Level: Advanced
Teaching Notes
Exploring Protein Structures in the RCSB PDB: Fluorescent Proteins
Learning Goals:
1. Visualize the structure of a given molecule using RCSB PDB resources.
2. Explore the structure to understand its structure function relationships
Educational Standards
A. Common Core
- Craft and Structure
- RI.9-10.4
- RI.11-12.4
- Integration of Knowledge and Ideas
- RI.9-10.7
- RI.11-12.7
B. Next Generation Science Standards
- Practices
- 8. Obtaining, Evaluating and Communicating Information
- Crosscutting Concepts
- 3. Scale, proportion and quantity
- 4. Systems and system models
- 6. Structure and function
- Disciplinary Core Ideas
- LS1.A: Structure and Function
- PS2.B: Types of Interactions
C. Advanced Placement Biology - Essential Knowledge (EK), Learning Objectives (LO), Science Practices (SP)
- EK 4.A.1
- LO 4.2, SP 1.3
- LO 4.3, SP 6.1, 6.4
Teaching Notes:
About Green Fluorescent Proteins:
Osamu Shimomura first discovered the Green Fluorescent Protein in 1960s. After its gene was cloned in the early 1990s Martin Chalfie and Roger Tsien figured out how to fuse this with other genes of interest and use it as a marker for gene expression and protein localization. In 2008 Shimomura, Chalfie and Tsien were awarded Nobel Prize in Chemistry for their contributions to this field.
Green Fluorescent Protein has been widely used as a tracking reagent in biology and biotechnology. The protein itself is shaped like a barrel made of a single beta sheet. In all GFP and its relative fluorescent proteins a special chemical group, called chromophore, is formed by the fusion of three consecutive amino acid residues. This absorbs and emits light at different wavelengths resulting in fluorescence.
To learn more about this protein and its various other fluorescent cousins, read the following Molecule of the Month features and their references:
http://pdb101.rcsb.org/motm/58
http://pdb101.rcsb.org/motm/174
Answers to the exercise questions:
1. Green fluorescent protein (GFP)
Q a. What can you learn about the structure from this image?
A a. The mage shows that the entire molecule is made up of a single protein chain.
Q b. Is the polymer chain seen in the graphical display in Chimera continuous? Explain what this means?
A b. No there is a break in the polymer chain in the middle of the structure. This is actually an artifact of the visualization software which does not show non-standard residues (chromophore) in the cartoon representation.
Q c. Where in the GFP structure is the chromophore located?
A c. The chromophore is located in the middle of the central helix of the GFP protein – in the hydrophobic core of the protein.
2. DsRed
Q a. Where do these aliphatic residues appear in the structure? Describe the distribution within each monomer of DsRed and also between different monomers.
A a. The aliphatic residues are located all over – specifically pointing towards the hydrophobic cores of each of the beta-barrels. Also they line the interfaces between pairs of chains A-B and chains C-D.
Q b. Do you see any patterns regarding the distribution of the aromatic residues?
A b. There are many aromatic side chains that are locate in the hydrophobic cores of the beta barrel. There is also a reasonably high number of aromatics at the interface between pairs of chains A-C and B-D.
Q c. Where are the polar residues located in the structure? Comment about the interaction interfaces between the 4 polymer chains in the structure.
A c. The polar residues are distributed all over the surfaces of the beta-barrel structures, except at the interface between pairs of chains A-B and C-D.
3. Compare the structures of Green Fluorescent Protein (GFP) and DsRed
Q a. How well do the 2 structures match?
A a. The backbone structures of the GFP and DsRed proteins align really well even though the sequences are very different.
Q b. Are the chromophores similar or different?
A b. The structure and location of the chromophores are very similar. The 3rd residue in the chromophore differs in the GFP and DsRed proteins so that part of the chromophore is different.
Q c. Where are the conserved residue’s side chains located with respect to the chromophores? Discuss the implications of your observation.
A c. They are located in the beta barrel pointing towards the chromophore, positioned above and below it. These residues play a key role in the initial cyclization step in the formation of the chromophore. Thus even though they are charged amino acids their position is conserved in the hydrophobic cores.
Developed as part of the RCSB Collaborative Curriculum Development Program 2015