FRET nanoscopy enables seamless imaging of molecular assemblies with sub-nanometer resolution

Budde, Jan-Hendrik; Nicolaas, van der Voort,; Felekyan, Suren; Folz, Julian; Kühnemuth, Ralf; Lauterjung, Paul; Köhler, M. K.; Schönle, Andreas; Sindram, Julian; Otten, Marius; Karg, Matthias; Herrmann, Christian; Barth, Anders; Seidel, Claus A. M.

doi:10.48550/arxiv.2108.00024

Cited by 3 publications

(3 citation statements)

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“…Although such studies are only initial steps toward the routine application of single-molecule spectroscopy in live cells, they demonstrate that exciting insights can be gained. Examples of promising current developments for further expanding the scope of single-molecule experiments in cells are the MINFLUX technique (59), the combination of amber codon suppression with photoactivable FRET dyes (119) to enable the precise tuning of concentrations akin to the procedures used in photoactivable localization microscopy (PALM) (117), and the direct combination of superresolution techniques with FRET (23,154).…”

Section: Toward Cellular Idp Interaction Dynamicsmentioning

confidence: 99%

Interaction Dynamics of Intrinsically Disordered Proteins from Single-Molecule Spectroscopy

Chowdhury

Nettels

Schuler

2023

Annu. Rev. Biophys.

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Many proteins contain large structurally disordered regions or are entirely disordered under physiological conditions. The functions of these intrinsically disordered proteins (IDPs) often involve interactions with other biomolecules. An important emerging effort has thus been to identify the molecular mechanisms of IDP interactions and how they differ from the textbook notions of biomolecular binding for folded proteins. In this review, we summarize how the versatile tool kit of single-molecule fluorescence spectroscopy can aid the investigation of these conformationally heterogeneous and highly dynamic molecular systems. We discuss the experimental observables that can be employed and how they enable IDP complexes to be probed on timescales from nanoseconds to hours. Key insights include the diverse structural and dynamic properties of bound IDPs and the kinetic mechanisms facilitated by disorder, such as fly-casting; disorder-mediated encounter complexes; and competitive substitution via ternary complexes, which enables rapid dissociation even for high-affinity complexes. We also discuss emerging links to aggregation, liquid–liquid phase separation, and cellular processes, as well as current technical advances to further expand the scope of single-molecule spectroscopy. Expected final online publication date for the Annual Review of Biophysics, Volume 52 is May 2023. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

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Section: Toward Cellular Idp Interaction Dynamicsmentioning

confidence: 99%

Interaction Dynamics of Intrinsically Disordered Proteins from Single-Molecule Spectroscopy

Chowdhury

Nettels

Schuler

2023

Annu. Rev. Biophys.

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“…In single molecule experiments, passivation allows fixing target molecules while at the same time limiting non-specific binding. This comes, however, at the expense of increasing surface roughness—whose heterogeneity can lead to height-related artifacts of ~24° inclination angle when using passivating proteins for immobilizing DNA constructs [ 50 ]—as well as potential fouling and autofluorescence due to non-specific interactions arising from an additional number of functionalization steps [ 51 ]. In this regard, a plasma-treatment approach for firmly immobilizing flat DNA origami constructs on the glass surface has been demonstrated offering direct interfacing with the surface, while keeping a low background noise suitable for super-resolution microscopy [ 18 , 52 ].…”

Section: Introductionmentioning

confidence: 99%

Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching

Cervantes-Salguero

Biaggne

Youngsman

et al. 2022

IJMS

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Nanoarchitectural control of matter is crucial for next-generation technologies. DNA origami templates are harnessed to accurately position single molecules; however, direct single molecule evidence is lacking regarding how well DNA origami can control the orientation of such molecules in three-dimensional space, as well as the factors affecting control. Here, we present two strategies for controlling the polar (θ) and in-plane azimuthal (ϕ) angular orientations of cyanine Cy5 single molecules tethered on rationally-designed DNA origami templates that are physically adsorbed (physisorbed) on glass substrates. By using dipolar imaging to evaluate Cy5′s orientation and super-resolution microscopy, the absolute spatial orientation of Cy5 is calculated relative to the DNA template. The sequence-dependent partial intercalation of Cy5 is discovered and supported theoretically using density functional theory and molecular dynamics simulations, and it is harnessed as our first strategy to achieve θ control for a full revolution with dispersion as small as ±4.5°. In our second strategy, ϕ control is achieved by mechanically stretching the Cy5 from its two tethers, being the dispersion ±10.3° for full stretching. These results can in principle be applied to any single molecule, expanding in this way the capabilities of DNA as a functional templating material for single-molecule orientation control. The experimental and modeling insights provided herein will help engineer similar self-assembling molecular systems based on polymers, such as RNA and proteins.

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“…In recent studies, it was demonstrated that hGBP1 can polymerize by detaching the E domain from the other two domains to swing it out, giving rise to disk-like structures, where the G domains are at the outside of the disk and the farnesyl groups assembled together inside the disk 8,59 . A conformation with the E domain in the folded-out position and dimerization via the G domains, as described here, could also tether two giant unilamellar vesicles together via the farnesyl lipid anchor 8,18,39 .…”

Section: Discussionmentioning

confidence: 99%

Domain motions, dimerization, and membrane interactions of the murine guanylate binding protein 2

Loschwitz

Steffens

Wang

et al. 2023

Sci Rep

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Guanylate-binding proteins (GBPs) are a group of GTPases that are induced by interferon-$$\gamma$$ γ and are crucial components of cell-autonomous immunity against intracellular pathogens. Here, we examine murine GBP2 (mGBP2), which we have previously shown to be an essential effector protein for the control of Toxoplasma gondii replication, with its recruitment through the membrane of the parasitophorous vacuole and its involvement in the destruction of this membrane likely playing a role. The overall aim of our work is to provide a molecular-level understanding of the mutual influences of mGBP2 and the parasitophorous vacuole membrane. To this end, we performed lipid-binding assays which revealed that mGBP2 has a particular affinity for cardiolipin. This observation was confirmed by fluorescence microscopy using giant unilamellar vesicles of different lipid compositions. To obtain an understanding of the protein dynamics and how this is affected by GTP binding, mGBP2 dimerization, and membrane binding, assuming that each of these steps are relevant for the function of the protein, we carried out standard as well as replica exchange molecular dynamics simulations with an accumulated simulation time of more than 30 μs. The main findings from these simulations are that mGBP2 features a large-scale hinge motion in its M/E domain, which is present in each of the studied protein states. When bound to a cardiolipin-containing membrane, this hinge motion is particularly pronounced, leading to an up and down motion of the M/E domain on the membrane, which did not occur on a membrane without cardiolipin. Our prognosis is that this up and down motion has the potential to destroy the membrane following the formation of supramolecular mGBP2 complexes on the membrane surface.

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FRET nanoscopy enables seamless imaging of molecular assemblies with sub-nanometer resolution

Cited by 3 publications

References 0 publications

Interaction Dynamics of Intrinsically Disordered Proteins from Single-Molecule Spectroscopy

Interaction Dynamics of Intrinsically Disordered Proteins from Single-Molecule Spectroscopy

Strategies for Controlling the Spatial Orientation of Single Molecules Tethered on DNA Origami Templates Physisorbed on Glass Substrates: Intercalation and Stretching

Domain motions, dimerization, and membrane interactions of the murine guanylate binding protein 2

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