Neoculin: A Sweet-Tasting, -Modifying Protein and its Interaction with T1R2 & T1R3 of the Sweet Taste Receptor Family

Jeffrey T. Kushner University of Pennsylvania Department of Chemistry and Masters in Chemistry Education Program Philadelphia, PA 19104-6228

ABSTRACT When discussing sweet tasting molecules, most individuals are more familiar with carbohydrate based sweeteners or artificial sweeteners. A group of sweet-tasting proteins are currently being studied for their potential as the new, low calorie, yet natural sweetener. Of this group, Neoculin, not only sweet, but has a taste-modifying property that enhances the sweet taste in a normally sour, or acidic, solution. Identifying how molecules interact with the T1R2+T1R3 complex is a beginning and important step to understanding the biochemical features required to generate an action potential. The use of X-ray crystallography, gel electrophoresis, and NMR analysis has only begun to enlighten the complex nature of sweet taste perception.

INTRODUCTION Taste is a critical sense for humans. Having the ability to recognize and determine the chemical consistency of a food and to perceive its nutritional value or potential for harm, is a valuable tool.(1) The perception of taste is first received by taste buds located in taste papillae on the , upper and lower palate, and the back of the throat.(1) Each contains about 150 cells. The cell membrane of these cells form microvilli where the nerve receptor is located.(1) These nerve receptors pick- up chemicals dissolved in saliva, oil, and water.(1) If a chemical interacts with a nerve receptor, an action potential is created.(1) This initial electrical impulse is then received by an afferent nerve fiber at the opposite end of the sensory neuron.(1) Afferent nerve pathways carry the electrical signal from the stimulus site to a specific region of the brain for interpretation.(1) Of the five major senses, the perception of sour and salt have similar mechanisms, while sweet, bitter, and unami have similar mechanisms.(2) Although certain taste mechanisms have similar patterns, sensory receptors on a taste bud are specific for one of the five taste senses. The sour taste is detected by direct interactions with hydrogen ion channels found in sensory neurons.(2) Varying concentrations of hydrogen ions determine the strength of the sour taste perceived.(3) Salty taste is detected by passage through sodium ion channels found in the cell membrane of sensory neurons.(2) Varying concentrations of sodium ions determines the strength of the salt taste perceived.(4) Passage of both types of ions produces a significant transmembrane current within the sensory neurons to create an action potential. Unami, the taste of ‘deliciousness’ evoked by the amino acid glutamate, and bitter, stimulated by alkaloids, use separate groups of the seven transmembrane protein 2 Kushner receptors found on the surface of sensory neurons.(2,5) These protein receptors have large numbers of amino acids that innervate with chemicals of each group. The unami taste, stimulated by the consumption of protein-rich foods such as meats and poultry, contain essential amino acids for the body.(2) Consuming potentially toxic chemicals produced by plants and other organisms provokes the bitter taste. Perceiving the bitter taste is a defense mechanism to avoid the consumption of poisons, which could harm the body.(4) Interestingly, many different chemicals contain alkaloid groups, but these tastants all stimulate the same neuron. Humans do not have the ability to distinguish different bitter chemicals, they can only determine different concentrations of the bitter taste.(4,6) A family of three taste receptors detects the sweet taste.(7,8) Prior to 2001, it was hypothesized by scientists that all sweet tasting chemicals were received by the sweet taste receptors the same way.(8) As technological capabilities in X-ray crystallography, NMR, and electrophoresis increased, it has now been determined that each receptor innervates differently with sweet tasting chemicals.(7,8) Sweet taste receptors can detect simple carbohydrates such as glucose and sucrose, which are easily digested and energy rich.(8) Simple peptide derivates, such as aspartame, also stimulate the sweet taste receptors, but do no offer any caloric value for the body. Other artificial, low calorie sweeteners, such as sucralose, a chlorocarbon, and saccharin, a sulfimide, articulate with the sweet taste receptors.(7) Finally, a small group of proteins also has the ability to interact with the sweet taste receptor family. Currently, these proteins are only found naturally in some tropical fruits, but they are a new research focus for the low calorie sweetener industry.(8) Neoculin, found in the Lembah fruit of West Malaysia, is one of the unique chemicals found in the category of sweet tasting proteins.(9-11) With the sweet-tasting characteristic, which is about 500 times sweeter than sucrose (on molar basis), this protein shows great promise as a low calorie, sugar substitute.(9-11) Neoculin has another unique characteristic, which distinguishes it from other sweet-tasting proteins. It has the ability to transform sour tasting chemicals to give the perception of a very sweet taste.(9-11) Current studies have focused on identifying key structural components of Neoculin. The use of X-ray crystallography, NMR, gel electrophoresis and mass spectroscopy are crucial to the research of proteins in taste chemistry.(12) Determining the three-dimensional shapes of Neoculin is aided by X-ray crystallography.(11) This technique first involves the crystallization of the desired chemical.(11) For Neoculin, the protein is first dissolved in an aqueous solution then slowly precipitated over a few weeks through the hanging-drop vapor-diffusion. This method allows a droplet of purified protein, buffer, and precipitant to equilibrate with a reservoir containing similar buffers and precipitants in high concentration.(11) Over time the individual molecules of Neoculin align themselves in a repeating series of unit cells to form a crystalline lattice. This lattice is held together by noncovalent interactions. The Neoculin crystals are then exposed to x-ray waves at a wavelength of 1.0Å.(11) The x-ray data for Neoculin is then collected and manipulated by computers (PHASER program). The Neoculin crystals obtained at the time of research belonged to the orthorhombic space group (P212121). The unit cell dimensions were a=48.0Å, b=101.1Å, and c=271.6Å at a resolution of 2.76Å.(11) 3 Kushner

