The Amgen Biotech Experience curriculum includes a series of cloning and protein purification experiments using red fluorescent protein (RFP). Fluorescent proteins have become a valuable tool in recent years among scientists in many different fields of biology.  Often, these glowing proteins are linked to other proteins of interest to confirm protein expression, to identify where specific proteins exist in the cell, and to track cell movement. Green fluorescent protein (GFP) is perhaps the most well-known fluorescent protein.  Isolated from the jellyfish Aequorea victoria, GFP is comprised of 238 amino acids that form 11 beta-sheets that roll in to a "beta-can," as seen in the image of GFP (right) from the European Bioinformatics Institute.

The PowerPoint presentation "Using and Analyzing Fluorescent Proteins" introduces students to some of the ways that fluorescent proteins are used in biology.  Protein Data Bank identifiers (PDB IDs) are provided in the PowerPoint for various fluorescent proteins.  Students can then visualize these proteins with the new Molecule World app.  After seeing the striking structural similarities of all fluorescent proteins, students learn how to compare DNA and protein sequences with the ubiquitous bioinformatics tool BLAST (Basic Local Alignment Search Tool). By the end of the activities, students will be able to answer the following questions:

1. Is red fluorescent protein (RFP) related to its famous cousin, GFP?  
2. What other fluorescent proteins, if any, are closely related to GFP and/or RFP?​

Student Resources:

• Comparing Sequences of Fluorescent Proteins Using BLAST Student Handout (pdf)
• DNA Sequences of Fluorescent Proteins 2014 (doc)
• Protein Sequences of Fluorescent Proteins 2014 (doc)
• Structure files of the fluorescent proteins covered in the PowerPoint and Student Handout
  • EGFP (enhanced Green Fluorescent Protein) - PDB IB 2Y0G
  • Citrine Fluorescent Protein - PDB ID 1HUY
  • mOrange Fluorescent Protein - PDB ID 2H5O
  • mCherry Fluorescent Protein - PDB ID 2H5Q
  • TagBFP (Blue Fluorescent Protein) - PDB ID 3M24
  • mTurquoise Fluorescent Protein - PDB ID 4AR7
  • Cerulean Fluorescent Protein - PDB ID 2Q57​

2Y0G in Molecule World

Coloring by hydrophobicity shows that hydrophobic residues (brown) are located in the interior of the protein with hydrophilic residues (blue) on the outside. The white residue in the center is the fluorophore.  

Teacher Resources:

• Background: Using and Analyzing Fluorescent Proteins PowerPoint (pptx)
• To obtain the Answer Key and Grading rubric and a PowerPoint of the Key for In-Class viewing, request an Educator account, then you'll be able to log in and access answer keys. 

Image by David S. Goodsell, RCSB Protein Data Bank (www.rcsb.org) of PDB IDs 3m24, 2q57, 4ar7, 2y0g, 1huy , 2h5o, 2h5q. For more information about GFP-like fluorescent proteins, visit the "GFP-like Proteins" page by Dr. Goodsell at the Research Collaboratory for Structural Bioinformatics (RCSB)'s PDB 101.

For more information about fluorescent proteins, including their discovery, molecular characterization, and use, visit "Fluorescent proteins" at Scholarpedia. 

Fluorescent Proteins References

1. PDB ID 2y0g: A. Royant & M. Noirclerc-Savoye (2011) Stabilizing role of glutamic acid 222 in the structure of enhanced green fluorescent protein. Journal of Structural Biology 174, 385-390.
2. PDB ID 3m24: O. M. Subach, V. N. Malashkevich, W. D. Zencheck, K. S. Morozova, K. D. Piatkevich, S. C. Almo & V. V. Verkhusha (2010) Structural characterization of acylimine-containing blue and red chromophores in mTagBFP and TagRFP fluorescent proteins. Chemistry & Biology 17, 333-341
3. PDB ID 2q57: G. D. Malo, L. J. Pouwels, M. Wang, A. Weichsel, W. R. Montfort, M. A. Rizzo, D. W. Piston & R. M. Wachter (2007) X-ray structure of Cerulean GFP: a tryptophan- based chromophore useful for fluorescence lifetime imaging. Biochemistry 46, 9865- 9873.
4. PDB ID 2h5o, 2h5q: X. Shu, N. C. Shaner, C. A. Yarbrough, R. Y. Tsien & S. J. Remington (2006) Novel chromophores and buried charges control color in mFruits. Biochemistry 45, 9639-9647.
5. PDB ID 1huy: O. Griesbeck, G. S. Baird, R E., Campbell, D. A. Zacharias & R. Y. Tsien (2001) Reducing the environmental sensitivity of yellow fluorescent protein. Journal of Biological Chemistry 276, 29188-29194.
6. PDB ID 4ar7: D. Von Stetten, M. Noirclerc-Savoye, J. Goedhart, T. W. J. J. Gadella & A. Royant (2012) Structure of a fluorescent protein from Aequorea victoria bearing the obligate- monomer mutation A206K. Acta Crystallographical Section F 68, 878.