Chromophore Formation

• Mechanism still debated

• Cis rotation around Y66 ? angle

• Reduction of S65 carbonyl oxygen cyclization

Tsien, R.Y., The green fluorescent protein. Annu
Rev Biochem, 1998. 67 p. 509-44.
8
Important Characteristics of GFP

• Post-translational chromophore is independence
  from cofactors and substrates.
• Can be fused to other proteins without affecting
  their function.
• Generally non-toxic.
• Resistant to heat, alkaline pH, detergents,
  photobleaching, chaotropic salts, organic salts
  and many proteases.
• A rainbow of exciting colors!

Zimmer, M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81. http//www.conncoll.edu/ccacad/zimmer/G
FP-ww/GFP2.htm
9
Creation/Discovery of GFP Variants

• Mutants developed, or new proteins sought to
  improve qualities observed with GFP and/or
  perform other functions
• Shift in emission spectra
• Increase in fluorescence
• Resistance to different physiological conditions
• Faster maturation

10
GFP and Related Variants

• Partial list of GFP Mutants and Analogues

Zimmer, M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81.
11
3º Structural Similarity
1ema GFP 1rrx EGFP
1cv7 CFP 2yfp YFP
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DsRed A Tetramer

• From corals of discosoma genus.
• Same structure as GFP, but only 26-30 sequence
  homology.
• Excellent FRET partner for YFP.
• Relatively pH insensitive.
• Some denaturation observed under mild acidic
  conditions (pH 4.0 to 4.8), but can be recovered
  by pH increase.

Zimmer, M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81.
13
Results of MSA Using ClustalW
14
GFP vs. DsRed Chromophores
GFP Chromophore
DsRed Chromophore Extra double bond causes red
shift
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Excitation and Emission Spectra
The excitation (A) and emission (B) spectra of
blue (BFP), cyan (CFP), green (GFP), yellow (
YFP), and red (mRFP1) fluorescent proteins.
Lippincott-Schwartz, J. and G.H. Patterson,
Development and Use of Fluorescent Protein
Markers in Living Cells. Science, 2003. 300 p.
87-91.
16
Protein(Acronym) ExcitationMaximum(nm) EmissionMaximum(nm) MolarExtinctionCoefficient QuantumYield in vivoStructure RelativeBrightness( of EGFP)
GFP (wt) 395/475 509 21,000 0.77 Monomer 48
Green Fluorescent Proteins Green Fluorescent Proteins Green Fluorescent Proteins Green Fluorescent Proteins Green Fluorescent Proteins Green Fluorescent Proteins Green Fluorescent Proteins
EGFP 484 507 56,000 0.60 Monomer 100
TurboGFP 482 502 70,000 0.53 Monomer 110
Blue Fluorescent Proteins Blue Fluorescent Proteins Blue Fluorescent Proteins Blue Fluorescent Proteins Blue Fluorescent Proteins Blue Fluorescent Proteins Blue Fluorescent Proteins
EBFP 383 445 29,000 0.31 Monomer 27
Cyan Fluorescent Proteins Cyan Fluorescent Proteins Cyan Fluorescent Proteins Cyan Fluorescent Proteins Cyan Fluorescent Proteins Cyan Fluorescent Proteins Cyan Fluorescent Proteins
ECFP 439 476 32,500 0.40 Monomer 39
Cerulean 433 475 43,000 0.62 Monomer 79
Yellow Fluorescent Proteins Yellow Fluorescent Proteins Yellow Fluorescent Proteins Yellow Fluorescent Proteins Yellow Fluorescent Proteins Yellow Fluorescent Proteins Yellow Fluorescent Proteins
EYFP 514 527 83,400 0.61 Monomer 151
Venus 515 528 92,200 0.57 Monomer 156
mCitrine 516 529 77,000 0.76 Monomer 174
Red Fluorescent Proteins Red Fluorescent Proteins Red Fluorescent Proteins Red Fluorescent Proteins Red Fluorescent Proteins Red Fluorescent Proteins Red Fluorescent Proteins
dTomato-Tandem 554 581 138,000 0.69 Monomer 283
DsRed 558 583 75,000 0.79 Tetramer 176
Weak Dimer Weak Dimer Weak Dimer Weak Dimer Weak Dimer Weak Dimer Weak Dimer
http//www.microscopyu.com/articles/livecellimagin
g/fpintro.html
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Limitations of GFP

• Slow post-translational chromophore formation (3
  hours).
• O2 requirement.
• difficulty distinguishing GFP from background
  fluorescence if GFP concentration low.