Nuclear magnetic resonance (NMR) spectroscopy is another technique used to provide information on protein shape and structure.(12-13) NMR uses radio frequencies in the presence of a magnetic field to provide information about a certain group of atoms.(12) Specific atoms that are affected by magnetic fields will align themselves with the strong magnetic field. An oscillating magnetic field, caused by radiowaves, is then applied to the sample.(12) Data is then collected as the specific atoms realign with the primary magnetic field. Over time, the characterization and three-dimensional structure can be determined.(12) At this time, NMR spectroscopy can only be utilized for proteins smaller than 25kDa.(12) Gel electrophoresis is a process that separates and compares proteins based on charged differences.(12) Protein samples can be separated using a polyacrylamide gel.(9) An electric charge is applied to opposite ends of the gel. Over a period of time the proteins can then migrate to one of the opposite poles.(12) Proteins of similar sizes and charges will migrate to similar regions of the gel.(12) Neoculin, the two subunits of Neoculin, and recombinant Neoculin are compared and analyzed by gel electrophoresis.(9) Mass spectroscopy is a newer method used to determine the amino acid sequence of proteins.(14) In this method, the entire peptide sequence or smaller segments of the peptide sequence are delivered to a spectrometer through electrospray ionization. As an enzyme breaks the peptide bonds, the mixture is passed through a high-pressure liquid chromatography column, exposed to a highly positive charge, and then into the mass spectrometer.(14) The mass spectrometer measures the mass- charge rations of amino acid fragments emitted from the column. A spectrum is collected and analyzed by computer to sequence the amino acids of the protein.(14) RESEARCH QUESTIONS: There are three driving questions behind this research. What features of Neoculin allow it to interact/bind with the T1R3 receptor and how is it different from other sweet tasting molecules? Gaining a better understanding of this interaction could clarify functional characteristics of the sweet taste receptor. What features/regions of Neoculin allow it to elicit a sweet taste response with the T1R2+T1R3 complex? The sweet taste receptor complex is known to have unique binding properties with a wide variety of molecules.(8) Neolculin, is one of the larger chemicals to elicit a sweet taste.(10) Finally, what causes the taste-modifying property between Neoculin and the T1R2+T1R3 complex? This property is unique to Neoculin and has not been addressed in detail through research. This paper attempts to, where possible, provide answers or hypotheses to these questions.

ANALYSIS Larger THE HUMAN SWEET TASTE RECEPTORS. To Smaller non-protein sweeteners sweetener begin to understand how Neoculin actively works with the human taste receptor complex, it is important to appreciate the dynamics of the human Seven transmembrane- taste receptor. The human taste receptor is a G helix receptor coupled protein receptor.(15) A G coupled protein Proteins receptor consists of a seven transmembrane-helix receptor that weaves through an area of the cell membrane (fig. 1).(8,16) This receptor protein is Fig. 1—seven transmembrane-helix coupled with T1R2+T1R3 sweet receptor complex.(8) 4 Kushner linked by gustducin, the important signal- transducing molecule of a receptor cell.(8) This protein senses molecules outside of the cell, binds with a molecule, changes conformation, and activates signal transduction pathways to yield a cellular response.(17) Although the full biochemical understanding for determining the sweet taste is still under investigation, a small family of seven transmembrane-helix receptors has been identified to respond to sweet compounds.(7,8,17) Each sweet Table 1—T1R Taste Receptor Function. T1R2 and T1R3 chemical group appears to function with this are primarily responsible for sweet taste detection.(7) family differently. The taste receptor (TR) family consists of three homodimers, which are individual protein groups identified as T1R1 (family 1; member 1), T1R2 (family 1; member 2) and T1R3 (family 1; member 3).(7) T1R2 and T1R3 are the two taste receptors responsible for identifying and binding with sweet chemicals to elicit a sweet response.(7) These two homodimers also form a heterodimer; a quaternary protein complex held together by disulfide bonds. As homodimers (see Table 1), T1R2 and T1R3 show only low binding attraction for sugar compounds.(7) As a heterodimer, they show high binding attraction for sugar compounds and artificial sweeteners. The T1R2+T1R3 complex has a large extracellular region called the Venus flytrap domain. This vase shaped receptor area contains an active site for binding with small ligands.(17-20) There is also a cysteine rich domain which, through recent studies, has shown to bind with sweet proteins.(21) NEOCULIN: Neoculin is a large heterodimeric (fig. 2 & fig. 3) protein consisting of an acid subunit and a basic subunit.(9-11) The Neoculin acidic subunit (NAS) consists of 113 amino acid residues, while the Neoculin basic subunit (NBS) consists of 114 amino acid residues (fig. 4).(11) Although these residues are different, 77% of the amino acid sequence is identical.(11) One of the most significant differences between the two subunits occurs at the outer loop region found on the outer face of each subunit. Loop regions are segments of amino acids usually found between beta-sheets or alpha- helixes. Although these regions do not take on a specific secondary structure, many active regions of proteins are found in these areas. In Neoculin, the loop region of the

Fig.2—Neoculin. Acid subunit in green; Fig.3—Neoculin rotated 180° along Y-axis and X- basic subunit in brown. C-terminus of axis.. Colors displaying amino acid both subunits identified by white sequence.(23) arrow.(22) 5 Kushner basic subunit is believed to be responsible for interacting with the taste receptor to produce a sweet taste response. Interestingly, computer-docking models have shown that the acidic subunit is not favored for bonding with the taste receptor to direct a sweet taste response.(10,11) This feature of taste reception between Neoculin and the taste receptor complex will be analyzed in more detail. Structurally, these two subunits consist of three, four- stranded β-sheets. Each β- sheet series makes up the face of a triangular pyramid.(11) These two complexes have four disulfide bonds between them, two that are intermolecular and two that are intramolecular.(9)

