Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel

Yong Wang,Hannah A Deberg,Yanxin Liu,Boris Martinac,Melinda Tonks Hoffman,Paul R Selvin,Klaus Schulten,Takeshi Nomura,Paul R Rohde

doi:10.7554/elife.01834

Abstract

The mechanosensitive channel of large conductance, which serves as a model system for mechanosensitive channels, has previously been crystallized in the closed form, but not in the open form. Ensemble measurements and electrophysiological sieving experiments show that the open-diameter of the channel pore is >25 Å, but the exact size and whether the conformational change follows a helix-tilt or barrel-stave model are unclear. Here we report measurements of the distance changes on liposome-reconstituted MscL transmembrane α-helices, using a 'virtual sorting' single-molecule fluorescence energy transfer. We observed directly that the channel opens via the helix-tilt model and the open pore reaches 2.8 nm in diameter. In addition, based on the measurements, we developed a molecular dynamics model of the channel structure in the open state which confirms our direct observations. DOI: http://dx.doi.org/10.7554/eLife.01834.001.

Highlights

Mechanosensitive (MS) channels are essential in both eukaryotes and prokaryotes (Haswell et al, 2011; Árnadóttir & Chalfie, 2010; Perozo, 2006)
The liposomes were immobilized on a glass coverslip, via biotin-avidin interaction, for single-molecule fluorescence resonance energy transfer (smFRET) measurements (Figure 1e)
The labeled proteins for patch-clamp experiments are from a different aliquot, the same batch, of the labeled proteins for smFRET experiments.) We emphasize that, smFRET has been applied to study the conformational changes of channels and transporters (Zhao et al, 2010, 2011; Akyuz et al, 2013; Choi et al, 2010), to our knowledge, it is the first time that smFRET has been used with channels reconstituted to liposomes

Summary

Introduction

Mechanosensitive (MS) channels are essential in both eukaryotes and prokaryotes (Haswell et al, 2011; Árnadóttir & Chalfie, 2010; Perozo, 2006) In eukaryotes, they are involved in diverse processes such as embryonic development, touch, pain, hearing, lung growth, and muscle homeostasis (Chalfie, 2009; Hamill & Martinac, 2001; Árnadóttir & Chalfie, 2010). They are involved in diverse processes such as embryonic development, touch, pain, hearing, lung growth, and muscle homeostasis (Chalfie, 2009; Hamill & Martinac, 2001; Árnadóttir & Chalfie, 2010) In bacteria, they are “safety valves”, opening their pores to release the pressure to protect cells from hypo-osmotic shock (Booth & Blount, 2012). TM1 and TM2 are primarily responsible for gating; it has been shown that complete deletion of the CP domain does not change the gating parameters substantially (Anishkin et al, 2003)

Methods

Results

Conclusion