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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

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.
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pone.0158372.g007: 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.

Mentions: The strong interaction between the carboxyl proton of E23 and the carbonyl oxygen in G30 contributes to maintain the integrity of the fold in MNEI by sealing a network of stabilizing H-bonds that ensures thermal stability. In order to recreate these interactions while removing pH dependancy, we analyzed the model of mutant MNEI-E23Q. As previously observed, removal of the ionizable side chain of E23 eliminates the dependancy of the stability from pH [24]. At the same time, the glutamine side chain should allow the formation of the favorable network of interactions observed in MNEI-GLH, likely increasing thermal stability at alkaline pH. Moreover, compared to the described alanine mutant [24], the presence of a side chain of comparable length in Q23 would avoid small distortion in the three dimensional structure, thus preserving the sweetness of MNEI. We know in fact that even slight deformations in the protein shape and local flexibility, such as those introduced by a single point alanine mutation, are able to impair the interaction with the sweet taste receptor and reduce the sweetness [81,82]. Again, we ran three independent simulations at 300 and 473 K. Analysis of the RMSD and RMSF plots shows that the structure is very stable at room temperature, with the area of highest flexibility localized in the L45 loop (residues 78–83), in accordance with what observed for the native structure. The mutant remains stable even after 10 ns simulations at 473 K, displaying minimal RMSD from the starting structure on the observed time scales even at such high temperatures (not shown). RMSF plots (Fig 5) show that, even at high temperature, molecular motions are limited, and deviations from the experimental structure remain below 2 Å. Loop L23 and, in general, the loops between the β-strands show comparable mobility as in MNEI-GLH. Moreover, loop Lα2 is globally more rigid than in the structures containing the glutamic acid in either protonation state. Secondary structure analysis confirms that the α and β elements are stable throughout the simulations (S2 and S3 Figs). In each of the three 300 K simulations, the side chain of Q23 establishes an interaction with the backbone oxygen of G30, helping retrace the stabilizing H-bonds between the helix and β2 of MNEI-GLH, thus incrementing resistance to unfolding at any pH. These interactions are listed in Table 2 and depicted in Fig 6. Water does not penetrate in the Lα2 loop, as proven by a RSA below 5% for Q23 during the whole simulation, very close to what obtained in the case of MNEI-GLH. A similar situation is also observed in the simulations at 473 K. Although MNEI-E23Q would not be affected by water penetration in loop Lα2, these data suggest that, despite increased molecular motions, the structure remains steadily in place, retarding unfolding. This situation is described in Fig 7, in which is reported the distribution of the RSA for residue 23 over the three simulations in the mutant and the parent protein in both protonation states, providing an indication of water penetration and flexibility of the hydrophobic pocket.


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

Leone S, Picone D - PLoS ONE (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0158372.g007: 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.
Mentions: The strong interaction between the carboxyl proton of E23 and the carbonyl oxygen in G30 contributes to maintain the integrity of the fold in MNEI by sealing a network of stabilizing H-bonds that ensures thermal stability. In order to recreate these interactions while removing pH dependancy, we analyzed the model of mutant MNEI-E23Q. As previously observed, removal of the ionizable side chain of E23 eliminates the dependancy of the stability from pH [24]. At the same time, the glutamine side chain should allow the formation of the favorable network of interactions observed in MNEI-GLH, likely increasing thermal stability at alkaline pH. Moreover, compared to the described alanine mutant [24], the presence of a side chain of comparable length in Q23 would avoid small distortion in the three dimensional structure, thus preserving the sweetness of MNEI. We know in fact that even slight deformations in the protein shape and local flexibility, such as those introduced by a single point alanine mutation, are able to impair the interaction with the sweet taste receptor and reduce the sweetness [81,82]. Again, we ran three independent simulations at 300 and 473 K. Analysis of the RMSD and RMSF plots shows that the structure is very stable at room temperature, with the area of highest flexibility localized in the L45 loop (residues 78–83), in accordance with what observed for the native structure. The mutant remains stable even after 10 ns simulations at 473 K, displaying minimal RMSD from the starting structure on the observed time scales even at such high temperatures (not shown). RMSF plots (Fig 5) show that, even at high temperature, molecular motions are limited, and deviations from the experimental structure remain below 2 Å. Loop L23 and, in general, the loops between the β-strands show comparable mobility as in MNEI-GLH. Moreover, loop Lα2 is globally more rigid than in the structures containing the glutamic acid in either protonation state. Secondary structure analysis confirms that the α and β elements are stable throughout the simulations (S2 and S3 Figs). In each of the three 300 K simulations, the side chain of Q23 establishes an interaction with the backbone oxygen of G30, helping retrace the stabilizing H-bonds between the helix and β2 of MNEI-GLH, thus incrementing resistance to unfolding at any pH. These interactions are listed in Table 2 and depicted in Fig 6. Water does not penetrate in the Lα2 loop, as proven by a RSA below 5% for Q23 during the whole simulation, very close to what obtained in the case of MNEI-GLH. A similar situation is also observed in the simulations at 473 K. Although MNEI-E23Q would not be affected by water penetration in loop Lα2, these data suggest that, despite increased molecular motions, the structure remains steadily in place, retarding unfolding. This situation is described in Fig 7, in which is reported the distribution of the RSA for residue 23 over the three simulations in the mutant and the parent protein in both protonation states, providing an indication of water penetration and flexibility of the hydrophobic pocket.

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