Other than the four disulfide Fig.4—Primary and Secondary sequence information of Neoculin Acidic bonds, there are eight hydrogen Unit (top) and Basic Unit (bottom).(22) PDB ID: 2D04 bonds to help stabilize the overall conformation of Neoculin.(11) These hydrogen bonds are believed to provide the molecule with the flexibility required to take on a number of different conformations. INTERACTION AND BINDING BETWEEN T1R2+T1R3 AND NEOCULIN: Conformational studies of the sweet taste receptors, T1R2 and T1R3, have lead to the development of both an open and closed conformation for each. The open conformations demonstrate an inactive receptor, waiting to bind with a sweet tasting molecule. The closed conformations demonstrate an active receptor, articulating with a sweet tasting chemical eliciting an action potential by the sensory neuron.(19) As a heterodimer, the T1R2+T1R3 complex can have two Fig. 5.—Taste Receptor Binding. Small molecular mass sweetener bond inside cavities of taste receptor different inactive conformations, based complex(top); sweet proteins external binding site(bottom). on which ‘wedge’ site is exposed.(19) Note change in conformation between inactive for (Roo) and Active form (Aoc) and change in equilibrium (arrows) when When the complex is not innervated with receptor is complexed.(8) a ligand, both binding sites are considered open, but due to positioning, only one site can actively participate in binding. The first heterodimer free form, called Roo_AB consists of both taste receptors in the resting state (R) and in the open (Roo) form. T1R2 is on the A chain and closed, T1R3 is on the B chain and open (see fig. 5).(19) The second heterodimer free form, called Roo_BA consists of both taste receptors in the resting state and open form, but T1R3 is 6 Kushner

on the A chain and closed and T1R2 is on the B chain and open.(16) Each inactive conformation has a specific bonded, active form. Aoc_AB matches with the first heterodimer free form where the taste receptor is actively (A) binding with a ligand. T1R2 is on the A chain and closed while T1R3 is on the B chain, in the open conformation, but bound with a ligand. Aoc_BA matches with the second heterodimer free form where the taste receptor is actively binding with a ligand. T1R2 is open on the B chain and actively binding with a ligand and T1R3 is closed on the A chain.(19) Through meticulous computer generated models, Morini, et al theorize that receptor-tastant binding occurs with T1R3 when it is in the open conformation to bind with ligands. This appears to be the more correct and most stable method of heterodimer articulation.(18) T1R2 and T1R3 both have a large negatively charged cavity to receive positively charged fragments of sweet compounds, but the T1R3 cavity appears to be larger and more easily accessible through the computer generated molecular models.(19) When binding with the taste receptor, Neoculin follows the ‘wedge’ site binding method.(17) When the protein complexes with the taste receptor, it stabilizes the receptor through a conformational change. The open, protonated form of Neoculin is the only conformation able to bind with the T1R2+T1R3 complex.(18) At equilibrium, only a fraction of Neoculin molecules adopt this open conformation, but as the pH is lowered, greater numbers of protonated molecules exist.(10) Binding of Neoculin in the open conformation causes the taste receptor to change conformation from inactive (Roo_AB) to active (Aoc_AB).(18) Jiang, et al proved through gene splicing that the cysteine-rich region of T1R3 significantly deters the response to sweet proteins.(21) Using human embryonic kidney cells that have been genetically altered to produce the human taste receptor complex, a group of scientists selectively ‘knocked-out’ the cysteine region (amino acids 536-545) of T1R3.(21) Systematically, certain cysteine amino acids were replaced with other amino acids that were either hydrophobic or contained strongly acidic or basic side groups.(21) This effectively altered the normally hydrophilic, neutral pH region. The altered human embryonic kidney cells were then exposed to many sweeteners including Brazzein, a sweet protein.(21) Although the small molecular weight sweeteners still produced a sweet response, Brazzein did not.(21) The amino acid, cysteine contains a thiol side-chain, which is nearly neutral (pH=5.02) and relatively hydrophilic.(12) It is possible that disulfide linkages are formed between thiol side-chains of T1R3 and other sulfur containing amino acids of Neoculin. There are six cysteine amino acids found between the acidic subunit and basic subunit of Neoculin that are located on an exterior face of the molecule.(22) The potential exists that one of these amino acids could form a disulfide bond with a cysteine amino acid from T1R3. This hypothesized formation of an intermolecular disulfide bond is unlikely though, because inactive T1R3 is regenerated to the inactive form to receive new molecules. Disulfide bonds are relatively strong covalent bonds, which would require a specific mechanism to break these bonds to release Neoculin. Hydrophilicity is another possible interaction between Neoculin and the cysteine-rich region of T1R3. There are a number of hydrophilic regions on both the acid and basic subunits of Neoculin. The basic subunit contains a patch of five amino acids (numbers 45-50) that

7 Kushner are significantly hydrophilic and located near an external face of the molecule. This could be one plausible mechanism for binding between T1R3 and the Neoculin basic subunit. Another potential chemical interaction between T1R3 and Neoculin could be an acidic/basic complementary pair. Since the cysteine region is slightly acidic, it could interact with a basic region located on an exterior surface of Neoculin. Both the acidic and basic subunits of Neoculin contain regions of basicity.(22) The largest of these patches is found on the Neoculin basic subunit, which contains a region of about five to six amino acids.(22) The amino acids are not found consecutively along the sequence, but form a localized region due to folding patterns of the secondary protein structure. This intermolecular interaction is another favorable, plausible mechanism for the interaction between T1R3 and Neoculin. It is an interaction that could occur if the molecules orient themselves correctly and it is an interaction that can be easily broken to regenerate T1R3. As previously mentioned, the external loop region of the Neoculin basic subunit appears to be the preferred region to bind with the T1R2+T1R3 complex.(10) Actual Fig. 6—Computer generated docking model between computer docking models provided about Neoculin (green) T1R2+T1R3 complex. T1R2 in magenta; T1R3 in green.(11) 10,000 possible results.(10) These results were narrowed down to nine preferable combinations where Neoculin and T1R2+T1R3 complex interacted the most. Achieving this maximum interaction could lead to greater stability because of increased hydrogen bonding, stronger affiliations between hydrophobic regions, and greater conformational stability between Neoculin and the T1R2+T1R3 complex.(10-11) From the nine models, one docking model has been selected as a representative hypothesis (see fig. 6).(11) This in silco (computer generated) hypothesis has not been proven or isolated in crystalline form for X-ray crystallography analysis. Current crystallization methods have not been successful for isolating a sweet protein combined with the T1R2+T1R3 complex.(11) When bound together, these chemicals may be too delicate to crystallize. The current method used for crystallization for X-ray analysis requires the chemical to be dissolved in distilled water.(11) It is possible that the complex may separate, deactivating the taste receptor, during dissolution. A sodium carodylate buffer with a pH of 7.4 is used in the handing-drop vapour-diffusion method of crystallization.(11) Since the conformation of Neoculin is pH sensitive, this could be another procedural step that affects crystallization. As technology and laboratory techniques improve, crystallization, X-ray analysis and a computer-generated model of Neoculin with the T1R2+T1R3 complex may be in the near future. FEATURES OF NEOCULIN AND SWEET TASTE PERCEPTION: Neoculin has a unique electrostatic potential difference believed to contribute to its function (see fig. 7).(17) The surface of the NBS contains thirteen basic residues, seven arginine, three lysine, and three histidine. Six of these compose a large cluster of basicity; two of which are histidine, which have a pKa=6.0, but the NBS only elicits a slight sweetness at neutral pH.(17) The basic residues found on the dimer interface are unprotonated at 8 Kushner

