Large-Scale All-Electron Quantum Chemical Calculation
Toward a Sweet-Tasting Protein, Brazzein, and Its Mutants
Yoichiro Yagi 1,2 and Yoshinobu Naoshima 1,212

1Institute of Natural Science, Okayama University of Science, Japan
Graduate School of Informatics, Okayama University of Science, Japan

Introduction

It had been recognized for many years that only small
molecules were capable of causing a sweet taste. The search forsweeteners, however, found out naturally occurring sweettasting macromolecules, namely sweet proteins, in a variety ofWest African and South Asian fruits. Thaumatin was firstidentified as one of the sweet proteins, and then monellin,mabinlin, pentadin, curculin, brazzein, and neoculin wereisolated sequentially. Sweet-tasting proteins are expected to be apotential replacement for natural sugars and artificial sweeteners.
The human sweet taste receptor is a heterodimer of two Gprotein coupled receptor subunits, T1R2 and T1R3, and broadly
responsive to natural sugars, artificial sweeteners, D-amino acids,and sweet-tasting proteins. The three-dimensional structure of
the sweet receptor protein T1R2/T1R3 is still unknown, and theexact mode for interaction of sweet-tasting proteins with theT1R2/T1R3 receptor has not yet been elucidated. Very recently
the recognition patterns of T1R2/T1R3 for small molecular
sweeteners were suggested [1].
We have recently been carrying out computational
simulations for saccharides and sweet-tasting proteins [2, 3].
The purpose of this study is to clarify characteristics of sweettasting materials and their functions and to search highly
functional materials for foods and pharmaceutical agents on thebasis of electronic state calculations. This work also aims toelucidate the mechanism for the expression of sweetness. A
small protein, des-pGlu brazzein, is one of the sweetest protein
sweeteners so far discovered and 4,000 times sweeter on aweight basis than a 2% sucrose solution. The protein iscomposed of a single polypeptide chain bearing 53 amino acidresidues and four disulfide bonds [4]. To examine a relationshipbetween the sweetness of protein sweeteners and their electronic
properties, we are currently working all-electron quantum
chemical calculations on des-pGlu brazzein and two different
mutants, Glu41Lys and Arg43Ala, using a density functional

method program, ProteinDF. The former mutant is sweeter thanthe brazzein and the latter mutant has a taste like water.
2Computational Methods

The NMR structure of brazzein was downloaded fromProtein Data Bank (PDB code: 2brz) and the structure of despGlu brazzein (Fig. 1(a)) obtained by removal of N-terminal
pyro-grutamate from brazzein. Since the structures of two
different mutants Glu41Lys and Arg43Ala are not available in
the Protein Data Bank, we mutated the amino acid residues
Glu41 and Arg43 in des-pGlu brazzein to Lys and Ala,
respectively, by using ProteinEditor implemented in ProteinDF
software package. Fig. 1(b) and (c) show the structures of bothmutants Glu41Lys and Arg43Ala.
2.1

Molecular Dynamics Calculation and Energy
Minimization
We used the commercial software package AMBER9 tocarry out molecular dynamics (MD) calculations and energy
minimizations on the structures of des-pGlu brazzein and twomutants Glu41Lys and Arg43Ala. All protein molecules weresolvated with TIP3P model water molecules within 8.0 Å ofrespective proteins, and MD calculations were conducted for
30ps at 300K to relax local structural distortions. The structures
thus obtained were optimized by an energy minimization. TheMD calculations and energy minimizations were performed on a
64bit Itanium2 system PC Linux Workstation.
2.2

All-Electron Quantum Chemical Calculation
After energy minimization, the surrounding water molecules
were removed and counterions were placed near each charged
amino acid residue to neutralize the system using ProteinEditor.
The positions of counterions were adjusted by the molecularArg43
Ala43

Glu41
(a)

Lys41
(b)

(c)

Fig. 1. Structures of des-pGlu brazzein (a) and Two Mutants Glu41Lys (b) and Arg43Ala (c).

