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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

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.
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pone.0158372.g004: 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.

Mentions: To understand the nature of these motions and their effect on the fold, we performed secondary structure calculations over time with DSSP [75,76]. Analysis of the simulations at 300 K shows no substantial deviation of secondary structure elements from their experimental description, a situation also observed for MNEI-GLH at 473 K (S2 and S3 Figs). When E23 side chain is ionized, though, we observe a partial disruption of the secondary structure starting as early as 4 ns, which explains the observed RMSD increase. Unfolding occurs to different extents in the three independent simulations, but it always involves residues at the C-terminal portions of the helix, as exemplified in Fig 3A, which shows the DSSP plot for one of the high temperature simulations on MNEI-GLU. While the helical structure is only marginally lost in the 473 K simulations on MNEI-GLH, the percentage of residual helical structure drops up to 20% of the starting value in MNEI-GLU (Fig 3B). To understand the contribution of E23 to the protein stability, we evaluated the intra-molecular interactions occurring in the native state from the simulations at 300 K. The protonated side chain can form a very stable H-bond with the carbonyl oxygen of G30: the average distance between the carboxyl in the side chain of E23 and G30(O) is 2.78 Å and the occurrence of such bond is 99.4% over the three simulations. By tightening the loop between the helix and β2, several non-secondary structure H-bonds between side chains are formed and show high occupancy, adding stability to the fold, as reported in Table 1. Despite being situated on a flexible loop, E23 remains stably buried throughout the room temperature simulations. A calculation of the relative surface accessibility (RSA) shows that on average E23 side chain is exposed to the solvent for only 4%. This value is well below the 20–25% threshold typically used to define solvent accessible residues in two states models [77–80]. This means that the flexibility of Lα2 is not enough to expose E23 side chain and the side chain can remain protonated (S3 Fig). In comparison, repulsive forces in MNEI-GLU result in a slight opening of the loop and consequent partial exposure of E23, which has an average RSA at 300 K around 20%, indicating that over time the residue can come in contact with the solvent [77,80]. At lower temperatures, the charge on the side chain of E23 can still be stabilized through hydration. Indeed, in each of the simulations at room temperature, a water molecule penetrates in the space at the C-terminal of the helix after few picoseconds, mediating the interactions between E23 and the amide protons of Y29 and Q28. This water mediated stabilization prevents the formation of the contacts observed in MNEI-GLH, but does not compromise the protein fold in the low temperature simulations. Nonetheless, in high temperature simulations, this results in faster unfolding, as evident in the secondary structure prediction plots (S3 Fig). The most relevant non-secondary structure interactions of the two states and their occurrence over time are reported in and Table 1 and represented in Fig 4.


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

Leone S, Picone D - PLoS ONE (2016)

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.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0158372.g004: 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.
Mentions: To understand the nature of these motions and their effect on the fold, we performed secondary structure calculations over time with DSSP [75,76]. Analysis of the simulations at 300 K shows no substantial deviation of secondary structure elements from their experimental description, a situation also observed for MNEI-GLH at 473 K (S2 and S3 Figs). When E23 side chain is ionized, though, we observe a partial disruption of the secondary structure starting as early as 4 ns, which explains the observed RMSD increase. Unfolding occurs to different extents in the three independent simulations, but it always involves residues at the C-terminal portions of the helix, as exemplified in Fig 3A, which shows the DSSP plot for one of the high temperature simulations on MNEI-GLU. While the helical structure is only marginally lost in the 473 K simulations on MNEI-GLH, the percentage of residual helical structure drops up to 20% of the starting value in MNEI-GLU (Fig 3B). To understand the contribution of E23 to the protein stability, we evaluated the intra-molecular interactions occurring in the native state from the simulations at 300 K. The protonated side chain can form a very stable H-bond with the carbonyl oxygen of G30: the average distance between the carboxyl in the side chain of E23 and G30(O) is 2.78 Å and the occurrence of such bond is 99.4% over the three simulations. By tightening the loop between the helix and β2, several non-secondary structure H-bonds between side chains are formed and show high occupancy, adding stability to the fold, as reported in Table 1. Despite being situated on a flexible loop, E23 remains stably buried throughout the room temperature simulations. A calculation of the relative surface accessibility (RSA) shows that on average E23 side chain is exposed to the solvent for only 4%. This value is well below the 20–25% threshold typically used to define solvent accessible residues in two states models [77–80]. This means that the flexibility of Lα2 is not enough to expose E23 side chain and the side chain can remain protonated (S3 Fig). In comparison, repulsive forces in MNEI-GLU result in a slight opening of the loop and consequent partial exposure of E23, which has an average RSA at 300 K around 20%, indicating that over time the residue can come in contact with the solvent [77,80]. At lower temperatures, the charge on the side chain of E23 can still be stabilized through hydration. Indeed, in each of the simulations at room temperature, a water molecule penetrates in the space at the C-terminal of the helix after few picoseconds, mediating the interactions between E23 and the amide protons of Y29 and Q28. This water mediated stabilization prevents the formation of the contacts observed in MNEI-GLH, but does not compromise the protein fold in the low temperature simulations. Nonetheless, in high temperature simulations, this results in faster unfolding, as evident in the secondary structure prediction plots (S3 Fig). The most relevant non-secondary structure interactions of the two states and their occurrence over time are reported in and Table 1 and represented in Fig 4.

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