The document provides an overview of green fluorescent protein (GFP) including its origins and history of study. It describes how GFP was cloned and sequenced in the 1990s, expressed in E. coli and C. elegans, and engineered into improved mutants with increased fluorescence. The document then discusses how the Protein Data Bank can be used to view 3D structures of proteins like GFP and how multiple sequence alignments and phylogenetic trees can reveal relationships between GFP derivatives.

1. GFP Workshop
Undergraduate Bioinformatics Club (UBIC) at UCSD
Alexander Niema Moshiri

2. Green Fluorescent Protein:
Origins
 Green Fluorescent Protein (GFP) is a naturally-occurring
protein in a species of jellyfish, Aequorea victoria
 When excited by blue or ultraviolet light, GFP
fluoresces a green color
A fluorescent Aequorea victoria

3. Green Fluorescent Protein:
A Brief History of wtGFP
 GFP has been studied as early as the 1960s
 However, its utility for molecular biologists was
not realized until the 1990s
 In 1992, Douglas Prasher cloned and
sequenced the wild-type GFP (wtGFP) gene
 “Wild-type” = Natural
 Prasher proposed using GFP as a biochemical
tracer that allows us to look at the inner
workings of cells
Douglas Prasher

4. Green Fluorescent Protein:
Recombination of wtGFP
 The lab of Martin Chalfie expressed wtGFP in E. coli and
C. elegans
 To their surprise, wtGFP was able to glow in both
species without needing any jellyfish cofactors
C. elegans expressing wtGFP

5. Green Fluorescent Protein:
Bioengineered
 In 1995, by changing a single amino
acid, Roger Tsien engineered the first
improved mutant of GFP with
increased fluorescence and
photostability
 Tsien was awarded the 2008 Nobel
Prize in chemistry for his GFP work
 He is currently a professor at UCSD
 Further improvements to GFP were
made over the next few years
Roger Tsien

6. Green Fluorescent Protein:
Current State of Mutants
 Today, many more derivatives have
been created from GFP and dsRed (a
red fluorescent protein)
 Researchers have access to a range
of colors, including green, yellow,
orange, red, violet, blue, and cyan
An illustration of a San Diego beach scene
drawn using 8 colors of FPs
Rainbow of FPs from the Tsien lab

7. Green Fluorescent Protein:
Experimental Uses
 We mentioned before that FPs can be used to track
cellular processes
 Researchers can simply attach an FP to some object of
interest and then they can visually follow the object
Mice expressing GFP next to normal mice GFP-expressing neurons

8. Protein Data Bank:
A Brief Overview
 The Protein Data Bank (PDB) is a
repository of 3D structural data
of large biological molecules
(e.g. proteins and nucleic acids)
 This structural data can be
downloaded and used to render
a 3D image of the molecule of
interest
3D rendering of GFP from PDB data

9. Protein Data Bank:
Step 1: Querying the PDB
 Open Mozilla Firefox and navigate to www.rcsb.org
 The search box on the top of the page allows you to
“Search by PDB ID, author, macromolecule, sequence,
or ligands”
 Search for the term Green Fluorescent Protein and hit
“Go”
 Scroll down and click on entry 4KW4: “Crystal Structure
of Green Fluorescent Protein”

10. Protein Data Bank:
Step 2: Questions About Results
 Who are the authors of the primary citation for 4KW4?
 What organism is this protein from?
 How long (in amino acids) is this protein?
 What method was used to produce this entry’s data?
 What is the resolution in Angstroms (Å)?

11. Protein Data Bank:
Step 3: Rendering 3D Structure
 Return to the PDB homepage: www.rcsb.org
 In the left-column panel, click “Visualize”
 In the box that says “Enter a PDB ID”, enter 4KW4 and
click “View Jmol”
 You should see a 3D rendering of GFP
 You can click and drag the 3D render to rotate it

12. Protein Data Bank:
Step 4: Display Customization
 Under “Select Display Mode,” click “Custom View”
 Cycle through the different Style options and choose
your favorite
 My personal favorite is the default, Cartoon
 Cycle through the different Color options
 You can also change the color(s) by Right-Clicking on the
3D render, going to Color, then Structures, then Cartoon
(assuming you’re still in Cartoon style), and choosing a
color
 You can also go to Color  Structures  Cartoon  By
Scheme and choose one of those options

13. Protein Data Bank:
Step 5: Exporting 3D Image
 Finish customizing the 3D image to your liking
 Feel free to play with the other options in the menu that
pops up when you Right-Click on the 3D image
 If you want to revert to the original settings, just refresh
the page and it will reload with the default settings
 When you are ready to export the final image, just click
the blue “Export 3D Image” button, specify a
destination, and click “Save”
 Enjoy your cool 3D image of GFP!

