Spatiotemporal Stop-and-go Dynamics of the Mitochondrial TOM Core Complex Correlates With Three-state Channel Activity

Abstract Single-molecule studies can reveal phenomena that remain hidden in ensemble measurements. Here, we show the correlation between lateral protein diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC) in membranes resting on ultrathin hydrogel films. Using electrode-free optical recordings of ion flux, we find that TOM-CC switches reversibly between three states of ion permeability associated with protein diffusion. Freely diffusing TOM-CC molecules are observed in a high permeability state, while non-moving molecules are in an intermediate and a low permeability state. We explain this behavior by the mechanical binding of the two protruding Tom22 subunits to the hydrogel and a concomitant combinatorial opening and closing of the two β-barrel pores of TOM-CC. TOM-CC could thus be the first β-barrel protein channel to exhibit membrane state-dependent mechanosensitive properties.

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Correlation of mitochondrial TOM core complex stop-and-go and open-closed channel dynamics

10.1101/2021.09.28.462098 ◽

2021 ◽

Author(s):

Shuo Wang ◽

Lukas Findeisen ◽

Sebastian Leptihn ◽

Mark I Wallace ◽

Marcel Hörning ◽

...

Keyword(s):

Protein Import ◽

Mitochondrial Protein ◽

Lateral Diffusion ◽

Protein Diffusion ◽

Channel Dynamics ◽

Stop And Go ◽

General Protein ◽

Opening And Closing ◽

The Relationship

The role of lateral diffusion of proteins in the membrane in the context of function has not been examined extensively. Here, we explore the relationship between protein lateral diffusion and channel activity of the general protein import pore of mitochondria (TOM-CC). Optical ion flux sensing through single TOM-CC molecules shows that TOM-CC can occupy three ion permeability states. Whereas freely diffusing TOM-CC molecules are preferentially found in a high permeability state, physical tethering to an agarose support causes the channels to transition to intermediate and low permeability states. This data shows that combinatorial opening and closing of the two pores of TOM-CC correlates with lateral protein diffusion in the membrane plane, and that the complex has mechanosensitive-like properties. This is the first demonstration of β-barrel protein mechanosensitivity, and has direct conceptual consequences for the understanding of the process of mitochondrial protein import. Our approach provides a novel tool to simultaneously study the interplay of membrane protein diffusion and channel dynamics.

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Faculty Opinions recommendation of General protein diffusion barriers create compartments within bacterial cells.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.717968591.793473080 ◽

2013 ◽

Author(s):

Mohan Balasubramanian ◽

Ramanujam Srinivasan ◽

Mithilesh Mishra

Keyword(s):

Diffusion Barriers ◽

Bacterial Cells ◽

Protein Diffusion ◽

General Protein

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Faculty Opinions recommendation of Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725442653.793522148 ◽

2016 ◽

Author(s):

Olivier Hyrien

Keyword(s):

Single Molecule ◽

Helicase Loading ◽

Origin Licensing ◽

Single Molecule Studies

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Faculty Opinions recommendation of Single-molecule studies of origin licensing reveal mechanisms ensuring bidirectional helicase loading.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725442653.793506614 ◽

2015 ◽

Author(s):

Antoine van Oijen

Keyword(s):

Single Molecule ◽

Helicase Loading ◽

Origin Licensing ◽

Single Molecule Studies

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Hopping and Flipping of RNA Polymerase on DNA during Recycling for Reinitiation after Intrinsic Termination in Bacterial Transcription

International Journal of Molecular Sciences ◽

10.3390/ijms22052398 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2398

Author(s):

Wooyoung Kang ◽

Seungha Hwang ◽

Jin Young Kang ◽

Changwon Kang ◽

Sungchul Hohng

Keyword(s):

Single Molecule ◽

Transcription Initiation ◽

Molecular Mechanisms ◽

Facilitated Diffusion ◽

One Dimensional ◽

Bacterial Transcription ◽

Fluorescence Measurements ◽

Dimensional Diffusion ◽

Single Molecule Studies ◽

Hopping Diffusion

Two different molecular mechanisms, sliding and hopping, are employed by DNA-binding proteins for their one-dimensional facilitated diffusion on nonspecific DNA regions until reaching their specific target sequences. While it has been controversial whether RNA polymerases (RNAPs) use one-dimensional diffusion in targeting their promoters for transcription initiation, two recent single-molecule studies discovered that post-terminational RNAPs use one-dimensional diffusion for their reinitiation on the same DNA molecules. Escherichia coli RNAP, after synthesizing and releasing product RNA at intrinsic termination, mostly remains bound on DNA and diffuses in both forward and backward directions for recycling, which facilitates reinitiation on nearby promoters. However, it has remained unsolved which mechanism of one-dimensional diffusion is employed by recycling RNAP between termination and reinitiation. Single-molecule fluorescence measurements in this study reveal that post-terminational RNAPs undergo hopping diffusion during recycling on DNA, as their one-dimensional diffusion coefficients increase with rising salt concentrations. We additionally find that reinitiation can occur on promoters positioned in sense and antisense orientations with comparable efficiencies, so reinitiation efficiency depends primarily on distance rather than direction of recycling diffusion. This additional finding confirms that orientation change or flipping of RNAP with respect to DNA efficiently occurs as expected from hopping diffusion.

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Breaking Down the Supramolecular Ensemble: Single-Molecule Studies of the Concentration Dependence of Main-Chain Supramolecular Polymer Molecular Weight Distributions on Surfaces

Australian Journal of Chemistry ◽

10.1071/ch09615 ◽

2010 ◽

Vol 63 (4) ◽

pp. 624

Author(s):

Michael J. Serpe ◽

Jason R. Whitehead ◽

Stephen L. Craig

Keyword(s):

Molecular Weight ◽

Single Molecule ◽

Monomer Concentration ◽

Supramolecular Polymer ◽

Force Microscopy ◽

Molecular Weight Distributions ◽

Atomic Force ◽

Multi Stage ◽

Single Molecule Studies ◽

Supramolecular Ensemble

Single molecule atomic force microscopy (AFM) studies of oligonucleotide-based supramolecular polymers on surfaces are used to examine the molecular weight distribution of the polymers formed between a functionalized surface and an AFM tip as a function of monomer concentration. For the concentrations examined here, excellent agreement with a multi-stage open association model of polymerization is obtained, without the need to invoke additional contributions from secondary steric interactions at the surface.

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