-0.2eV

0.2eV

Asp29

Asp29

Tyr8

His31

Asp29

His31

His31

Tyr8
Arg33

Tyr8

Arg33

Asn44

Arg33

Asn44
Asn44
Arg43

Arg43

Lys6

Lys6

Ala43

Lys6
Lys41

Glu41

Glu41(b)(a)(c)

Fig. 2. Electrostatic Potential Maps of des-pGlu brazzein (a) and Two Mutants Glu41Lys (b) and Arg43Ala (c).
Color Code: Red Negative Charge, Green Neutral, and Blue Positive Charge.
mechanics calculation using AMBER9. The resulting structures
were subsequently subjected to an all-electron quantum
chemical calculation with the Slater and Vosko-Wilk-Nusair(SVWN) functional using ProteinDF. These ProteinDF
calculations were carried out on a Core2Duo system PC Linux
Cluster (16 Cores) and gave the electrostatic potential (ESP)
map and the highest occupied and lowest unoccupied molecularorbitals (HOMO and LUMO).
3

Results and Discussion

3.1

des-pGlu brazzein
As shown in Fig. 2(a), the calculated ESP map for des-pGlu
brazzein showed that the basic amino acid residues such as Lys6,
His31, Arg33, and Arg43 have positive charge, while the acidic
amino acid residues such as Asp29 and Glu41 and the Cterminal Tyr54 residue have negative charge. In addition, we
found that positive charge is evident on the neutral amino acidresidue Tyr8. A comparison of our computational results withexperimental results on the sweetness of mutants [4] suggeststhat these charged amino acid residues are involved in changing
and/or eliciting the sweetness of des-pGlu brazzein.
3.2

Two Mutants Glu41Lys and Arg43Ala
The analogous ProteinDF calculations on two mutantsGlu41Lys and Arg43Ala were carried out to examine arelationship between the sweetness of protein sweeteners andtheir electronic properties. The calculated ESP maps for twomutants Glu41Lys and Arg43Ala are shown in Fig. 2(b) and (c),respectively. Table 1 illustrates the electric charge of theselected amino acid residues in des-pGlu brazzein and Glu41Lysand Arg43Ala mutants. For Glu41Lys mutant, Fig. 2(b) andTable 1. Electric Charge of the Selected Amino AcidResidues in des-pGlu brazzein and TwoMutants.
Amino AcidResiduedes-pGlu
brazzein

Tyr8+++

His31
Arg33

Arg43+−+

Asn440Glu41++: Strongly Positive−: Negative

Table 1 show that the electric charge of the basic amino acidresidues Arg33 and Arg43 and the neutral amino acid residue
Tyr8 became more strongly positive; the neutral amino acidresidue Asn44 became positively charged. Consequently, thenumber of amino acid residues bearing positive charge increased
and the positive charge widely spread over the mutant. On theother hand, for Arg43Ala mutant, Fig. 2(c) and Table 1 clearly
reveal that the positive charge of the basic amino acid residue
Arg33 and the neutral amino acid residue Tyr8 was neutralized.
Thus, the number of positively charged amino acid residues
evidently decreased in this mutant.
The neutral amino acid residues such as Tyr8 and Asn44 andthe basic amino acid residues such as His31, Arg33, and Arg43
are expected to be closely associated to the interaction with thehuman sweet receptor protein T1R2/T1R3.
4

The all-electron quantum chemical calculations toward thesweet-tasting protein des-pGlu brazzein and two different
mutants Glu41Lys and Arg43Ala were performed to explore therelationship between the sweetness of protein sweeteners andtheir electronic properties. The ProteinDF computations indicatethat the charged amino acid residues and charge distribution ofsweet-tasting proteins can play important roles in the interaction
between the proteins and the human sweet receptor protein
T1R2/T1R3.
Acknowledgements
We wish to thank Mr. Yohei Shibasaki for his
computational works and great assistance. We would
also like to thank the members of Naoshima laboratory
for their helpful advices and discussions.
References[1]Glu41LysArg43Ala++
++
++

0+

[2]0−

[3]++
++
+: Positive00: NeutralConclusion[4]K. Masuda, A. Koizumi, K. Nakajima, T. Tanaka, K. Abe, T.
Misaka, and M. Ishiguro, Characterization of the Modes ofBinding between Human Sweet Taste Receptor and LowMolecular-Weight Sweet Compounds, PLoS ONE, 7, e35380(2012).
Y. Shibasaki, Y. Fujimoto, and Y. Naoshima, MolecularComputational Simulations for Sugars –Studies Toward theDevelopment of Frontier Sweetening Agents–, Proceedings of the28th Japan Simulation Conference, 151-154, 2009.
Y. Yagi and Y. Naoshima, Electronic State Calculation toward
Sweet-Tasting Proteins, The 2011 Joint Conference of CBI & JSBi,CBI-P1-6, 2011.
G. Hellekant and V. Danilova, Brazzein a Small, Sweet Protein:
Discovery and Physiological Overview, Chemical Senses,
30(suppl 1): i88-i89, 2005.