Green Fluorescent Protein GFP

1
Green Fluorescent Protein- GFP

• By Mylinh Ngo, Laurie Brier, and Hemal Patel

2
A Cool Protein

• Give off the green light in the presence of UV
  light
• Marker of the twenty-first century
• Nobel Prize in Chemistry 2008

3
Nobel Prize in 2008

• Osamu Shimomura
• Martin Chalfie
• Roger Y. Tsien

http//nobelprize.org/nobel_prizes/chemistry/laure
ates/2008/
4
Origin of GFP

• Jellyfish, Aequorea victoria in 1962
• Modified version of GFP
• Cat
• Pig
• Rabbit

http//www.cbc.ca/news/yourview/2007/12/uv_light_m
akes_fluorescent_fel.html
5
Scientific Impact of GFP

• Nontoxic
• No significant physiological effects
• Can be used to
• Watch process that was previously invisible
• How cancer cell spreads
• Alzheimer's disease

6
Common Use of GFP

• Monitor changes in a cell

http//nobelprize.org/nobel_prizes/chemistry/laure
ates/2008/chemadv08.pdf
7
Excitation Spectrum vs. Emission Spectrum

• Chromophore p-hydroxylbenzylideneimidazolinone
• Ser65-Tyr66-Gly67
• Excitation Spectrum
• Absorb at maximum at 400nm
• and 470nm
• Conjugation
• Emission Spectrum
• Shift to 505nm and 540nm
• Responsible for the color

http//nobelprize.org/nobel_prizes/chemistry/laure
ates/2008/chemadv08.pdf
8
Mechanisms

• GFP folds autocatalytically
• Only needs oxygen
• Nucleophilic attach of the amide of Gly67 on the
  carbonyl of Ser65
• Imidazolinone
• Dehydration of Tyr66
• Maturation of GFP Chromophore

http//nobelprize.org/nobel_prizes/chemistry/laure
ates/2008/chemadv08.pdf.
9
Structure of GFP
10
Crystal Structure Of GFP

• PDB number 3CD9
• Method used to determine structure X-Ray
  diffraction

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General Structure Information

• Number of chains 1 chain
• Composed of 226 residues (excluding gaps)
• Total of 7 helices
• 12 strands
• No disulfide bridges

12
Helices

• CHOFAS

• Predicts 8 helices

• GOR4

• Predicts 3 helices

• PELE (JOI)

• Predicts 8 helices

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Helices
7 Helices (Magenta) 1 beta barrel (Yellow)
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Sheets

• CHOFAS

• Predicts 9 sheets

• GOR4
• Predicts 14 sheets

• PELE (JOI)

• Predicts 11 sheets

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Sheets
Top view
Side view
16
Compiling information from Chofas, GOR4, PELE,
Cn3D and Protein explorer

• Notice that each method shows a different number
  of helices

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Ligands
Carbon- Grey Oxygen- Red Nitrogen- Blue
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Other Ligands
Carbon- Grey Oxygen- Red Nitrogen- Blue
BFP
RFP
YFP
GFP
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Ligand Interaction
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TMAP, GREASE, TMHMM

• Reveals no transmembrane segment

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Phylogeny
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Homologs

• BlastP
• Star coral CFP
• A. macrodactyla OFP
• Common jellyfish YFP
• Strawberry anemone RFP
• Flat lettuce coral GFP-like
• Snakelocks anemone NRP

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MSA

• MVIEW
• ClustalW

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Unrooted Tree

• ClustalW
• ClustalTree

100
100
76.3
100
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Unrooted Tree

• ClustalW
• ClustalTree

100
100
76.3
100
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Unrooted Tree

• ClustalW
• ClustalTree

100
100
76.3
100
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Unrooted Tree

• ClustalW
• ClustalTree

100
100
76.3
100
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Unrooted Tree

• ClustalW
• ClustalTree

100
100
76.3
100
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Rooted Tree
http//www8.nos.noaa.gov/biogeo_public/habitat_pho
tos.aspx
http//www.dailymail.co.uk/sciencetech/article-108
4261/Aliens-deep-The-weird-wonderfully-wobbly-anim
als-discovered-sea-census.html
http//www.wetwebmedia.com/pectiniids.htm
http//www.obs-vlfr.fr/laval/images/Bougisia20or
nata20-20Adult20female20staying20on20a20Phi
alidium20sp..jpg
http//www.horozbina.com/icerik/44/Anemonia-sulcat
a---Mediterranean-Anemone.html
http//www.ajtai.org/page/uwphoto.php?fotoCorynac
tis_californica
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Comparing Structures

• Aequoria victoria GFP (3CD9)
• Snakelocks Anenome NRP (1XMZ)

Key -fully conserved -not conserved
-no alignment due to indel -ligands
http//www.ncbi.nlm.nih.gov/Structure/vast/vastsrv
.cgi?sdid286101
31
References

• Biology Workbench. 1999. University of Illinois.
  20 Nov. 2008 http//seqtool.sdsc.edu/cgi/bw.cgi
• "GFP-Like Protein." Evolution of Structure 21
  (2004) 841.
• "Green Fluorescent Protein." Royal Swedish
  Academy (2008) 1-17.
• NCBI Homepage. 23 July 2008. U.S. National
  Library of Medicine. 20 Nov. 2008
  lthttp//www.ncbi.nlm.nih.gov/gt.
• "Nobel Prize In Chemistry 2008." Chemistry 2008.
  2008. Nobel Foundation. 25 Nov. 2008
  lthttp//nobelprize.org/nobel_prizes/chemistry/laur
  eates/2008/gt.
• Nobel Foundation. "Green Fluorescent Protein
  Pioneers Share 2008 Nobel Prize In
• Chemistry." ScienceDaily 8 October 2008. 1
  December 2008 lthttp//www.sciencedaily.com
  /releases/2008/10/081008100616.htmgt.
• Protein Explorer FrontDoor. 15 Nov. 2008
  lthttp//www.umass.edu/microbio/chime/pe_beta/pe/pr
  otexpl/frntdoo2.htmgt.
• "UV light makes fluorescent felines glow." 14
  Dec. 2007. CBS News. 20 Nov. 2008
  lthttp//www.cbc.ca/news/yourview/2007/12/uv_light_
  makes_fluorescent_fel.htmlgt.

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Q and A

• Any Questions?