Soft Drinks

Soft Drinks

Sugary foods Registered trademarks of PepsiCo, 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 (>30BMI) 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 Sweetn Low Registered trademark of Cumberland Packing Corp. 2003 (

www.sweetnlow.com); The Merisant Company, 2006 (www.equal.com); McNeil 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 T1R2 & T1R3 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 T1R3 receptor 2. Regions of Neoculin that elicit sweet taste response with T1R2+T1R3 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, 2nd 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, pp751.

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 T1Rs Heterodimer

Small sweeteners Large nonprotein sweeteners T1R2+T1R3 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: 2d04. 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 180 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 T1R3 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 T1R3 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

101LWPLGLNGCR SLN Neoculin Basic Subunit--Chain B 1 DNVLLSGQTL HADHSLQAGA YTLTIQNKCN LVKYQNGRQI WASNTDRRGS 51 GCRLTLLSDG NLVIYDHNNN DVWGSACWGD NGKYALVLQK DGRFVIYGPV 101LWSLGPNGCR RVNG Adapted from: PDB ID: 2d04. 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 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 1st Fold Hydrophobic (Tyr, Gly)

Hydrophilic (His, Asp) Hydropathicity change 3rd Fold Neutral pH (Gly, Gln, Gln)

Basic pH (Arg, Arg) Change in pH character Docking Model Neoculin bonded to T1R3 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 Conclusions Binding between NBS & T1R3 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 References (1) Marieb, Elaine, N. Chemical senses: taste and smell. Human nd Ed. Benjamin Publishing Company. New York, 1992; pp 496-499. Anatomy and Physiology, 2nd (2) Shallengerger, Robert, S. Taste recognition chemistry. Pure Appl. Chem. 1997, 69, pp 659-666.

(3) Brownlee, Christen. Sweet finding. Sci News. 2006, 170, p 9. (4) Turnbull, Bianca; Matisoo-Smith, Elizabeth. Taste sensitivity to 6-npropylthiouracil predicts acceptance of bitter-tasting spinach in 3-6y-old children. Am. J .Clin. Nutr. 2002, 76, pp 1101-1105. (5) Margolskee, Robert, F. Molecular mechanisms of bitter and sweet taste Transduction. J. of Biol. Chem. 2002, 277, pp 1-4. (6) Reinberger, Stefanie. Bitter could be better. Sci. Am. Mind. 2006, 17, p 3. (7) Matsunami, Hiroaki; Amrein, Hubert. Taste perception: how to make a gourmet mouse. Cur. Biol. 2004, 14, pp R118-R120. (8) Temussi, Pierandrea. The history of sweet taste: not exactly a piece of cake. J. Mol. Recog. 2006, 19, pp 188-199. (9) Nakajima, Ken-ichiro.; Askura, Tomiko.; Maruyama, Jun-ichi.; Morita, Yuji.; Oike, Hideaki.; Shimizu-Ibuka, Akiko.; Misaka, Takumi.; Sormachi, Hiroyuki.; Arai, Soichi.; Kitamoto, Katsuhiko.; Abe, Keiko. Extracellular production of Neoculin, a sweet-tasting heterodimeric protein with taste-modifying activity, by Aspergillus oryzae. Appl.

Environ. Microbiol. 2006, 72, pp 3716-3723. References (cont.) (10) Nakajima, Ken-ichiro.; Askura, Tomiko.; Oike, Hideaki.; Morita, Yuji.; Shimizu-Ibuka, Akiko.; Misaka, Takumi.; Sormachi, Hiroyuki.; Arai, Soichi.; Abe, Keiko. Neoculin, a tastemodifying protein, is recognized by human sweet taste receptor. Chem. Senses. 2006, 17, pp 1241-1244. (11) Shimizu-Ibuka, Akiko.; Morita, Yuji.; Terada, Tohru.; Asakura, Tomiko.; Nakajima, Ken-ichiro.; Iwata, So.; Misaka, Takumi.; Sorimachi, Hiroyuki.; Arai, Soichi.; Abe, Keiko. Crystal structure of Neoculin: insights into its sweetness and taste-modifying activity. J Mol. Biol. 2006, 359, pp 148158. (12) Garrett, R. H.; and Grisham, C.M. Amino acids and proteins. Principles of Biochemistry with a Human Focus, updated Third Edition. Brooks/Cole. Belmont, CA, 2007. pp 590-627. (13) McMurry, John.; Casellion, Mary, E. Amino acids and proteins. Fundamentals of General, Organic, and Biological Chemistry; Fourth Edition. Pearson Education, Inc. New Jersey. 2003, pp 500-559.

