Soft Drinks

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Sugary foods
Registered trademarks of PepsiCo, Inc., 2007
(www.pepsi.com www.mtdew.com www.mugrootbeer.com
), Coca-Cola Company, 2007 (www.thecoca-colacompan
y.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 )
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Sweet Chemicals
Chemical Name Structure
Glucose
Sucrose
Fructose
www.chemfinder.com
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Obesity Map--2006
Percentage of obese adults (gt30BMI)
Overweight and Obesity. Center for Disease
Control and Prevention. 2007. (accessed
8/5/07) http//www.cdc.gov/nccdphp/dnpa/obesity/tr
end/maps/index.htm
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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)
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Artificial Sweeteners
Sweetener Structure
Aspartame
Saccharin
Sucralose
www.chemfinder.com
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Synthesis of Sucralose
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Artificial sweeteners

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

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Dietary market

• In need of a natural low calorie or zero calorie
  sweetener
• Sweet Proteins?

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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
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Research Focus

• Features of Neoculin and interaction with T1R3
  receptor
• Regions of Neoculin that elicit sweet taste
  response with T1R2T1R3 complex
• Cause of Taste Modifying Function

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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.
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Taste Overview
Sour
Salt

• Different mechanisms for simple tastes vs.
  complex tastes

Adapted from Lindeman, Bernd. Taste Reception.
Physiological Reviews. 1996, 76, pp751.
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Taste Overview

• Series of events to elicit an action potential

University of Arizona, 2007 (www.neurobio.arizona.
edu)
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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.
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Taste Receptor Overview

• Homodimers
• T1Rs
• Heterodimer
• T1R2T1R3
• Sweet taste receptor complex
• Venus flytrap domain
• Sweeteners
• Proteins

Large non-protein sweeteners
Small sweeteners
7-transmembrane helix-receptor
proteins
Adapted from Temussi, Pierandrea. J. Mol. Recog.
2006, 19, pp 188-199.
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Neoculin Overview

• Acidic Subunit
• 114 amino acids

• From Curculigo latifolia plant (West Malaysia)

• Basic Subunit
• 113 amino acids

PDB ID 2d04.
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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)
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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

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Protonations
Histidine
Aspartate
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Features of the T1R3
Inactive
Active

• Cysteine-rich region
• Negatively charged
• cavity

Adapted from Temussi, Pierandrea. J. Mol. Recog.
2006, 19, pp 192
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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, 621 (2005)
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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

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Amino Acid Difference
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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
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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
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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
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Taste-Modifying

• Possibly due to drastic change in electrostatic
  potential of heterodimer
• OR
• Protonation of only acidic unit

PDB ID 2d04.
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Conclusions

• Binding between NBS T1R3 at loop region
• NAS aids conformational change
• Protonated form of Neoculin changes conformation
  for Taste-Modifying activity

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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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References

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References (cont.)

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References (cont.)

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