Sugary foods Registered trademarks of Pepsi Co Inc

Sugary foods Registered trademarks of Pepsi. Co, Inc. , 2007 (www. pepsi. com; www. mtdew. com; www. mugrootbeer. com), Coca-Cola Company, 2007 (www. thecoca-colacompany. com), The Beverage Partners Worldwide, 2007 (www. nestea. com) and Snapple Beverage Corporation, 2006 (www. snapple. com), The Hershey Company, 2007 (www. hersheys. com), Mars, Inc. , 2006 (www. snickers. com; www. m-ms. com; www. milkywaybar. com; )

Sweet Chemicals Chemical Name Glucose Sucrose Fructose www. chemfinder. com Structure

Obesity Map--2006 Percentage of obese adults (>30 BMI) Overweight and Obesity. Center for Disease Control and Prevention. 2007. (accessed: 8/5/07) http: //www. cdc. gov/nccdphp/dnpa/obesity/trend/maps/index. htm

Dietary options § Control consumption of sugars § Simple carbohydrates § Usage of low calorie or zero calorie sweeteners § Splenda § Equal § Sweet’n Low Registered trademark of Cumberland Packing Corp. 2003 (www. sweetnlow. com); The Merisant Company, 2006 (www. equal. com); Mc. Neil Nutritionals, LLC, 2007 (www. splenda. com)

Artificial Sweeteners Sweetener Structure Aspartame Saccharin Sucralose www. chemfinder. com

Synthesis of Sucralose

Artificial sweeteners § Consumers are hesitant due to health issues § Cancer concerns (saccharin/aspartame) § Side effects § Headaches § Diarrhea

Dietary market § In need of a natural low calorie or zero calorie sweetener § Sweet Proteins?

Neoculin: A Sweet-Tasting, Taste -Modifying Protein and its Interaction with T 1 R 2 & T 1 R 3 of the Sweet Taste Receptor Family Jeffrey T. Kushner University of Pennsylvania MCE Program Cohort 6 Thesis Presentation

Research Focus 1. Features of Neoculin and interaction with T 1 R 3 receptor 2. Regions of Neoculin that elicit sweet taste response with T 1 R 2+T 1 R 3 complex 3. Cause of Taste Modifying Function

Taste Overview • Taste Map No Longer • Refined knowledge since 2001 • Five Major Tastes Marieb, Elaine, N. “Chemical senses: taste and smell. ” Human Anatomy and Physiology, 2 nd Ed. Benjamin Publishing Company. New York, 1992; pp 496.

Taste Overview Sour Salt § Different mechanisms for simple tastes vs. complex tastes Adapted from: Lindeman, Bernd. Taste Reception. Physiological Reviews. 1996, 76, pp 751.

Taste Overview § Series of events to elicit an action potential University of Arizona, 2007 (www. neurobio. arizona. edu)

Testing Taste Human Embryonic Kidney Cells Taste Testing Panel Nakajima, et al. Appl. Environ. Microbiol. 2006, 72, pp 3720. Electric Tongue Lotus Bakeries NV, 2007 (www. lotusbakeries. com) Legin, et al. Anal. Bioanal. Chem. 2004, 380, pp 42.

Taste Receptor: Overview § Homodimers § T 1 R’s § Heterodimer Small sweeteners Large nonprotein sweeteners § T 1 R 2+T 1 R 3 § Sweet taste receptor complex § Venus flytrap domain § Sweeteners § Proteins 7 -transmembrane helix-receptor proteins Adapted from: Temussi, Pierandrea. J. Mol. Recog. 2006, 19, pp 188 -199.

Neoculin: Overview §From Curculigo latifolia plant (West Malaysia) §Basic Subunit § Acidic Subunit § 114 amino acids § 113 amino acids PDB ID: 2 d 04.

Features of Neoculin § Side-by-side comparison Yellow line--hydrophobicity purple line--hydrophilicity Acidic Subunit Basic Subunit Bowen, R. Protein hydropathicity plots. 1998. http: //arbl. cvmbs. colostate. edu/molkit/hydropathy/ (accessed: 7/22/07)

Features of Neoculin Rotated 180º Adapted from: Esposito, et al. J. Mol. Biol. 2006, 360, pp 452. § Electrostatic Potential § Potential energy associated with electric fields § NAS--yellow; NBS--orange § Blue--basic; red--acidic

Protonations Histidine Aspartate

Features of the T 1 R 3 Inactive Active § Cysteine-rich region § Negatively charged cavity Adapted from: Temussi, Pierandrea. J. Mol. Recog. 2006, 19, pp 192

Specifics of Neoculin § Acidic Loops § Stability § Taste Modifying Basic Loops Bind with T 1 R 3 to elicit sweet response Moreland, J. L. ; Gramada, A. ; Buzko, O. V. ; Zhang, Qing; Bourne, P. E. Molecular biology toolkit (MBT): a modular platform for developing molecular visualization applications. BMC Bioinformatics, 6: 21 (2005)

Loop Differences Neoculin Acidic Subunit--Chain A 1 DSVLLSGQTL YAGHSLTSGS YTLTIQNNCN LVKYQHGRQI WASDTDGQGS 51 QCRLTLRSDG NLIIYDDNNM VVWGSDCWGN NGTYALVLQQ DGLFVIYGPV 101 LWPLGLNGCR SLN Neoculin Basic Subunit--Chain B 1 DNVLLSGQTL HADHSLQAGA YTLTIQNKCN LVKYQNGRQI WASNTDRRGS 51 GCRLTLLSDG NLVIYDHNNN DVWGSACWGD NGKYALVLQK DGRFVIYGPV 101 LWSLGPNGCR RVNG Adapted from: PDB ID: 2 d 04. § 56 amino acids found in four loops § Only 10 residue differences

Amino Acid Difference

Important Amino Acids Amino Acid Structure Hydropathicity Basicity Tyrosine Hydrophobic Neutral Glycine Neutral Glutamine Hydrophilic Neutral Histidine Hydrophilic Slightly Basic Aspartic Acid Strongly hydrophilic Acidic Arginine Hydrophilic Basic www. chemfinder. com

Important differences Acidic Subunit Basic Subunit Outcome 1 st Fold Hydrophobic (Tyr, Gly) Hydrophilic (His, Asp) Hydropathicity change 3 rd Fold Neutral p. H (Gly, Gln) Basic p. H (Arg, Arg) Change in p. H character

Docking Model § Neoculin bonded to T 1 R 3 utilizing ‘wedge site binding’ § Types of interactions § Number of surface interactions determines sweetness Shimizu-Ibuka, et, al. Crystal structure of Neoculin: insights into its sweetness and taste-modifying activity. J Mol. Biol. 2006, 359, pp 155

Taste-Modifying § Possibly due to drastic change in electrostatic potential of heterodimer OR § Protonation of only acidic unit PDB ID: 2 d 04.

Conclusions § Binding between NBS & T 1 R 3 at loop region § NAS aids conformational change § Protonated form of Neoculin changes conformation for Taste-Modifying activity

Future Research in Taste § Crystallize tastant bonded with taste receptor § Identify specific amino acid regions on Taste Receptor involved in binding § bonding patterns and behaviors between taste receptor and tastants § Pursue mass extracellular production of Neoculin

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