Zimmer, M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81.
18
Faster Maturing Fluorescent Proteins

• GFP has a slow maturation time (3 h at room
  temperature) and its efficiency further decreases
  at 37 C.
• Faster maturation and decrease in temperature
  dependence would improve FP for biological
  applications.
• TurboGFP
• variant of the green fluorescent protein CopGFP
  cloned from copepoda Pontellina plumata
• Venus
• A variant of EYFP

http//www.evrogen.com/TurboGFP.shtml Rekas, A.,
et al., Crystal structure of venus, a yellow
fluorescent protein with improved maturation and
reduced environmental sensitivity. J Biol Chem,
2002. 277(52) p. 50573-8.
19
TurboGFP

• A dimer with bright green fluorescence.
• Faster maturation than EGFP when expressed in
  eukaryotic cells.
• It is specially recommended for cell and
  organelle labeling and for tracking the promoter
  activity.

http//www.evrogen.com/TurboGFP.shtml
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TurboGFP vs. EGFP
http//www.evrogen.com/TurboGFP.shtml
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Comparison of Maturation Rates
http//www.evrogen.com/TurboGFP.shtml
(A) Refolding kinetics. (B) Chromophore
maturation kinetics.
22
Creation of Venus

• EYFP (S65G/V68L/S72A/T203Y).
• Enhanced fluorescence.
• Citrine (Griesbeck et al), EYFP mutations and
  Q69M.
• Reduced pH and halide sensitivity and facilitated
  expression at 37 C.
• Venus replaced the Q69M mutation used in citrine
  by five other mutations (F46L, F64L, M153T,
  V163A, S175G) in addition to the original four
  mutation of EYFP.
• F46L accelerates the oxidation step in
  chromophore formation.
• The other four mutations improved folding at 37
  C, and one or more of these was responsible for
  decreasing proton and chloride sensitivity.

Rekas, A., et al., Crystal structure of venus, a
yellow fluorescent protein with improved
maturation and reduced environmental sensitivity.
J Biol Chem, 2002. 277(52) p. 50573-8.
23
Summary of Data Analysis

• Lower affinity for Cl- ion binding than EYFP
• More resistant to protonation at lower pH
• Faster folding

Rekas, A., et al., Crystal structure of venus, a
yellow fluorescent protein with improved
maturation and reduced environmental sensitivity.
J Biol Chem, 2002. 277(52) p. 50573-8.
24
Summary of Venus Mutations

• F64L
• Conformational change resulting in reduction in
  halide binding affinity due to solvent
  inaccessibility.
• M153T, V163A
• Believed to increase maturation rate, but no data
  cited to directly support this.
• S175G
• Removal of the hydroxyl group of serine
  drastically alters the backbone conformation of
  the region spanning residues 173175.
• This mutation in cycle-3 GFP has been shown to
  facilitate in vivo protein folding and enhance
  fluorescence intensity.
• Structural basis of this increased folding rate
  is unclear.

Rekas, A., et al., Crystal structure of venus, a
yellow fluorescent protein with improved
maturation and reduced environmental sensitivity.
J Biol Chem, 2002. 277(52) p. 50573-8.
25
FP General Applications

• Protein dynamics/markers in living cells
• Fluorescence microscopy
• Real-time molecular and cellular analysis
• Fusion constructs in gene therapy
• Tool to detect apoptosis

Zimmer, M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81.
26
FP Applications Fusion Tags

• Most common application of GFP.
• FPs can be fused to most proteins using standard
  subcloning techniques and used to map geography
  of cell.
• Resulting chimera can be expressed in a cell or
  organism.
• Chromophore is produced in vivo.