Fig. 7—Computer generated model of polar residues in Neoculin. Acid subunit in yellow; basic subunit in orange. Acidic regions are red; basic regions are blue.(11) neutral pH. The unprotonated form of Neoculin takes on a closed conformation. This conformation is less flexible, has minimal hydrogen bonding and surface interaction with the taste receptor complex. The resulting sweet taste perception is relatively weak. (11) Under acidic conditions, pH near 2.5, the basic residues of Neoculin are all protonated. This causes a conformational change in the overall structure of Neoculin. The protonated form of Neoculin takes a wide-open conformation. This conformation allows for more flexibility and increase in hydrogen bonding and surface interaction with the taste receptor complex; therefore, sweetness perception is strong.(11) Table 2—Hydrophilicity and Hydrophobicity of NAS and NBS(24) Neoculin Acidic Subunit Neoculin Basic Subunit

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A hydrophathicity plot (see Table 2) of Neoculin was performed to analyze any possible correlation between hydrophilicity and hydrophobicity of the acidic and basic subunits.(24) These diagrams can reveal important information about the polar and non-polar regions of a specific protein sequence. The Hopp-Woods scale graphs the hydrophilicity of an amino acid sequence. Hydrophilic regions are commonly exposed on the folded surface of proteins.(24) The Neoculin acidic subunit contains only two large hydrophilic regions whereas the basic subunit contains six regions. The most hydrophilic region of those identified is found on an exterior surface of the basic subunit (amino acids 45-50). It is also relevant to note that the location of this region is found within a looped area. This will be examined and explained in a later section of this paper. The Kyte-Doolittle scale graphs the hydrophobicity of an amino acid sequence. Hydrophobic regions tend to show where the protein may interact with a lipophilic region of another protein.(24) With a sequence homology of 77%, there are similarities between the regions of greater hydrophobicity (>0.5) found between the two subunits.(10-11) The hydrophobic regions could provide areas of intermolecular interaction to stabilize a certain conformation. These regions could also serve to bind or stabilize an interaction with the human taste receptor. LOOP IMPORTANCE IN NEOCULIN: Loop regions of proteins are caused by secondary folding patterns and intermolecular attractions, like hydrogen bonding, to form the tertiary protein structure. (12) The two main loop regions (see fig. 8) of both the acidic subunit and basic subunits of Neoculin are strikingly similar. They both occur at about the same region of their respective subunits Fig. 8—Loop regions of the Neoculin acidic unit (on left in green) and Neoculin basic unit (on right in brown).(23) and they contain nearly the same peptide sequence.(22) Interestingly, only the basic subunit is known to cause an action potential of the T1R2 + T1R3 complex. It is believed that the Neoculin basic subunit is directly bonded with T1R3 while the Neoculin acidic subunit stabilizes the conformation of the heterodimer when bound to the T1R2+T1R3 complex.(11) The acidic subunit serves a significant role in the taste-modifying ability discussed in the next section. There are only ten different amino acids of the twenty-eight found in the amino acid sequence of both folded regions (fig. 9).(22) These subtle changes in the loop sequence could be responsible for the different functions between the two subunits. Table 3 displays the different amino acids, by position, within the folded region of each chain of the acidic and basic subunit. 10 Kushner

Analysis of the amino acid sequence of both folded regions on the Neoculin acidic and basic subunits has lead to several conclusions. Substitutions found in the first fold, changes the Fig. 9—Amino acid sequence of Neoculin acidic unit (chain A) and Neoculin basic hydropathicity of this subunit chain B. Amino acids found in folded region are highlighted in blue.(22) folded region. In the acidic subunit, only one of the four residues (histadine) has an affinity for water, while the rest of the loop is primarily hydrophobic. The substituted amino acids in the basic residue, histidine and aspartic acid are highly hydrophilic. The sequence difference changes the affinity for water between the two loop regions. Analysis of the second loop region identifies the substitution of asparagine, in the acidic subunit, for lysine, in the basic subunit. This is almost an equal trade off between the two regions. Both amino acids are amines of approximately the same length and molecular mass; they each have a high affinity for water. The only slight difference noted is asparagine, in the acidic subunit, has a neutral pH whereas lysine is slightly basic. Another sharp contrast occurs in the third fold of the subunits. Between the different residues, the acidic subunit contains three of four amino acids that are neutral and hydrophilic in character. The basic subunit replaces two of those residues with amino acids that are basic and hydrophilic in character. This change in pH character could be responsible for the binding between the basic subunit and the cysteine-rich region of T1R3, which is acidic in nature. An acidic/basic complimentary pair is a favorable bonding pair between the T1R3 and the basic subunit. The final two differences between the subunits exchange neutral amino acid groups. Methionine and valine, on the acidic subunit are replaced with one acidic group, aspartic acid, and one neutral group, asparagine, on the basic subunit. The overall changes in amino acid character are responsible for the bonding pattern differences with the T1R3 receptor. Table 3—Amino acid difference between folded regions of NAS and NBS(22) Subunit Acidic Subunit—Chain A Basic Subunit—Chain B Position Symbol Name Symbol Name Fold 1; position 1 Y Tyrosine H Histidine Fold 1; position 3 G Glycine D Aspartic Acid Fold 2; position 3 N Asparagine K Lysine Fold 3; position 3 D Aspartic Acid N Asparagine Fold 3; position 6 G Glycine R Arginine Fold 3; position 7 Q Glutamine R Arginine Fold 3; position 10 Q Glutamine G Glycine Fold 4; position 2 D Aspartic Acid H Histidine Fold 4; position 5 M Methionine N Asparagine Fold 4; position 6 V Valine D Aspartic Acid