14. Multiple Sequence Alignment:
The FASTA Format
 The FASTA format is a text-based format for
representing DNA, RNA, or Protein sequences
 A sequence in the FASTA format begins with a single-line
description (beginning with the ‘>’ character), followed
by line(s) of sequence data

15. Multiple Sequence Alignment:
Sequence Alignment
 A sequence alignment is a way of arranging biological
sequences (DNA, RNA, or Protein) to identify regions of
similarity between the sequences
 Gaps can be inserted between characters in the
sequences so that identical or similar characters can be
aligned in the same column
An example multiple sequence alignment

16. Multiple Sequence Alignment:
GFP and its Derivatives
 In the following activity, we will
align the sequences of GFP and some
of its derivative fluorescent proteins
 These proteins’ sequences are
provided in the file named
protein_sequences.fasta
 Using the results from the multiple
sequence alignment, we will be able
to construct a phylogenetic tree
 This tree will provide us information
about the pairwise “closeness”
between the protein sequences

17. Multiple Sequence Alignment:
ClustalW2
 ClustalW2 is a popular multiple sequence alignment tool
 Download protein_sequences.fasta from:
 http://ubic.ucsd.edu/gfp/
 Go to the ClustalW2 website:
 http://www.ebi.ac.uk/Tools/msa/clustalw2/
 Under “STEP 1 – Enter your input sequences”, upload
protein_sequences.fasta by clicking “Choose File”
 Under “STEP 4 – Submit your job”, click “Submit”

18. Multiple Sequence Alignment:
ClustalW (Continued)
 After you click Submit, ClustalW2 will redirect you to
the results of the multiple sequence alignment
 The IDs of the sequences are to the left of the alignment,
and each row of the alignment corresponds to a single
sequence (e.g. the first row of every chunk is
“GFP(4KW4)”)
 If the alignment doesn’t make sense to you, be sure to
ask one of the UBIC officers any questions you have!

19. Evolutionary Relationships:
Phylogenetic Tree
 A phylogenetic tree is a branching diagram (or “tree”)
that shows relationships of “closeness” between
different biological species or other entities
 Elements that are closer together on the tree have
“closer” (more similar) sequences
 In the ClustalW2 results page, click “Send to
ClustalW2_Phylogeny”
 On the resulting page, under “STEP 3 – Submit your
job”, click “Submit”
 Draw out the phylogenetic tree (questions will be asked
about it on the Extra Credit assignment)

20. Phylogenetic Trees:
Biological Importance
 The information provided by phylogenetic trees is
extremely valuable and is even applicable to medicine
 In 1994, Richard Schmidt, an American physician, used a
sample of blood from one of his AIDS-infected patients to
inject into his ex-lover and former colleague, Janice
Trahan, infecting her with HIV
 HIV DNA was collected from the victim, from the putative
patient source, and from thirty-two other unrelated, HIV-
positive individuals
 Scientists concluded that of all the samples they tested,
the two viruses' DNA from the victim and the patient
matched almost exactly, even with HIV's potential to
mutate very rapidly

21. Phylogenetic Tree from the
HIV Court Case

22. GFP Workshop:
Summary
 Congratulations on finishing the GFP Workshop!
Throughout the workshop, you learned the following:
 GFP’s history and uses
 How to use the PDB (and rendering 3D protein structures)
 Multiple Sequence Alignment using ClustalW2
 Phylogenetic Tree Construction from a Multiple Sequence
Alignment using ClustalW2_Phylogeny
 We hope you enjoyed the workshop, and we hope you
have found interest in the field of Bioinformatics!