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

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yong Wang ◽  
Yanxin Liu ◽  
Hannah A DeBerg ◽  
Takeshi Nomura ◽  
Melinda Tonks Hoffman ◽  
...  

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.

2015 ◽  
Vol 184 ◽  
pp. 117-129 ◽  
Author(s):  
M. Beckers ◽  
F. Drechsler ◽  
T. Eilert ◽  
J. Nagy ◽  
J. Michaelis

Single-molecule studies can be used to study biological processes directly and in real-time. In particular, the fluorescence energy transfer between reporter dye molecules attached to specific sites on macromolecular complexes can be used to infer distance information. When several measurements are combined, the information can be used to determine the position and conformation of certain domains with respect to the complex. However, data analysis schemes that include all experimental uncertainties are highly complex, and the outcome depends on assumptions about the state of the dye molecules. Here, we present a new analysis algorithm using Bayesian parameter estimation based on Markov Chain Monte Carlo sampling and parallel tempering termed Fast-NPS that can analyse large smFRET networks in a relatively short time and yields the position of the dye molecules together with their respective uncertainties. Moreover, we show what effects different assumptions about the dye molecules have on the outcome. We discuss the possibilities and pitfalls in structure determination based on smFRET using experimental data for an archaeal transcription pre-initiation complex, whose architecture has recently been unravelled by smFRET measurements.


2016 ◽  
Vol 110 (3) ◽  
pp. 194a
Author(s):  
Aaron Keller ◽  
Matt DeVore ◽  
Dung Vu ◽  
Tim Causgrove ◽  
James Werner

Biosensors ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 168
Author(s):  
Heyjin Son ◽  
Woori Mo ◽  
Jaeil Park ◽  
Joong-Wook Lee ◽  
Sanghwa Lee

Single-molecule fluorescence energy transfer (FRET) detection has become a key technique to monitor intra- and intermolecular distance changes in biological processes. As the sensitive detection range of conventional FRET pairs is limited to 3–8 nm, complement probes are necessary for extending this typical working range. Here, we realized a single-molecule FRET assay for a short distance range of below 3 nm by using a Cy2–Cy7 pair having extremely small spectral overlap. Using two DNA duplexes with a small difference in the labeling position, we demonstrated that our assay can observe subtle changes at a short distance range. High sensitivity in the range of 1–3 nm and compatibility with the conventional FRET assay make this approach useful for understanding dynamics at a short distance.


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