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Breaking the hydrophobicity of the MscL pore: insights into a charge-induced gating mechanism.

Chandramouli B, Di Maio D, Mancini G, Barone V, Brancato G - PLoS ONE (2015)

Bottom Line: Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed.Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties.In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.

View Article: PubMed Central - PubMed

Affiliation: Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126, Pisa, Italy.

ABSTRACT
The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.

No MeSH data available.


Related in: MedlinePlus

Pore radius, hydration along the channel and intersubunit contacts.Pore radius along the axial positions as a function of simulation time for (A) WT, (B) WT_1e and (C) NL models, obtained considering the backbone atoms. (D) Distribution of water molecules in the pore. For clarity, water molecules at the periplasmic vestibule of the pore are shown in black. (E) Inter-helix (TM1 vs TM1) contacts in WT_1e simulation relative to the starting configuration as a function of time.
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pone.0120196.g006: Pore radius, hydration along the channel and intersubunit contacts.Pore radius along the axial positions as a function of simulation time for (A) WT, (B) WT_1e and (C) NL models, obtained considering the backbone atoms. (D) Distribution of water molecules in the pore. For clarity, water molecules at the periplasmic vestibule of the pore are shown in black. (E) Inter-helix (TM1 vs TM1) contacts in WT_1e simulation relative to the starting configuration as a function of time.

Mentions: In order to better investigate, at molecular level, the present gating mechanism, we conceived an additional model (WT_1e) in which the same negatively charged group was directly attached to the constricted site (V21) in a single subunit (subunit 1) of the WT protein, therefore mimicking the light-actuation of a single subunit of MscL. Inspection of the average pore radius along the channel axis revealed a clear expansion of the WT_1e model with respect to the WT protein (S4 Fig.), albeit to a lesser extent compared to the NL model. This can be better analyzed following the time evolution of the pore radius along the axial position, as shown in Fig. 6. In the WT simulation, the channel is constricted around the mutated site (-1< z <1) with a pore radius of < 4.5 Ang that is maintained throughout the considered time interval (Fig. 6A), while an expansion up to 5.5 Ang and 6.5 Ang is observed for the WT_1e and NL models (Fig. 6B,C). In the latter cases, a proportional expansion is observed in the regions flanking the functionalized site. The partially expanded WT_1e model was sufficient to allow ion permeation, as reported in Table 2.


Breaking the hydrophobicity of the MscL pore: insights into a charge-induced gating mechanism.

Chandramouli B, Di Maio D, Mancini G, Barone V, Brancato G - PLoS ONE (2015)

Pore radius, hydration along the channel and intersubunit contacts.Pore radius along the axial positions as a function of simulation time for (A) WT, (B) WT_1e and (C) NL models, obtained considering the backbone atoms. (D) Distribution of water molecules in the pore. For clarity, water molecules at the periplasmic vestibule of the pore are shown in black. (E) Inter-helix (TM1 vs TM1) contacts in WT_1e simulation relative to the starting configuration as a function of time.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4380313&req=5

pone.0120196.g006: Pore radius, hydration along the channel and intersubunit contacts.Pore radius along the axial positions as a function of simulation time for (A) WT, (B) WT_1e and (C) NL models, obtained considering the backbone atoms. (D) Distribution of water molecules in the pore. For clarity, water molecules at the periplasmic vestibule of the pore are shown in black. (E) Inter-helix (TM1 vs TM1) contacts in WT_1e simulation relative to the starting configuration as a function of time.
Mentions: In order to better investigate, at molecular level, the present gating mechanism, we conceived an additional model (WT_1e) in which the same negatively charged group was directly attached to the constricted site (V21) in a single subunit (subunit 1) of the WT protein, therefore mimicking the light-actuation of a single subunit of MscL. Inspection of the average pore radius along the channel axis revealed a clear expansion of the WT_1e model with respect to the WT protein (S4 Fig.), albeit to a lesser extent compared to the NL model. This can be better analyzed following the time evolution of the pore radius along the axial position, as shown in Fig. 6. In the WT simulation, the channel is constricted around the mutated site (-1< z <1) with a pore radius of < 4.5 Ang that is maintained throughout the considered time interval (Fig. 6A), while an expansion up to 5.5 Ang and 6.5 Ang is observed for the WT_1e and NL models (Fig. 6B,C). In the latter cases, a proportional expansion is observed in the regions flanking the functionalized site. The partially expanded WT_1e model was sufficient to allow ion permeation, as reported in Table 2.

Bottom Line: Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed.Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties.In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.

View Article: PubMed Central - PubMed

Affiliation: Scuola Normale Superiore, Piazza dei Cavalieri 7, I-56126, Pisa, Italy.

ABSTRACT
The mechanosensitive channel of large conductance (MscL) is a protein that responds to membrane tension by opening a transient pore during osmotic downshock. Due to its large pore size and functional reconstitution into lipid membranes, MscL has been proposed as a promising artificial nanovalve suitable for biotechnological applications. For example, site-specific mutations and tailored chemical modifications have shown how MscL channel gating can be triggered in the absence of tension by introducing charged residues at the hydrophobic pore level. Recently, engineered MscL proteins responsive to stimuli like pH or light have been reported. Inspired by experiments, we present a thorough computational study aiming at describing, with atomistic detail, the artificial gating mechanism and the molecular transport properties of a light-actuated bacterial MscL channel, in which a charge-induced gating mechanism has been enabled through the selective cleavage of photo-sensitive alkylating agents. Properties such as structural transitions, pore dimension, ion flux and selectivity have been carefully analyzed. Besides, the effects of charge on alternative sites of the channel with respect to those already reported have been addressed. Overall, our results provide useful molecular insights into the structural events accompanying the engineered MscL channel gating and the interplay of electrostatic effects, channel opening and permeation properties. In addition, we describe how the experimentally observed ionic current in a single-subunit charged MscL mutant is obtained through a hydrophobicity breaking mechanism involving an asymmetric inter-subunit motion.

No MeSH data available.


Related in: MedlinePlus