Zimmer, M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81.
Lippincott-Schwartz, J. and G.H. Patterson,
Development and Use of Fluorescent Protein
Markers in Living Cells. Science, 2003. 300 p.
87-91
27
FP Applications FRET

• FRET Fluorescent resonance energy transfer.
• nonradiative exchange of energy from excited
  donor to acceptor fluorophore within 10-100 Å
  from donor.
• Emission spectrum must overlap excitation
  spectrum of acceptor.
• BFP and GFP
• CFP and YFP
• Used to study protein/protein interactions
  energy transfer dependent on distance between
  fluorophores
• FRET used to monitor conformational changes in
  metal release of metallathionein.

Zimmer, M., Green fluorescent protein (GFP)
applications, structure, and related
photophysical behavior. Chem Rev, 2002. 102(3)
p. 759-81.
28
Examples of FRET Applications
Giepmans, B.N., et al., The fluorescent toolbox
for assessing protein location and function.
Science, 2006. 312(5771) p. 217-24.
29
Bimolecular and Unimolecular Indirect FRET
Basic Designs of Fluorescent Indicators Employing
GFP-Based FRET (A) Bimolecular
fluorescent indicators. CFP and YFP are fused to
domains A and B, respectively, which interact
with one another depending upon the
ligand binding or modification of domain A. (B)
Unimolecular fluorescent indicators.
Miyawaki, A., Visualization of the spatial and
temporal dynamics of intracellular signaling.
Dev Cell, 2003. 4(3) p. 295-305.
30
FP Applications FRAP

• FRAP Fluorescence recovery after photobleaching
• bleach area with high-intensity light
• monitor recovery of resultant fluorescence loss
  in the bleached area using time-lapse microscopy
• used to determine mobility of GFP chimera in cell
• Alters the fluorescence steady state in a cell
  without disrupting protein pathways or creating
  protein gradients.

Lippincott-Schwartz, J. and G.H. Patterson,
Development and Use of Fluorescent Protein
Markers in Living Cells. Science, 2003. 300 p.
87-91
31
FP Applications FLIP

• FLIP Fluorescence loss in photobleaching.
• Repeatedly photobleaching fluorescence in one
  area of the cell while collecting images of the
  entire cell.
• By monitoring the fluorescence in the
  nonphotobleached regions, the mobility of a
  fluorescently tagged protein can readily be
  observed and the continuity of cellular
  environments determined.

Lippincott-Schwartz, J. and G.H. Patterson,
Development and Use of Fluorescent Protein
Markers in Living Cells. Science, 2003. 300 p.
87-91
32
FRAP and FLIP
Fig. 3. Kinetic microscopy techniques. (A) In
FRAP, a region of the cellis selectively
irradiated to photobleach fluorescent molecules.
The recovery of fluorescent molecules into that
region is assessed quantitatively to determine
diffusion coefficients and mobile fractions. (B)
In FLIP, a region of the cell is repeatedly
photobleached. Movement of fluorescent molecules
into the region being photobleached results in
loss of fluorescence from areas outside the box
and can be used to access the boundaries for a
proteins diffusional movement within a cell.
Lippincott-Schwartz, J. and G.H. Patterson,
Development and Use of Fluorescent Protein
Markers in Living Cells. Science, 2003. 300 p.
87-91.
33
FP Applications BIFC

• Bimolecular Fluorescence Complementation (BiFC)
  is a means of observing protein-protein
  interactions.
• Two proteins of interest are tagged with one half
  of a fluorescent protein.
• If the two proteins come together and the tags
  unite then the full fluorescent protein can form.
  
• However, this process can take 60 seconds, a
  limiting factor for applications of this approach.

Giepmans, B.N., et al., The fluorescent toolbox
for assessing protein location and function.
Science, 2006. 312(5771) p. 217-24.
34
Future Directions for Fluorescent Proteins

• Fluorescence detection in clinical assays like
  protein activity, profiling in patient cells and
  biopsies
• Monitoring effects of drugs on cell signaling in
  individual patients.
• High-throughput drug screening in protein
  microarrays.
• Polychromatic imaging of targeted proteins.

Giepmans, B.N., et al., The fluorescent toolbox
for assessing protein location and function.
Science, 2006. 312(5771) p. 217-24.
35
Finis