(14) Pavia, Donald, L.; Lampman, Gary, M.; Kriz, George, S. Mass spectrometry and NMR. Introduction to Spectroscopy, Third Edition. Thompson Learning, Inc. 2001, pp 10; 102; 256; 390. (15) Sugita, M. Taste perception and coding in the periphery. Cell. Mol. Life Sci. 2006, 63, pp 2000-20155. (16) Berg, Jeremy, M.; Tymoczko, John, L.; Lubert, Stryer. Responding to environmental changes: taste is a combination of senses that function by different mechanisms. Biochemistry, 5thth Edition. W.H. Freeman and Co. 2002. (Accessed: 6/5/07) http://www.ncbi.nlm.nih.gov/books/bv.fcgi?highlight=32.2&rid=stryer.section.4589#top (17) Esposito, Veronica.; Gallucci, Roberta.; Picone, Delia.; Saviano, Gabriella.; Tancredi, Teodorico.; Temussi, Piero, A. The importance of electrostatic potential in the interaction of sweet proteins with the sweet taste receptor. J. Mol. Biol. 2006, 360, pp 448-456. References (cont.) (18) Morini, Gabriella.; Bassoli, Angela.; and Temussi, Piero, A. From small

sweteners to sweet proteins: anatomy of the binding sites of the human T1R2_T1R3 receptor. J Med. Chem. 2005, 48, pp5520-5529. (19) Nie, Yiling.; Vigues, Stephan.; Hobbs, Jeanette, R.; Conn, Graeme, L.; Munger, Steven, D. Distinct contributions of T1R2 and T1R3 taste receptor subunits to the detection of sweet stimuli. Curr. Biol. 2005, 15, pp 1948-1952. (20) Walters, D. Eric.; Hellekant, Goran. Interactions of the sweet protein Brazzein with the sweet taste receptor. J. Agric. Food Chem. 2006, 54, pp 10129-10133. (21) Jiang, Peihua.; Ji, Qingzhou.; Liu, Zhan.; Snyder, Lenore, A.; Benard, Lumie, M.J.; Margolskee, Robert, F.; Max, Marianna. The cysteine-rich region of T1R3 determines responses to intensely sweet proteins. J. Biol. Chem. 2004, 279, pp 45068-45075. (22) PDB ID: 2d04. Shimizu-Ibuka, A.; Morita, Y.; Terada, T.; Asakura, T.; Nakajima, K.; Iwata, S.; Misaka, T.; Sorimachi, H.; Arai, S.; Abe, K. Crystal structure of Neoculin, a sweet protein with taste-modifying activity. J. Mol. Biol. 2006, 359, pp 148-158. (23) 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)

(24) Bowen, R. Protein hydropathicity plots. 1998. http://arbl.cvmbs.colostate.edu/molkit/hydropathy/ (accessed: 7/22/07) (25) Cook, David, J.; Hollowood, Tracey, A.; Linsorth, Roberts, S.T.; Taylor, Andrew, J. Correlating instrumental measurements of texture and flavour release with human perception. Int. J. Food Sci. Tech. 2005, 40, pp 631-641. References (cont.) (26) Froloff, Nicolas.; Faurion, Annick.; MacLeod, Patrick. Multiple human taste receptor sites: a molecular modeling approach. Chem. Senses. 1997, 21, pp 425-445. (27) Beauchamp, Gary, K.; Reed, Danielle, R.; Tordoff, Michael, G.; Bachmanov, Alexander, A. Genetics of taste. Amer.Chem.Soc. 2002, Chapter. 4, pp 40-51. (28) Cambell, Neil, A. Recombinant DNA. Biology, Third Edition. The Benjamin/Cummings Publishing Company, Inc. 1993, pp 390399. (29) Roy, Glenn.; McDevitt, John, T. In vitro taste sensors: technology and applications. Amer.Chem.Soc. 2002, Chapter. 20, pp 262275. (30)Riul, Antonio, Jr.; Malmegrim, Roger, R.; Fonseca, Fernando, J.; Mattoso, Luiz, H.C. Nano-assembled films for taste sensor

