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. 2016 Jun 24;11(6):e0158372.
doi: 10.1371/journal.pone.0158372. eCollection 2016.

Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein

Serena Leone  1 , Delia Picone  1
Affiliations

Molecular Dynamics Driven Design of pH-Stabilized Mutants of MNEI, a Sweet Protein

Serena Leone et al. PLoS One. .

Abstract

MNEI is a single chain derivative of monellin, a plant protein that can interact with the human sweet taste receptor, being therefore perceived as sweet. This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry. Unfortunately, applications of MNEI have been so far limited by its intrinsic sensitivity to some pH and temperature conditions, which could occur in industrial processes. Changes in physical parameters can, in fact, lead to irreversible protein denaturation, as well as aggregation and precipitation. It has been previously shown that the correlation between pH and stability in MNEI derives from the presence of a single glutamic residue in a hydrophobic pocket of the protein. We have used molecular dynamics to study the consequences, at the atomic level, of the protonation state of such residue and have identified the network of intramolecular interactions responsible for MNEI stability at acidic pH. Based on this information, we have designed a pH-independent, stabilized mutant of MNEI and confirmed its increased stability by both molecular modeling and experimental techniques.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Comparison of available experimental structures of MNEI.
Overlay of experimental structures of MNEI derived from NMR (1FA3, green) and X-rays crystallography (2O9U, red). The image focuses on loop Lα2 and on the distances E23-Q28 and E23-29 (dotted lines), defining the opening of the loop and allowing for water penetration from the bulk of the solvent.
Fig 2
Fig 2. Cα-Root Mean Square Fluctuation for the different MD simulations.
The figure shows the Cα-RMSD for the various runs at 300 K (A) and 473 K (B) on MNEI-GLU (MNEI-GLU_1, black; MNEI-GLU_2, red; MNEI-GLU_3, green) and MNEI-GLH (MNEI-GLH_1, blue; MNEI-GLH_2, cyan; MNEI-GLH_3, magenta). In all the simulation at room temperature, the structure remains stable and the highest flexibility is observed at the loops between the secondary structure elements. When the temperature is increased, the N-terminal portion exhibits a wider displacement from the experimental fold if E23 side chain is deprotonated.
Fig 3
Fig 3. Time evolution of secondary structure.
The figure displays the change over time of secondary structure for run MNEI-GLU_473_2 (A) and the percentage of residual helical structure (B) for MNEI-GLU (MNEI-GLU_1, black; MNEI-GLU_2, red; MNEI-GLU_3, green) and MNEI-GLH (MNEI-GLH_1, blue; MNEI-GLH_2, cyan; MNEI-GLH_3, magenta). Secondary structure elements were calculated with DSSP and are color coded (blue, α-helix; red, β-sheets; yellow, turns; green, bend; grey, 3-helix). During high temperature simulations, MNEI-GLU consistently looses secondary structure of portions of the helix, which remains structured for as little as 20% of the starting value. Residues in proximity of the Lα2 loop become partially unstructured after only 2 ns. Secondary structure plots for all the simulations are reported in the supplementary material.
Fig 4
Fig 4. Main stabilizing interactions from MD simulations.
Snapshots of MD trajectories at 300 K describing the principal non-secondary structure interactions in MNEI-GLU (blue) and MNEI-GLH (pink). Penetration of a water molecule in the loop does not compromise the stability of the structure at room temperature, but disrupts the zipping network of H-bonds occurring when E23 side chain is protonated, leading to faster unfolding of the helix in the simulations at 473 K.
Fig 5
Fig 5. Cα-Root Mean Square Fluctuation of MNEI-E23Q.
Cα-Root Mean Square Fluctuation for the three independent 10 ns MD simulations at 300 K (A) and 473 K (B) on MNEI-E23Q (MNEI-E23Q_1, pink; MNEI-E23Q_2, green; MNEI-E23Q_3, orange). Increasing the temperature of the MD runs leads to more flexibility at the loops, but stability is comparable to the simulations at room temperature.
Fig 6
Fig 6. Stabilizing interactions in MNEI-E23Q.
Snapshot of MD trajectory at 300 K describing the principal non-secondary structure interactions in MNEI-E23Q. These hydrogen bonds recreate the network stabilizing MNEI-GLH, allowing for the same thermal stability at any pH.
Fig 7
Fig 7. Relative Surface Accessibility from MD simulations.
Distribution of relative surface accessibility of E23/Q23 in the three MD simulations for MNEI-GLU (black), MNEI-GLH (red), MNEI-GLN (green) at 300 (A) and 473 K (B). The figure shows that residue 23 remains stably buried in loop Lα2 in the simulations on MNEI-GLH and MNEI-E23Q. When the residue is deprotonated, average RSA is around 20%, a value that represents partial accessibility to the bulk solvent. At high temperature, partial unfolding of the helix C-terminal completely exposes E23 in MNEI-GLU, whereas the residue remains inaccessible to the bulk water in MNEI-GLH and MNEI-GLU.
Fig 8
Fig 8. Thermal stability of MNEI and MNEI-E23Q.
Comparison of CD unfolding curves for MNEI (A) and MNEI-E23Q (B) at pH 3.5 (red), 5.1 (orange), 6.8 (green) and 8.0 (blue). The image shows that melting temperature of MNEI drops about 15°C at neutral to alkaline pHs. On the contrary, MNEI-E23Q exhibits comparable stability to MNEI at acidic pH, and preserves this quality at neutral and alkaline pH.
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Grants and funding

This work was supported by grant 2011-PDR-19 by Fondazione con il SUD (www.fondazioneconilsud.it).

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