Dimeric interface of His11Phe mutant.

<p>Dotted lines show hydrogen bonds at the interface.</p

Dissociation of neoculin oligomer in MD calculations at acidic pH.

<p>A. An early stage (2 ns). B. A late stage (10 ns). Line color is as defined for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126921#pone.0126921.g011" target="_blank">Fig 11</a>. C. Distance between the centroids of each monomer in the 10 ns MD trajectory. Red, blue, purple and orange lines to represent the centroid distances between pairs of monomers represented in green and grey (GG), green and black (GB), green and yellow (GY), and yellow and black (YB), respectively.</p

Hydrogen bond interactions of the Gln90Lys mutant under the neutral condition.

<p>Two initial structures of monomers at the interface.</p

Hydrogen bond network at the dimeric interface of the Gln90Lys mutant under the acidic condition.

<p>Hydrogen bond network at the dimeric interface of the Gln90Lys mutant under the acidic condition.</p

Structural Basis of pH Dependence of Neoculin, a Sweet Taste-Modifying Protein

<div><p>Among proteins utilized as sweeteners, neoculin and miraculin are taste-modifying proteins that exhibit pH-dependent sweetness. Several experiments on neoculin have shown that His11 of neoculin is responsible for pH dependence. We investigated the molecular mechanism of the pH dependence of neoculin by molecular dynamics (MD) calculations. The MD calculations for the dimeric structures of neoculin and His11 mutants showed no significant structural changes for each monomer at neutral and acidic pH levels. The dimeric structure of neoculin dissociated to form isolated monomers under acidic conditions but was maintained at neutral pH. The dimeric structure of the His11Ala mutant, which is sweet at both neutral and acidic pH, showed dissociation at both pH 3 and 7. The His11 residue is located at the interface of the dimer in close proximity to the Asp91 residue of the other monomer. The MD calculations for His11Phe and His11Tyr mutants demonstrated the stability of the dimeric structures at neutral pH and the dissociation of the dimers to isolated monomers. The dissociation of the dimer caused a flexible backbone at the surface that was different from the dimeric interface at the point where the other monomer interacts to form an oligomeric structure. Further MD calculations on the tetrameric structure of neoculin suggested that the flexible backbone contributed to further dissociation of other monomers under acidic conditions. These results suggest that His11 plays a role in the formation of oligomeric structures at pH 7 and that the isolated monomer of neoculin at acidic pH is responsible for sweetness.</p></div

Dimeric structure of His11Tyr mutant under neutral condition.

<p>A. Hydrogen bond interactions shown as dotted lines at the interface. B. Time dependent distances between Tyr11_A and Asp91_B, His14_A and Tyr21_B and Ser15_A and Gln35_B of the mutant under neutral conditions in red, purple and green traces, respectively. C. RMSD values for the main chain atoms of the His11Tyr mutant the 10 ns MD trajectory from the initial structure under the neutral condition (left) and the acidic condition (right). Line color is as defined for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126921#pone.0126921.g002" target="_blank">Fig 2B</a>.</p

Tetrameric structure of neoculin monomers in the crystal structure.

<p>A. The surfaces of Pro103A, Leu106A and Asn44B are colored orange. Another monomer (yellow ribbon) has contact with neoculin near these three residues. The other two neoculin molecules are shown as a grey surface and a black ribbon. B. Tetrameric structure rotated 90° clockwise from the structure depicted in A (left). Rotation of the structure on the left 180° about a vertical axis to show the structures on the other side of the dimer (right).</p

Location of residues showing mobile main chains on the neoculin monomer.

<p>A. The mobile residues are colored in red based on the difference in RMSD value for the main chain atoms of each residue in the MD trajectory. Residues in NAS and NBS are indicated by A and B with the residue number, respectively. B. The difference in RMSD value for the main chain atoms of each residue in the MD trajectory. Bars for mobile residues with RMSD values of ≥0.1 are colored in red and immobile residues with RMSD values of <0.1 are colored in grey. The regions of secondary structures are indicated in blue below the graph, and the yellow-colored regions are loop structures.</p

The dissociated monomers of neoculin His11Ala mutant.

<p>A. Dissociated structures after MD calculations under neutral conditions. B. RMSD values for the main chain atoms of the neoculin mutant in the 10 ns MD trajectory compared to the initial structure (MD under the neutral pH condition). C. RMSD values for the main chain atoms of the neoculin mutant in the MD trajectory from the initial structure (MD under the acidic condition). Line color is as defined for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126921#pone.0126921.g002" target="_blank">Fig 2B</a>.</p

Dissociated monomers of the His11Phe mutant in MD calculations under the acidic condition.

<p>Dissociated monomers of the His11Phe mutant in MD calculations under the acidic condition.</p