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TASTE-MODIFYING: The taste-modifying function of Neoculin appears to be due to the electrostatic potential difference between the two subunits.(17) At neutral pH, the Neoculin acidic subunit has a -3 charge whereas the Neoculin basic subunit has a +3 charge.(11) But, under acidic conditions, the acidic subunit carries a +8 charge and the basic subunit carries a +15 charge.(11) This drastic change in electrostatic potential is due to the protonation of the aspartate and histidine residues found between the subunits.(11) The electrostatic potential difference could also be due to the changes in the amino acid sequence of the folded regions (see fig. 10) discussed in the previous section. Since the basic unit is responsible for binding to T1R3 to elicit the initial sweet response, a change in pH could affect the acidic subunit, causing a change in conformation to increase the number of interactions between the basic subunit and T1R3 F ig. 10—Folded regions of Neoculin Acidic Subunit (top) and Basic to produce a stronger sweet taste. Subunit (bottom). Both ribbon structures have exploded amino acid views to show potential interactions of various amino acids side groups.(23)

Testing Tastants Many products are tested for specific taste characteristics that consumers desire. Before the final product can be tested, scientists have to test the initial ingredients to determine if the human tongue can sense them, and if they have the precise qualities and features necessary. There are three methods scientists use to test for taste: human taste panels, human embryonic kidney cells, and the recently developed, electric tongue. Each of these three methods has their own specific advantages and disadvantages for testing taste. Human taste panels are commonly used to get direct feedback and input regarding certain tastes of chemicals in foods.(25) A panel usually consists of three to fifteen members who are considered taste specialists.(9,25) A taste specialist is an individual who studies the differences in flavor and concentration.(9,25) They learn flavors and tastes similar to how students may study for an exam. By practicing and reviewing known flavors and concentrations, the panel members can then analyze an 12 Kushner unknown sample by attempting to compare it to the known flavors and concentrations they studied. Panel members may even become specialized in one specific taste or flavor, such as sweetness or bitterness.(25) Human taste subjects can provide important insight on the flavors of specific chemicals or food products.(27) There are a few disadvantages to using taste panels though. Taste specialists can only provide qualitative feedback.(9,25) They are unable to give accurate, quantitative data that can be reproduced. Also, to use human test subjects, scientists have to perform detailed studies using animals before receiving permission by the U.S. Food and Drug Administration to begin research with humans.(27) This can be a time-consuming and arduous process to provide enough data to prove that the chemical would be safe for humans. Since the advent of recombinant DNA technology, human embryonic kidney cells have been used to resemble taste tissue found in the human tongue.(9-11) Human embryonic kidney cells are undifferentiated cells. This means they are cells that have not developed kidney tissue function yet, but they have the necessary proteins and enzymes to develop into specialized tissues. Scientists will use these cells to splice out the segments of human DNA responsible for directing kidney specialization and replace them with the genes responsible for a specific human taste receptor.(28) (see recombinant DNA textbox for more information) The cells are allowed to replicate and the genes are turned on Fig 11—Human embryonic kidney cells with human taste so they can be translated into the taste receptors integrated. A) showing inactive cells B) showing cells receptor proteins by the thousands of activated with addition of Neoculin; sweet tasting protein . White and colored arrows showing responding cells of culture.(9) human embryonic kidney cells.(28) This provides scientists with a thin plate, or culture, of taste receptor cells. Certain chemicals can then be applied to the tissue, allowed to incubate, and, after a period of time, examined for interactions.(9) A florescent dye treatment can also be used in conjunction with the taste receptor kidney cells to verify if the chemical activates the cells.(9) (see Fig. 11) This method allows scientists to determine if specific chemical concentrations can be received by the human taste receptor. Although very effective, human embryonic kidney cell tests do not provide any further or more detailed information other than if an interaction occurred or not. Recently, scientists have been experimenting with the development of an electric tongue.(29-33) This device could soon have the ability to test a wide variety of chemicals for taste qualities and concentrations. It could also possibly determine the level of chemical interaction with specific taste receptors and determine the human perception of the taste. An electronic tongue consists of three distinct parts, which parallel human taste function. Thin membranes made with either chalcogenide glass*, plasticized PVC membranes, or silicon transistors with organic coatings found on the electrical probes pick up chemicals in solution similar to how a human tongue receives chemicals.(32) Transducer circuits quantify and digitize the chemical information received and then transmit those signals to the computer. This process mirrors the

*chalcogenide glass—glass containing sulfur, selenium, or tellurium as a substantial constituent; used to allow transmittance of materials. 13 Kushner pathway of nerves from the tongue to the taste location in the brain. Finally, electric have computer software that interprets the sensory data to determine taste patterns and perceptions.(29-33) Like the human brain, sensory chemical information is quickly and carefully analyzed to determine the perceived taste. (see fig. 12) Although still being refined, electric tongue technology has distinct advantages in scientific analysis. Scientists can use small amounts of chemicals to determine taste qualities.(29-33) These chemicals can be analyzed without fear of harming human test subjects. Electric tongue software can process both qualitative Fig 12—Electric Tongue Model; diagram shows 1) sensors; 2) transducer circuits; 3) magnetic solution stirrer. information and quantitative data to give Note—computer with chemical analysis software.(32) scientists a larger perspective of the taste of a chemical.(32) Finally, precision electric tongues can sense, separate, and perceive complex mixtures of a variety of blended tastes, which human taste testers cannot separate.(29-33) This allows scientists to experiment taste chemistry further by studying the impact of changing the chemical make-up of the mixture.

