St. Dominic SMART Team: Elana Baltrusaitis, Allyson Bigelow, Rachel Brielmaier, Pamela Burbach, Jake Dowler, Johnny Fuller, Caroline Hildebrand, Teagan Jessup, Molly Jordan, Josh Kramer, Jenna Lieungh, Alex Mikhailov, Pat O’Grady, Andrew Pelto, Quin Rowen, Rachelle Schmude, Bobby Schultz, Katherine Seubert, Alex Sherman, Parker Sniatynski, Alex Venuti, Erin Verdeyen, and Molly Wetzel Teacher Advisor Donna LaFlamme Mentors: Nathan Duncan and Françoise Van den Bergh, Ph.D., Medical College of Wisconsin
Luciferase is the generic name for an enzyme responsible for bioluminescence reactions and is commonly associated with fireflies. It is also found in many other organisms including bacteria, fungi, anemones, and dinoflagellates. Since the gene for the North American firefly (Photinus pyralis) luciferase was cloned in 1985, scientists have been genetically engineering the gene into living cells. The luciferase reaction is now widely used in scientific research to study protein production in cells, to analyze gene promoter activity, to study stem cell function in vivo, and in cancer studies, to trace the metastasis of cancer cells in living test animals. The scientific study of the luciferase enzymes themselves is also continuing. In recent research, single amino acid mutations to the active site cause the emission of different colored light in a predictable way. The uses of and improvements in bioluminescent imaging are increasing exponentially in cell biology, molecular biology, and in medical research.
LUCIFERASE FACTS TO KNOW LUCIFERASE IN RESEARCH • Luciferase is an enzyme found in the lanterns of fireflies in cells called photocytes. Fat Cell Growth Over 12 Weeks • Its function is to catalyze a bioluminescence reaction in which the substrate luciferin is oxidized to oxyluciferin. ACTIVE SITE OF FIREFLY LUCIFERASE • Bioluminescence is the production of light by a chemical reaction within (Luciola cruciata) living organisms. • Fireflies use bioluminescence for communicating, attracting mates, luring in prey, and self-defense.
Firefly Luciferase (Luciola cruciata) Japanese Firefly (Luciola cruciata) Physical Model based on PDB File: 2D1R Scientists discovered a fat progenitor cell with the help of luciferase. The progenitor IVIS® imaging system which is used cells were injected into mice genetically to pick up the faint light given off unable to produce fat. The luciferase gene by the oxyluciferin in the cells turned on in mature fat cells and allowed the expressing luciferase. researchers to track the development of the fat cells over time in the living animal. [2]
http://2.bp.blogspot.com/_VVjUm4hzpvo/SSPbxn1OnJI/AA [1] AAAAAABd8/UnJeVFSM9RI/s400/firefly.jpg ACTIVE SITE OF WILD TYPE AND MUTANT S286N LUCIFERASE FIREFLY BIOLUMINESCENCE REACTION Ile 288
DLSA RasMol Image based on PDB File: 2D1R
The luciferase active site provides a hydrophobic pocket for Ile 288 producing the electronically unstable oxyluciferin. Single amino acid mutations of Ile 288 to Val288 and to Ala288 and [1] of Ser 286 to Asn 286 results in the emission of lower energy S286N light. [1] See below. Wild-Type DLSA Mutant Step 1: Luciferin and magnesium-ATP react to form the luciferyl-AMP intermediate. [1] Ile 288 is being rotated so it is not packing the intermediate analogue, DSLA ,tightly in the mutant active site. This changes the color of the Step 2: Luciferin-AMP reacts with O2 to form excited oxyluciferin which Wild Type(yellow) I288V (orange) l288A (reddish-pink) S286A(red) emits a photon of light as it returns to its ground energy state. emitted light from green to lower energy red light because excited oxyluciferin is allowed to lose energy before it emits light. [1]
1961-The structure and synthesis of 1985-Marlene DeLucca and 1885- Raphael DuBois ground up beetle 1976- DeLucca shows that the firefly luciferin was determined colleagues cloned the gene for lanterns in hot and cold water obtaining bioluminescence reaction is a two step Present- luciferase/luciferin is 1955-Luciferase was purified as by White, Field, McElroy, firefly luciferase no luminescence in the hot water. He named luciferin and process requiring ATP, O2, and used to locate tumors and monitor the a protein by Green and McElroy and McCapra.[4] and expressed the luciferase. Hot water destroyed luciferase. [3] luciferin. spread of cancer in test animals gene in E. coli bacteria.
1947- W.D. McElroy discovered that ATP is an 1959- McElroy and Seliger discovered the 1967- Determination of picogram amounts 1990/91-Mutant luciferases 1996- E. Conti, N. Frank, 2010-St.Dominic essential requirement for the bioluminescence quantum yield of the oxidation of of ATP using luciferin-luciferase enzyme discovered that produce and P. Brick crystallized SMART Team designs reaction and light production is proportional to ATP. [3] firefly luciferin to be 90% efficient.[3] system by G.E. Lyman and different colors of light firefly luciferase and physical model of firefly bioluminescencehttp://upload.wikimedia.org/wikipedia/en/2/27/William_David_McElroy.jpg http://upload.wikimedia.org/wikipedia/commons/f/f7/Luciferase.png http://hongkiat.s3.amazonaws.com/colorfulwp/Rainbow_Ocean__by_Thelma1.j obtained an x-ray structure J.P. DeVincenzo. [4] pg luciferase (2d1r) [1]Primary Citation : T. Nakatsu, et. al. Structural basis for the spectral difference in luciferase bioluminescence. Nature, 2006, 440, 372-376 at 2 angstroms resolution. [2] M. S. Rodeheffer et. al. Identification of White Adipocyte Progenitor Cells In Vivo. Cell, 2008, 135240-249 [3] H. Fraga, Firefly luminescence: A historical perspective and recent developments. Photochemical and Photobiological Sciences, 2008, 7, 146-158 [4] J. Lee, Bioluminescence Timelines, University of Georgia. A SMART Team project supported by the National Institutes of Health (NIH)-National Center for Research Resources Science Education Partnership Award (NCCR-SEPA).