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Site-directed spin-labeling analysis of reconstituted Mscl in the closed state.

Perozo E, Kloda A, Cortes DM, Martinac B - J. Gen. Physiol. (2001)

Bottom Line: In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer.Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions.However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22906, USA. eperozo@virginia.edu

ABSTRACT
The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O(2) or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21-26 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.

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Intersubunit electron spin–spin coupling in MscL. (A) selected residues along TM1 showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (mole/mole labeling ratio, 10:1 spin label/subunit). Traces in red were obtained from underlabeled channels and represent unbroadened spectra (mole/mole labeling ratio, 1:10 spin label/subunit). (B) Pattern of intersubunit proximities derived from the Ω parameter (top, TM1; bottom, TM2). The double arrow line points to a stretch of hydrophobic residues with restricted mobility. (C) Data in B as mapped onto the equivalent positions in the Tb-MscL structure.
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Figure 9: Intersubunit electron spin–spin coupling in MscL. (A) selected residues along TM1 showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (mole/mole labeling ratio, 10:1 spin label/subunit). Traces in red were obtained from underlabeled channels and represent unbroadened spectra (mole/mole labeling ratio, 1:10 spin label/subunit). (B) Pattern of intersubunit proximities derived from the Ω parameter (top, TM1; bottom, TM2). The double arrow line points to a stretch of hydrophobic residues with restricted mobility. (C) Data in B as mapped onto the equivalent positions in the Tb-MscL structure.

Mentions: Maximally labeled and underlabeled spectra from selected residues along TM1 (Fig. 9 A) illustrate the point. When labeled at a 10:1 molar ratio of MscL monomer to spin label, the vast majority of the labeled channels are predicted to have a single nitroxide group and, hence, represents the unbroadened spectra (Fig. 9 A, red traces). Reversing the labeling ratio (1:10 molar ratio, monomer to spin label) maximizes the likelihood that all available cysteines will be modified with a spin label, thus, assuring broadened spectra (Fig. 9 A, black traces). Application of this type of analysis generates an intersubunit interaction profile (Fig. 9) pointing to residues likely to be located near the symmetry axis of MscL (its permeation path). Results from the TM1 segment (Fig. 9, top) reveal a prominent cluster of interacting residues at positions 20–26, in remarkable agreement with the mobility and NiEdda accessibility data (Fig. 3, top and bottom), suggesting a hydrophobic constriction among these residues. Significant levels of spectral broadening were also observed, with an impressive α-periodicity, at both sides of the major cluster. Surprisingly, we were able to detect small, but significant, levels of spin–spin interactions among residues in the COOH-terminal half of TM2, suggesting that this side of the TM2 helix is likely to face the symmetry axis (Fig. 9, bottom). The spatial distribution of these interacting residues within MscL is illustrated in Fig. 9 C, where Ω values obtained for residues in both TM segments are mapped into the Tb-MscL structure, either in ribbon representation or in a cross-section of an accessible surface representation. It is worth noting that the strongest Ω values from the 20 to 26 cluster forms an unmistakable ring around the narrowest section of the MscL permeation path, strongly suggesting that this section of the channel physically contributes to restrict solute permeation in the closed state.


Site-directed spin-labeling analysis of reconstituted Mscl in the closed state.

Perozo E, Kloda A, Cortes DM, Martinac B - J. Gen. Physiol. (2001)

Intersubunit electron spin–spin coupling in MscL. (A) selected residues along TM1 showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (mole/mole labeling ratio, 10:1 spin label/subunit). Traces in red were obtained from underlabeled channels and represent unbroadened spectra (mole/mole labeling ratio, 1:10 spin label/subunit). (B) Pattern of intersubunit proximities derived from the Ω parameter (top, TM1; bottom, TM2). The double arrow line points to a stretch of hydrophobic residues with restricted mobility. (C) Data in B as mapped onto the equivalent positions in the Tb-MscL structure.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2233830&req=5

Figure 9: Intersubunit electron spin–spin coupling in MscL. (A) selected residues along TM1 showing different degrees of spectral broadening due to spin–spin coupling. Traces in black were obtained from fully labeled channels (mole/mole labeling ratio, 10:1 spin label/subunit). Traces in red were obtained from underlabeled channels and represent unbroadened spectra (mole/mole labeling ratio, 1:10 spin label/subunit). (B) Pattern of intersubunit proximities derived from the Ω parameter (top, TM1; bottom, TM2). The double arrow line points to a stretch of hydrophobic residues with restricted mobility. (C) Data in B as mapped onto the equivalent positions in the Tb-MscL structure.
Mentions: Maximally labeled and underlabeled spectra from selected residues along TM1 (Fig. 9 A) illustrate the point. When labeled at a 10:1 molar ratio of MscL monomer to spin label, the vast majority of the labeled channels are predicted to have a single nitroxide group and, hence, represents the unbroadened spectra (Fig. 9 A, red traces). Reversing the labeling ratio (1:10 molar ratio, monomer to spin label) maximizes the likelihood that all available cysteines will be modified with a spin label, thus, assuring broadened spectra (Fig. 9 A, black traces). Application of this type of analysis generates an intersubunit interaction profile (Fig. 9) pointing to residues likely to be located near the symmetry axis of MscL (its permeation path). Results from the TM1 segment (Fig. 9, top) reveal a prominent cluster of interacting residues at positions 20–26, in remarkable agreement with the mobility and NiEdda accessibility data (Fig. 3, top and bottom), suggesting a hydrophobic constriction among these residues. Significant levels of spectral broadening were also observed, with an impressive α-periodicity, at both sides of the major cluster. Surprisingly, we were able to detect small, but significant, levels of spin–spin interactions among residues in the COOH-terminal half of TM2, suggesting that this side of the TM2 helix is likely to face the symmetry axis (Fig. 9, bottom). The spatial distribution of these interacting residues within MscL is illustrated in Fig. 9 C, where Ω values obtained for residues in both TM segments are mapped into the Tb-MscL structure, either in ribbon representation or in a cross-section of an accessible surface representation. It is worth noting that the strongest Ω values from the 20 to 26 cluster forms an unmistakable ring around the narrowest section of the MscL permeation path, strongly suggesting that this section of the channel physically contributes to restrict solute permeation in the closed state.

Bottom Line: In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer.Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions.However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA 22906, USA. eperozo@virginia.edu

ABSTRACT
The mechanosensitive channel from Escherichia coli (Eco-MscL) responds to membrane lateral tension by opening a large, water-filled pore that serves as an osmotic safety valve. In an attempt to understand the structural dynamics of MscL in the closed state and under physiological conditions, we have performed a systematic site-directed spin labeling study of this channel reconstituted in a membrane bilayer. Structural information was derived from an analysis of probe mobility, residue accessibility to O(2) or NiEdda and overall intersubunit proximity. For the majority of the residues studied, mobility and accessibility data showed a remarkable agreement with the Mycobacterium tuberculosis crystal structure, clearly identifying residues facing the large water-filled vestibule at the extracellular face of the molecule, the narrowest point along the permeation pathway (residues 21-26 of Eco-MscL), and the lipid-exposed residues in the peripheral transmembrane segments (TM2). Overall, the present dataset demonstrates that the transmembrane regions of the MscL crystal structure (obtained in detergent and at low pH) are, in general, an accurate representation of its structure in a membrane bilayer under physiological conditions. However, significant differences between the EPR data and the crystal structure were found toward the COOH-terminal end of TM2.

Show MeSH
Related in: MedlinePlus