DISCUSSION ADVANCES IN TASTE CHEMISTRY: There have been many significant advances in understanding the chemistry of taste. Prior to 2001, the accepted model for taste perception throughout the tongue was a tongue map.(1) Researchers believed that taste buds were localized to a specific region of the tongue according to the perceived taste. Bitter taste buds were thought to be located in the back of the tongue; sour taste buds located on the lateral sides of the tongue.(1) On the anterior portion of the tongue, salt taste buds were thought to be more medial, while sweet taste buds were thought to be concentrated along the lateral sides of the anterior portion of the tongue.(1) It was also believed that all chemicals shared a common method of stimulating a sweet taste receptor.(1) No matter the size or molecular mass of the molecule, all sweet tasting chemicals were believed to somehow articulate with the inner cavity of a sweet taste receptor. Larger molecules were believed to contain ‘sweet fingers’; molecular extensions that protruded into the small cavity of the sweet taste receptor to elicit a taste response.(9-11,15,18-19) Increased resolution in X-ray crystallography, and advances in NMR technology now provide more detail and insight to receptor-tastant interactions. Molecular images, enhanced by computers, were used to disprove the ‘sweet fingers’ theory.(10-11) Large molecular mass molecules, like Neoculin, have separate binding patterns, sites and properties with superficial aspects of the sweet taste receptor.(18) ELICITING A SWEET TASTE: Although ligand activation varies widely in chemical constitution, there are a few key characteristics that appear to be relevant in the binding

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of a sweet tasting molecule to the taste receptors.(19) The presence of Fig. 13—Ribbon structure of complementary pairs between the Brazzein, tastant and the taste receptor appear isolated from the Pentadiplandra crucial for the binding and change brazzeana fruit. conformation of the complex.(19) Approximatly 500-2000 times There are two important features for sweeter than taste recognition to occur. First, sucrose.(34) hydrogen bonding, with hydrogen bond donors matching with hydrogen bond acceptors, is critical for ligand- receptor stability and conformational change. Second, hydrophobic regions on the tastant matching with

Fig. 14—Ribbon other hydrophobic regions of the Structure of Hen taste receptor appears to allow for Egg White lysozyme. proper tastant orientation within the Approximately taste receptor binding site.(19) It is 200 times sweeter than also noted that most sweet tasting Thaumatin and proteins have a basicity factor which Monellin.(35) appears relevant in interaction with the T1R2+T1R3 complex to activate the sweet taste perception.(9) OTHER SWEET TASTING Fig. 15—Ribbon PROTEINS: Neoculin, as one affiliate structure of of a small group of sweet tasting MNEI; one of two synthetic proteins, also has the property of versions of modifying a sour taste to perceive it Monellin. Approximatetly as sweet.(9-11) However, it is not 90,000 times the only member of the sweet tasting sweeter than sucrose (molar protein group. Brazzein (fig. 13), egg basis).(36) white lysozyme (fig. 14), Monellin (fig. 15), and Thaumatin (fig. 16), are other members of this small

group.(20-21,34-39) Structurally, there are few similarities between Fig. 16—Ribbon structure of these chemicals which helps Thaumatin, demonstrate the diversity in activity of isolated from Thaumatoccus the T1R2+T1R3 complex.(38) There daniellii of West are no tertiary similarities, but all Africa. Approximately sweet tasting proteins do have a 100,000 times richness of β-sheets.(38) The only sweeter than sucrose (molar other common similarity is each does basis).(37) contain disulfide bonds, although the number of disulfide bonds differs between the molecules.(38)

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Brazzein (see fig. 13), at fifty-four amino acid residues long, is the smallest of the sweet tasting proteins.(20,34,38) It follows similar receptor binding patterns to that of simple carbohydrates. This molecule has a high binding affinity for the T1R2 dimer and binds inside the open subunit.(20) Brazzein forms a greater number of hydrogen bonds with the T1R2 receptor because the glutamine side chain found in Brazzein forms compatible ligands with the arginine side chain in the receptor.(20,34) There are some favorable bonds formed with the T1R3 diemer, but it is believed that these help stabilize the active conformation.(20) Egg white lysozyme (fig. 14), from the eggs of hens, contains an amino acid sequence of 129 residues, which elicits a sweet taste.(35,39) The basicity of this molecule is due to lysine regions and arginine regions that are found in the amino acid chain.(39) These critical residues for sweetness are located on a given surface of lysozyme which interacts with the T1R2+T1R3 complex.(39) The other sweet proteins, Monellin (fig. 15) and Thaumatin (fig. 16), are found naturally in various fruits. Thaumatin is a single chain of 202 amino acid residues long.(36-38) The amino acid sequence does not contain any carbohydrate or modified amino acids.(38) It is a basic protein with a high electrostatic point.(38) Monellin, in its natural form, has a low melting point, making it difficult to work with in biochemical applications. Two synthetic forms, SCM and MNEI, have been produced to yield a basic protein with a high electrostatic point (36,38). The synthetic forms of Monellin contain 94 and 96 amino acids respectively. Both of these synthetic forms contain two separate chains which link without disulfide bonds.(38) Through hydrogen bonding and van der Waals forces the two chains interact closely to elicit the sweet taste.(19) Due to these interactions the natural form is unstable when heated. The synthetic forms were joined at the N terminus of one chain and the C terminus of the other chain through a covalent linkage to make them more stable.(36,38) ARTIFICIAL SWEETENERS: The sweet tasting proteins interact with the T1R2+T1R3 complex quite differently than carbohydrate sweeteners and artificial sweeteners. Aspartame is a very flexible molecule, allowing it to interact with the T1R2+T1R3 complex because it can take on a number of available conformations to bind with the sweet taste receptor.(40,41) The L-shaped conformation appears to be the preferred conformation because it possesses a hydrophobic region, which articulates nicely with the hydrophobic region of the T1R2+T1R3 complex.(40,41) The hydrogen bonding nature of the carboxyl ester group also stabilizes the ligand with the taste receptor.(40,41) Saccharin, although not nearly as flexible, also utilizes the hydrophobic interaction and the hydrogen bonding ability of the carboxyl ester group to bond and activate with the T1R2+T1R3 complex.(26) Sucralose, being a small molecule similar to sucrose, can interact by hydrogen bonding in the low molecular weight binding sites of the T1R3 dimer, but the T1R2 dimer helps stabilize the active form conformation.(41) CARBOHYDRATE SWEETENERS: Simple carbohydrates are small, low molecular weight compounds, which utilize the T1R2+T1R3 complex to trigger a sweet taste.(42) Since these molecules are small, they utilize more homodimeric ligand binding to either the T1R2 or T1R3 binding site.(42) It is hypothesized that the other receptor proteins not actively binding with the molecule are used to stabilize the overall conformational change to stimulate an action potential.(18) Glucose, the less sweet of the simple

