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New research reveals how E. coli relieves the pressure

2/24/2014

Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel. (A) Labeled MscL proteins were reconstituted into liposomes, which were then immobilized on a coverslip (top). Via smFRET experiments, the FRET efficiency were measured (bottom). (B) The addition of LPC traps the protein in the open conformation (top), resulting in a shift in the FRET efficiency (bottom). (C) Translational movements of six residues on the two transmembrane helices (TM1 and TM2) measured via smFRET. All the residues move away from the pore center, arguing in favor of the helix-tilt model. (D) The structure of MscL in the open state (right) was developed based on the smFRET measurements, satisfying all the distance constraints measured from smFRET experiments, with the structure of MscL in the closed state (left) as a reference.
Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel. (A) Labeled MscL proteins were reconstituted into liposomes, which were then immobilized on a coverslip (top). Via smFRET experiments, the FRET efficiency were measured (bottom). (B) The addition of LPC traps the protein in the open conformation (top), resulting in a shift in the FRET efficiency (bottom). (C) Translational movements of six residues on the two transmembrane helices (TM1 and TM2) measured via smFRET. All the residues move away from the pore center, arguing in favor of the helix-tilt model. (D) The structure of MscL in the open state (right) was developed based on the smFRET measurements, satisfying all the distance constraints measured from smFRET experiments, with the structure of MscL in the closed state (left) as a reference.
Bacterial cells would pop like overfilled water balloons if they had no means of regulating fluid pressure buildup. Unlike water balloons, however, the cell membranes of bacteria have “safety valves”, channels that open in response to increased mechanical stress caused by excessive fluid buildup. The mechanosensitive channel of large conductance, or MscL, is a non-selective pore that is permeable to ions, water, and small proteins when open.

Now a team of two University of Illinois physics professors, working with two post-docs in the Center for the Physics of Living Cells, showed that in MscL, two helices tilt towards the membrane and open the channel.  Professor Paul Selvin and post-doc Yong Wang, used a technique called single-molecule Fluorescence Resonance Energy Transfer, or smFRET, to measure distances that moved a nanometer –or less—as the channel opened in response to pressure changes.

The technique, originally invented by U of I Physics professor Taekjip Ha, Selvin and others, has now become a regular tool in biophysics to measure incredibly small distances.

The pore, they found, went from a mere one-half of one nanometer when closed, to 2.8 nanometers when opened.

While 2.8 nanometers may not seem large, it is plenty of space for water, ions and small proteins, to escape, relieving the pressure that threatens to blow the cell up.

But how exactly did the channel open? Two models—a helix-tilt and a barrel-stave model—had been proposed. In the barrel-stave model, a helix called TM1 moves, while the other helix, called TM2, remains stationary. This results in an open pore that is lined with TM1 and TM2 helices in the same way that wooden staves line a barrel. In the helix-tilt model, both helices tilt towards the membrane to open the channel.

Selvin explains, “Using smFRET, we found that both helices moved, which is exactly what the helix-tilt model predicts.”

To get a more detailed look, Selvin turned to Klaus Schulten and post-doc Yanxin Liu, who made a computer model and “watched” the protein move in silico. The model not only reproduced the experimental details, but also found that the protein helices lay down in the membrane with increased pressure, a motion which was difficult to ascertain from the experiments.

“It’s sort of like a pancake,” says Schulten. “The water pressure squeezes down, flattening the MscL.”

The findings have implications for medical research into the rise in antibiotic resistance as well as many ailments that afflict people.

“These mechanosensitive channels play a critical role in bacterial adaptation,” comments Selvin, “and our understanding of their structure and mechanics may support the development of MscL-targeted drugs.”

The team's findings have been published in the online journal eLIFE.

link to article: http://elife.elifesciences.org/content/3/e01834

 

 

This work was funded by the National Institutes of Health through grant number R01 GM068625 (PRS) and grant numbers R01 GM067887, U54 GM087519, and 9P41GM104601 (KS); by the National Science Foundation through grant number PHY0822613 (PRS and KS), and by the National Health and Medical Research Council through grant number 635525 (BM). The conclusions presented are those of the scientists and not necessarily those of the funding agencies.