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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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Residue environmental parameter profiles for TM1 mapped on equivalent positions of the Tb-MscL crystal structure. (A) Mobility parameter ΔHo−1; (B) oxygen accessibility parameter ΠO2; and (C) NiEdda accessibility parameter ΠNiEdda. Two views of the mapped parameters of TM1 are shown rotated 180°. In each case, surface accessible representation of whole MscL and of TM1 only was done using the program Grasp (Nicholls et al. 1991).
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Figure 4: Residue environmental parameter profiles for TM1 mapped on equivalent positions of the Tb-MscL crystal structure. (A) Mobility parameter ΔHo−1; (B) oxygen accessibility parameter ΠO2; and (C) NiEdda accessibility parameter ΠNiEdda. Two views of the mapped parameters of TM1 are shown rotated 180°. In each case, surface accessible representation of whole MscL and of TM1 only was done using the program Grasp (Nicholls et al. 1991).

Mentions: The correlation between the EPR-derived environmental parameters and their location within the crystal structure is shown in Fig. 4, where the unprocessed data for ΔH0−1, ΠO2, and ΠNiEdda are mapped onto their equivalent positions in Tb-MscL. In each case, the property mapped is shown in the context of a solvent-accessible surface representation (1.4-Å probe size) of the full-length channel, and as solvent-accessible surface of TM1 only (in two orientations, rotated 180°).


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

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

Residue environmental parameter profiles for TM1 mapped on equivalent positions of the Tb-MscL crystal structure. (A) Mobility parameter ΔHo−1; (B) oxygen accessibility parameter ΠO2; and (C) NiEdda accessibility parameter ΠNiEdda. Two views of the mapped parameters of TM1 are shown rotated 180°. In each case, surface accessible representation of whole MscL and of TM1 only was done using the program Grasp (Nicholls et al. 1991).
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Residue environmental parameter profiles for TM1 mapped on equivalent positions of the Tb-MscL crystal structure. (A) Mobility parameter ΔHo−1; (B) oxygen accessibility parameter ΠO2; and (C) NiEdda accessibility parameter ΠNiEdda. Two views of the mapped parameters of TM1 are shown rotated 180°. In each case, surface accessible representation of whole MscL and of TM1 only was done using the program Grasp (Nicholls et al. 1991).
Mentions: The correlation between the EPR-derived environmental parameters and their location within the crystal structure is shown in Fig. 4, where the unprocessed data for ΔH0−1, ΠO2, and ΠNiEdda are mapped onto their equivalent positions in Tb-MscL. In each case, the property mapped is shown in the context of a solvent-accessible surface representation (1.4-Å probe size) of the full-length channel, and as solvent-accessible surface of TM1 only (in two orientations, rotated 180°).

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