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Table 4—Natural and Artificial Sweeteners.

Aspartame Saccharin Sucralose Glucose Sucrose carbohydrates, utilizes hydrogen bonding to the N-terminal domain of the T1R2 receptor. In comparison, sucrose utilizes hydrogen bonding to the T1R3 N-terminal domain.(18) Sucrose binds more effectively to the T1R3 binding site than does glucose to the T1R2 binding site.(42) It is hypothesized that the more effective the binding of the tastant to the taste receptor a greater sweet perception will be established.(8,15,26) This hypothesis can be used to explain the drastic differences in sweet perception of small carbohydrates, when compared to artificial sweeteners, and to sweet tasting proteins. Since sucrose is the main common sugar used by most individuals as table sugar, it is given a sweetness factor of one and is used in molar mass comparison for determining the sweet taste perception.(38) When compared to the molar mass of sucrose, glucose is about 0.74 times sweeter than sucrose.(42) Since sucrose binds to the taste receptor more effectively, it has a greater sweet taste perception. It can be assimilated that artificial sweeteners like aspartame (160 times sweeter), saccharin (450 times sweeter), and sucralose (600 times sweeter) effectively bind in higher affinity to the T1R2+T1R3 complex than does sucrose.(26,40- 42) Even more dramatic are the sweet tasting proteins. On average, sweet tasting proteins are perceived as 37,500 to 100,000 times sweeter than sucrose.(20,38) This drastic increase could be related to the greater number of hydrogen bond donor- acceptor pairs found between the two groups and/or the interactions of hydrophobic regions found between the two groups.(19) COMMON PATTERNS FOR SWEET TASTE: Although drastically different, there are a few common trends found between sweet tasting chemicals and their interaction with the T1R2+T1R3 complex. The primary requirement to bind with either the T1R2 and/or T1R3 receptor is some sort of complementary base pairing. Hydrogen bonding appears to be the primary interaction for small molecules while either acid/base pairs, hydrophobicity or hydrophilicity interactions seems to be the primary interaction for large molecules. Either pattern also requires that the ligand be easily separated for other sweet tasting molecules to interact and produce a new action potential. A final trend found with all sweet molecules is the ability to stabilize the conformational change that occurs to elicit a sweet response. Whether through hydrogen bonding, hydrophobic/hydrophilic interactions, or acid/base pairings, the taste receptor-tastant complex must remain stable in either conformation.

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Natural or Artificial? Recombinant DNA Technology used in the Production of Neoculin Found in the Lembah fruit of West Malaysia, the sweet tasting protein, Neoculin, shows promise as a suitable low calorie sweetener. The disadvantage of this protein is its remote isolated location and limited capability as a producible crop. Using recombinant DNA technology scientists have developed a method to produce Neoculin. Aspergillus oryzae, a type of fungus commonly used in Japan as a recombinant host, can be used to synthesize Neoculin while maintaining both its sweet taste and taste- modifying ability.(9) Through extensive research and testing using gel electrophoresis and X-ray crystallography, scientists have determined that the Neoculin produced by Aspergillus oryzae is structurally identical to the natural form of Neoculin found in the Lembah fruit.(9-11) Recombinant DNA technology, also known as gene cloning, is a process that has been used by scientists for centuries.(28) Initially, crude methods involved limited technology and selective breeding of organisms for desired traits. As vast knowledge has Fig 17—A bacterial plasmid in comparison to a bacterial chromosome. been gained regarding DNA sequences of organisms, Plasmids contain accessory genes not biotechnologists can now more clearly examine and vital to bacterial function.(28) specify a gene sequence or DNA region to be cloned. Using bacterial plasmids*(see fig.17), biotechnologists insert a specific DNA sequence or gene into the ring of DNA. The plasmids are then reintroduced into the bacterial cell for amplification. After the amplification process, the specified gene is repeatedly translated by the newly formed colonies into a protein sequence.(28) The following describes this process for Neoculin in more detail. Researchers at the Department of Applied Biological Chemistry at the University of Tokyo in Japan have been one of the first groups to successfully produce Neoculin through the use of recombinant DNA produced by Aspergillus oryzae, a fiber-like fungus similar to bread mold. Recent attempts have proven to be unsuccessful when researchers have used bacterial cells because of the complex, two-part subunits of Neoculin.(9) Bacterial cells could produce the Neoculin subunits individually in independent cells, but the formation of the correct disulfide linkages between the two subunits could not be bonded in the lab. Using complex, nucleus-containing cells, the correct disulfide bonds could be formed by Aspergillus oryzae.(9) One of the most important steps in recombinant DNA technology is the ability to find and utilize the correct restriction enzyme to isolate the desired Neoculin gene.(9) A restriction enzyme is a complex protein that is used to break phosphodiester (peptide) bonds at a specific segment before the desired gene.(28) This sequence has to be found at or near both the beginning and the end of the gene to be cleaved correctly.(28) Also, the sequence cannot be found within the gene or the gene will be spliced. Restriction enzymes extract the double helix sequence with single-stranded ends called ‘sticky ends’.(28) These ‘sticky ends’ allow the DNA fragment to be correctly inserted

