An
            <i>in vivo</i>
            assay identifies changes in residue accessibility on mechanosensitive channel gating

Bartlett, Jessica L.; Levin, G. M.; Blount, Paul

doi:10.1073/pnas.0402040101

Cited by 58 publications

(88 citation statements)

References 17 publications

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“…1A) (21), strongly supporting tilting. The two tilted models are not fully compatible because the EPR-based model implies a large rotation of the TM1 helix, contradicting the observed cysteine cross-links (22) and methanethiosulfonate accessibility data (23).…”

Section: Mscl Structure and Character Of Transitionmentioning

confidence: 61%

“…This thickness mismatch may exert a closing force. The second factor is high hydrophobicity of the gate formed by the rings of Val 23 and Leu 19 in E. coli MscL, which is dehydrated according to molecular dynamics (28). The vapor bubble in the gate makes MscL leak-proof, whereas surface tension of watervapor interfaces above and below the constriction additionally tightens the gate.…”

Section: How Can the High Energy Of Mscl Activation Be Combined With mentioning

confidence: 99%

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State-stabilizing Interactions in Bacterial Mechanosensitive Channel Gating and Adaptation

Anishkin

Sukharev

2009

Journal of Biological Chemistry

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We outline several principles that we believe define the gating of two bacterial mechanosensitive channels, MscL and MscS. Serving as turgor regulators in bacteria and other walled cells, these molecules are tangible models for studying conformational transitions in membrane proteins driven directly by membrane tension. MscL, a compact pentamer, reversibly opens a gigantic 30-Å pore at near-lytic tensions. MscS, a heptameric complex, exhibits transient activation of a smaller pore at moderate tensions, thereby entering a tension-insensitive inactivated state. By comparing the structures and predicted transitions in these channels, we concluded that opening is commonly achieved through tilting and outward motion of the porelining helices, which is kinetically limited by hydration of the pore. The intricate adaptive behavior in MscS appears to depend on specific interhelical associations and the flexibility of the pore-lining helices. We discuss physical factors that may direct the transitions and stabilize main functional states in these channels.

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Section: Mscl Structure and Character Of Transitionmentioning

confidence: 61%

Section: How Can the High Energy Of Mscl Activation Be Combined With mentioning

confidence: 99%

State-stabilizing Interactions in Bacterial Mechanosensitive Channel Gating and Adaptation

Anishkin

Sukharev

2009

Journal of Biological Chemistry

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“…15 In addition, all current models for the structural changes that occur upon gating include a tilting of both transmembrane domains within the plane of the membrane. In addition to tilting, for TM1 several lines of evidence also suggest a clockwise "corkscrewing" of TM1 of almost 180°, as would be observed from the periplasm; these lines of evidence include EPR experiments, 20 disulfide trapping experiments within the pore 9 and between TM1 and TM2 upon gating, 17 accessibility and gating influences of the sulfhydryl reagent MTSET + on TM cysteine mutants, 21,22 and the engineering of heavy metal binding sites within the pore region. 8 The S1 amphipathic helix, with its two conserved phenylalanines, is joined to this dynamic TM1 by a conserved flexible glycine residue at position 14 that has been shown to have functional significance.…”

Section: Discussionmentioning

confidence: 99%

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

Iscla¹,

Wray

Blount³

2012

Channels

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“…Previously, charge-induced activation of MscL has been shown in intact E. coli cells (11,41), with spheroplasts (12,42) and with reconstituted systems (6,7,43). In all of these cases, MscL was embedded in a lipid bilayer environment.…”

Section: Mtset-induced Increase In the Size Of Mscl Obtained From Im-mentioning

confidence: 99%

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

Konijnenberg

Yilmaz²,

Ingólfsson

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Mechanosensitive ion channels are sensors probing membrane tension in all species; despite their importance and vital role in many cell functions, their gating mechanism remains to be elucidated. Here, we determined the conditions for releasing intact mechanosensitive channel of large conductance (MscL) proteins from their detergents in the gas phase using native ion mobilitymass spectrometry (IM-MS). By using IM-MS, we could detect the native mass of MscL from Escherichia coli, determine various global structural changes during its gating by measuring the rotationally averaged collision cross-sections, and show that it can function in the absence of a lipid bilayer. We could detect global conformational changes during MscL gating as small as 3%. Our findings will allow studying native structure of many other membrane proteins.ion mobility mass spectrometry | MscL | membrane proteins | structure function | ion channel gating O ne of the best candidates to explore the gating of mechanosensitive channels is the mechanosensitive channel of large conductance (MscL) from Escherichia coli. The crystal structure of MscL in its closed/nearly closed state from Mycobacterium tuberculosis revealed this channel as a homopentamer (1). Each subunit has a cytoplasmic N-and C-terminal domain as well as two α-helical transmembrane (TM) domains, TM1 and TM2, which are connected by a periplasmic loop. The five TM1 helices form the pore and the more peripheral TM2 helices interact with the lipid bilayer.MscL detects changes in membrane tension invoked by a hypoosmotic shock and couples the tension sensing directly to large conformational changes (1, 2). On the basis of a large body of structural and theoretical data, numerous gating models of MscL have been proposed (3-9). These models agree upon (i) the hydrophobic pore constriction of the channel and (ii) the channel opens by an iris-like rotation-i.e., a tilting and outward movement of transmembrane helices that make the channel wider and shorter (5). This mechanism is supported by patchclamp (10), disulfide cross-linking (11), FRET spectroscopy (12), and site-directed spin labeling EPR experiments (6, 7), as well as computational studies (13-15). So far, direct experimental results have only been observed for short-range local structural changes, and no measure of the overall global structural changes during channel gating have been reported. Because there is no crystal structure available for the open MscL channel, elucidating overall global structural changes from the onset of channel activation is of utmost importance for our understanding of the gating mechanism of mechanosensitive channels. Here, we provide direct experimental evidence for the key areal changes occurring during channel gating by combining our ability to activate MscL in a controlled manner to different subopen states (16) with a native ion mobility-mass spectrometry (IM-MS) approach. Results and DiscussionDetergents that are Unstable in the Gas Phase Allow Detection of MscL in Its Native Form. Native mass ...

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An in vivo assay identifies changes in residue accessibility on mechanosensitive channel gating

Cited by 58 publications

References 17 publications

State-stabilizing Interactions in Bacterial Mechanosensitive Channel Gating and Adaptation

State-stabilizing Interactions in Bacterial Mechanosensitive Channel Gating and Adaptation

The dynamics of protein-protein interactions between domains of MscL at the cytoplasmic-lipid interface

Global structural changes of an ion channel during its gating are followed by ion mobility mass spectrometry

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