application. Artif. Organs. 2003, 27, pp 469-472. (31) Jensen, Ric. Electronic tongue measure food flavors and water chemistry. Envir. Health, 1999, pp 38. (32) Legin, Andrey.; Rudnitskaya, Alisa.; Clapham, David.; Seleznev, Boris.; Lord, Kevin.; Vlasov, Yuri. Electronic tongue for pharmaceutical analytics: quantification of tastes and masking effects. Anal. Bioanal. Chem. 2004, 380, pp 36-45. (33) Murray, Owen, J.; dang, Wenbin; Bergstrom, David. Using an electronic tongue to optimize taste-masking in a lyophilized orally disintegrating tablet formulation. Pharm. Tech. 2004, pp 42-52. (34) PDB: ID: 2brz. Caldwell, J.E.; Abildgaard, F.; Dzakula, Z.; Ming, D.; Hellekant, G.; Markley, J.l. Solution structure of the thermostable sweet-tasting protein Brazzein. Nat.Struct.Biol. 1998, 5, pp 427-431. References (cont.) (35) PDB ID: 1gxv. Refaee, M.; Tezuka, T.; Akasak, K.; Williamson, M. Pressure-dependent changes in the solution structure of hen egg-white lysozyme. J.Mol.Biol. 2003, 327, pp 857.

(36) PDB ID: 1fa3. Spadaccini, R.; Crescenzi, O.; Tancredi, T.; DeCasamassimi, N.; Saviano, G.; Scognamiglio, R.; DiDonato, A.; Temussi, P.A. Solution structure of a sweet protein: NMR study of MNEI, a single chain Monellin. J.Mol.Biol. 2001, 305, pp 505-514. (37) PDB ID: 2blr. Nanao, M.H.; Sheldrick, G.M.; Ravelli, R.B. Improving radiation-damage substructure of Rip. Acta.Crystallogr. 2005, 61, pp 1227. (38) Masada, Tetsuya.; Kitabatake, Naofumi. Developments in biotechnological production of sweet proteins. J Biosci. Bioeng. 2006, 102, pp 375-389. (39) Masuda, Tetsuya.; Ide, Nobuyuki.; Kitabatake, Naofumi. Structure-sweetness relationship in egg white lysozyme: role of lysine and arginine residues on the eliciatation of lysozyme sweetness. Chem. Senses. 2005, 30; pp 667-681. (40) De Capua, Antonia.; Goodman, Murray.; Amino, Yusuke.; Saviano, Michele.; and Benedetti, Ettore. Conformational analysis of aspartame-based sweeteners by NMR spectroscopy, molecular dynamics simulations, and x-ray diffraction studies. ChemBioChem. 2006, 7, pp 377-387. (41) Vepuri, Suresh, B.; Tawari, Nilesh, R.; Degani, Mariam, S. Quantitative structure-activity relationship study of some aspartic acid

analogues to correlate and predict their sweetness potency. QSAR Comb. Sci. 2007, 26, pp 204-214. References (cont.) (42) Birch, Gordon, G. Role of water in sweet taste chemoreception. Pure Appl. Chem. 2002, 74, pp 11031108. (43) Chemical Reference Database: ChemFinder. CambridgeSoft Corporation, 2004. www.chemfinder.com (accessed: 7/28/07) (44) Tully, William; Vernon, Nicholas, M.; Walsh, Peter, A. Process for the preparation of 1,6-dichloro-1,6dideoxy-.beta.-D-fructofuranosyl-4-chloro-4-deoxy-.alpha. United States Patent:#4,801,700. January 31, 1989 (45) Acid-Base Properties of Proteins. Chemistry; A Project of the American Chemical Society. Freeman. New York, 2005, pp 619-621.

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