*plasmid—accessory DNA found in a bacterial cell that is not responsible for primary cell processes; plasmids can be exchanged with other bacterial cells. 18 Kushner

into the plasmid. (see fig. 18) DNA ligase, another enzyme forms the covalent bonds between the complementary base pairs between the plasmid and gene sequence.(28) Once the recombinant plasmid has been formed, it is necessary to replicate the plasmid as many times as possible, as fast as possible. (see fig. 19) Researchers use Escherichia coli, a bacteria, to take up the plasmid from cultures through the process of transformation**.(28) E. coli is only used to replicate, or clone, the plasmid containing the recombinant Neoculin DNA.(9) As E. coli replicates into many cells it also replicates copies of the recombinant plasmid. Since the Neoculin protein cannot be successfully complexed with bacterial cells, the replicated plasmids from E. coli have to be isolated and restriction enzymes are once again required to splice the desired gene sequence.(9) Aspergillus oryzae is treated with the same restriction enzyme to form complementary ‘sticky Fig 18—insertion of a gene sequence using ends’ to accept the Neoculin gene.(9) The restriction enzymes and ‘sticky ends’.(28) filamentous fungus is then placed in cultures for about 72 hours to grow and produce

Neoculin. Once the Neoculin protein is

translated, it then needs to be isolated and purified before it is tested against the natural form.(9) The final step in producing recombinant Neoculin is to test the recombinant samples to the natural samples for the sweet taste and the taste- modifying properties.(9) The first test method involves comparing the activation of human embryonic kidney cells that have been engineered to contain the human taste receptor protein.(9) These cells are exposed to natural Neoculin and recombinant Neoculin in separate cultures, and at the time of testing, both were found to activate the human taste receptor protein.(9) The second testing method consists of a three member taste-testing panel. This panel evaluates the similarity between sweet tastes of natural Neoculin Fig 19—the recombinant DNA process using bacteria.(28)

**transformation—process by which extracellular DNA is incorporated into bacterial DNA. 19 Kushner compared to recombinant Neoculin.(9) Once the sweet taste is verified between the two samples, the taste-modifying property will be confirmed.(9-11) Taste testers are given a slightly sour solution of citrate buffer (pH=4) 30 seconds after being given samples of natural Neoculin and recombinant Neoculin.(9-11) Both protein samples should produce a stronger, sweet taste perception to the taste testers instead of a slightly sour taste. The incurring debate is whether Neoculin that is produced using recombinant DNA technology is to be considered natural or artificial. The protein sequence, conformation, and taste quality is exactly identical to that of natural Neoculin, and it is produced from living organisms rather than through a series of chemical reactions like other artificial sweeteners. So, you decide; is this to be considered a natural process or an artificial process?

CONCLUSION Due to the strong preference and over-consumption of sugary foods, diabetes and obesity are reaching pandemic proportions in the United States and other prosperous countries.(21) In part, obesity can be related to the sedentary life styles of children and adults, but foods and soft drinks that are high in sugar content are significantly contributing to the problem. The use of artificial, low calorie or zero calorie sweeteners has become a dietary option in many nutritional guides and diet plans.(21) Many people are hesitant to consume these sweeteners because of health related fears, such as cancer or adverse side effects, such as diarrhea or headaches. The dietary market is in drastic need of a natural, low calorie or zero calorie sweetener. A great potential exists with sweet proteins, such as Neoculin, to fill this multi-million dollar market.(21) Through the use of computer generated molecular modeling, X-ray crystallography, and NMR analysis the specific features of interactions between tastants and taste receptors can be theorized.(9,18,25,40) Although these technologies are highly sophisticated and detailed, they are still limited in their ability to analyze a diverse array of chemical natures. Computer generated molecular modeling, or in silco as described, only theorizes how certain chemical groups may interact. These methods use broad generalizations and low resolution modes which could lead to significant errors in structural conformations of individual molecules. These errors might not allow molecules to form complexes with other molecules.(18) X-ray crystallography and NMR analysis are limited by the chemical species required for analysis. Currently, crystallizing Neoculin at a low pH is not possible due to the nature of the protonated form.(10,11) Further advances in technology may allow for more comprehensive explanations in the chemistry of taste. Recent research studies have revealed the complex and dynamic interactions between the human sweet taste receptor and various sweeteners and sweet proteins. These studies have determined many different bonding patterns and bonding sites that exist to elicit a sweet response. One of the most intriguing chemicals of this group, Neoculin, not only tastes sweet, but has a sweet modifying function that can enhance the stimulation of the sweet taste receptor in the presence of a sour (low pH) solution. It has been determined through experimentation that the sweet taste perception of

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Neoculin is linked to the basic subunit. The sweet response could potentially be due to different types of intermolecular interactions that may occur between the Neoculin basic subunit and the T1R3 protein receptor. Currently, research studies are attempting to determine the factors contributing to the taste-modifying property of Neoculin. A conformational change of Neoculin during its interaction with the T1R2+T1R3 complex could be due to polar residues on the acidic subunit. The taste-modifying ability, along with the low calorie characteristic of this molecule, makes it a possible substitute for carbohydrate sweeteners and artificial sweeteners. Current studies have revealed how this molecule interacts with the human T1R2+T1R3 complex to trigger an action potential for determining a sweet taste. Future studies may reveal in more detail how the taste modifying properties trigger a stronger action potential, and how Neoculin can be produced synthetically in mass quantities for distribution.

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