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

Leone S, Picone D - PLoS ONE (2016)

Bottom Line: This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Sciences, University of Naples Federico II, Naples, Italy.

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.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0158372.g002: 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.

Mentions: The values obtained for the pKa of E23, either from existing literature and from the NMR structures, point toward the occurrence of a switch in its protonation state at neutral to mildly alkaline pHs. This is indeed the pH range at which irreversible denaturation and precipitation of MNEI has been observed [14,31,43,44]. As previously noted, MNEI does not contain histidines and E23 is the only residue that can undergo a protonation change in this range of pH. To investigate the consequences at the atomic level of this event, we performed three independent 10 ns MD simulations for each of the two protonation states of E23 on PDB model 1FA3 (MNEI-GLU/ MNEI-GLH). The simulations were carried out at room temperature (300 K) and high temperature (473 K), to accelerate potential local unfolding events in consequence of different stabilities, according to well established protocols [64–72]. Although this approach may not allow exhaustive sampling of all the conformations assumed by the partially unfolded protein, which would require, for instance, longer simulations at lower temperature [73,74], it is nonetheless suitable to highlight the weak spots within the structure where unfolding initiates, suggesting potential sites for genetic manipulations. The simulations at room temperature confirmed the structure stability at every pH, in accordance with experimental data [24,43,44]. The Cα-RMSD plot for each simulation reaches a plateau within 2 Å from the NMR structure (S1 Fig), whereas the plot of the RMS fluctuation for each residue shows that the regions of higher flexibility are localized, as expected, at the N- and C-termini and at the loops between the strands (Fig 2A). The helix appears stably positioned in the β-grasp and the mobility of its C-terminus, where E23 is located, is substantially unaffected by its protonation state. Upon increase of the simulation temperature to 473 K, the structure of MNEI-GLH is substantially unaffected: the RMSD remains within 3 Å of the NMR structure for the first 7 ns of the simulation and unfolding begins only at the end of the MD run (S1 Fig). This is consistent with the experimentally observed thermostability at acidic pH of MNEI [24,43,44]. When E23 is deprotonated, the RMSD diverges, reaching values above 5 Å after only 4 ns, and the protein proceeds toward fast unfolding. A plot of the RMSF shows that N-terminal of the protein, up to residue 40, becomes increasingly mobile (Fig 2B), with the helix being displaced from its original position up to 7 Å. The remaining portion of the protein is more stable, but either the loops and the β-strands have higher mobility compared to MNEI-GLH, suggesting that the destabilization is conveyed through the entire protein structure.


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

Leone S, Picone D - PLoS ONE (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920389&req=5

pone.0158372.g002: 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.
Mentions: The values obtained for the pKa of E23, either from existing literature and from the NMR structures, point toward the occurrence of a switch in its protonation state at neutral to mildly alkaline pHs. This is indeed the pH range at which irreversible denaturation and precipitation of MNEI has been observed [14,31,43,44]. As previously noted, MNEI does not contain histidines and E23 is the only residue that can undergo a protonation change in this range of pH. To investigate the consequences at the atomic level of this event, we performed three independent 10 ns MD simulations for each of the two protonation states of E23 on PDB model 1FA3 (MNEI-GLU/ MNEI-GLH). The simulations were carried out at room temperature (300 K) and high temperature (473 K), to accelerate potential local unfolding events in consequence of different stabilities, according to well established protocols [64–72]. Although this approach may not allow exhaustive sampling of all the conformations assumed by the partially unfolded protein, which would require, for instance, longer simulations at lower temperature [73,74], it is nonetheless suitable to highlight the weak spots within the structure where unfolding initiates, suggesting potential sites for genetic manipulations. The simulations at room temperature confirmed the structure stability at every pH, in accordance with experimental data [24,43,44]. The Cα-RMSD plot for each simulation reaches a plateau within 2 Å from the NMR structure (S1 Fig), whereas the plot of the RMS fluctuation for each residue shows that the regions of higher flexibility are localized, as expected, at the N- and C-termini and at the loops between the strands (Fig 2A). The helix appears stably positioned in the β-grasp and the mobility of its C-terminus, where E23 is located, is substantially unaffected by its protonation state. Upon increase of the simulation temperature to 473 K, the structure of MNEI-GLH is substantially unaffected: the RMSD remains within 3 Å of the NMR structure for the first 7 ns of the simulation and unfolding begins only at the end of the MD run (S1 Fig). This is consistent with the experimentally observed thermostability at acidic pH of MNEI [24,43,44]. When E23 is deprotonated, the RMSD diverges, reaching values above 5 Å after only 4 ns, and the protein proceeds toward fast unfolding. A plot of the RMSF shows that N-terminal of the protein, up to residue 40, becomes increasingly mobile (Fig 2B), with the helix being displaced from its original position up to 7 Å. The remaining portion of the protein is more stable, but either the loops and the β-strands have higher mobility compared to MNEI-GLH, suggesting that the destabilization is conveyed through the entire protein structure.

Bottom Line: This unusual physiological activity makes MNEI a potential template for the design of new sugar replacers for the food and beverage industry.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical Sciences, University of Naples Federico II, Naples, Italy.

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.

No MeSH data available.


Related in: MedlinePlus