Ralph Wieneke

Goethe University Frankfurt

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Robert Tampé

Goethe University Frankfurt

Mike Heilemann

Goethe University Frankfurt

Alina Kollmannsperger

Goethe University Frankfurt

Anika Raulf

Goethe University Frankfurt

Armin Geyer

Philipps University of Marburg

Federica Gasparri

University of Copenhagen

Stephan A. Pless

University of Copenhagen

Ahmad Reza Mehdipour

Ghent University

Miriam Klausberger

BOKU University

Nikolaus F. Kienzl

BOKU University

Cooperative Institutions

Goethe University Frankfurt

Max Planck Society

Centre National de la Recherche Scientifique

University of Copenhagen

Heidelberg University

University Hospital Frankfurt

Philipps University of Marburg

Johannes Kepler University of Linz

Institut de génétique et de développement de Rennes

ETH Zurich

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Chemical modification of proteins by insertion of synthetic peptides using tandem protein trans-splicing

Zenodo (CERN European Organization for Nuclear Research) (2020)

Keith K. Khoo Iacopo Galleano Federica Gasparri Ralph Wieneke Hendrik J. Harms

Source data underlying Figs 2c-e, 3c,e-f, 6c,d,f, and Supplementary Figs S1c, S3a, S4c-e,h-j, S5c, S8b, S9d-e, S10d-e, S11d

Tandem

10.5281/zenodo.3712821

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Live-cell labeling of endogenous proteins with nanometer precision by transduced nanobodies

Chemical Science (2018)

Alina Klein S. Hank Anika Raulf Eike F. Joest F. Tissen

Using engineered nanobodies with bright organic dyes (fluorescent nanobodies) and subsequent microfluidic cell manipulation, controlled nanobody delivery was achieved, allowing the multiplexed imaging and super-resolution of endogenous protein networks in living cells.

Live cell imaging

10.1039/c8sc02910e

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Citations (29)

Multivalent Chelators for In Vivo Protein Labeling

Angewandte Chemie (2019)

Ralph Wieneke Robert Tampé

Abstract With the advent of single‐molecule methods, chemoselective and site‐specific labeling of proteins evolved to become a central aspect in chemical biology as well as cell biology. Protein labeling demands high specificity, rapid as well as efficient conjugation, while maintaining low concentration and biocompatibility under physiological conditions. Generic methods that do not interfere with the function, dynamics, subcellular localization of proteins, and crosstalk with other factors are crucial to probe and image proteins in vitro and in living cells. Alternatives to enzyme‐based tags or autofluorescent proteins are short peptide‐based recognition tags. These tags provide high specificity, enhanced binding rates, bioorthogonality, and versatility. Here, we report on recent applications of multivalent chelator heads, recognizing oligohistidine‐tagged proteins. The striking features of this system has facilitated the analysis of protein complexes by single‐molecule approaches.

Chemical Biology

Crosstalk

10.1002/ange.201811293

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Citations (1)

Author Correction: Force-tuned avidity of spike variant-ACE2 interactions viewed on the single-molecule level

Nature Communications (2023)

Rong Zhu Daniel Canena Mateusz Sikora Miriam Klausberger Hannah Seferovic

Avidity

10.1038/s41467-023-36905-2

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Formation of Silica Precipitates on Membrane Surfaces in Two and Three Dimensions

Langmuir (2010)

Anja Bernecker Joanna Ziomkowska Svenja Heitmüller Ralph Wieneke Armin Geyer

Ether lipids with alkyl chains of uniform length and varying amine headgroups were synthesized and assembled into bilayer structures in aqueous solution, which served as templates for the formation of silica in two and three dimensions produced under ambient conditions. Dynamic light scattering revealed that unilamellar vesicles of the aminolipids are formed by the extrusion method. The alkylation of the polar amine headgroup was systematically increased from a primary, secondary, and tertiary amine to a quaternary ammonium salt, and the amount of silica was quantified by the beta-silicomolybdate method as a function of the headgroup. A lysinol-connected ether lipid harboring two primary amine groups was also investigated. This variation enabled us to compare the influence of the headgroup on the properties of the precipitated silica in detail. By spreading of unilamellar aminolipid vesicles onto planar silicon substrates, two-dimensional planar bilayers can be produced. By means of ellipsometry in conjunction with atomic force microscopy, we were able to demonstrate that very thin silica layers with a thickness of a few nanometers are formed within minutes on the surface of the aminolipid bilayers. All layers are composed of silica nanospheres, and the thickness turned out to be independent of the amine headgroup.

10.1021/la1021627

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Live-cell protein labelling with nanometre precision by cell squeezing

Nature Communications (2016)

Alina Kollmannsperger Armon Sharei Anika Raulf Mike Heilemann Róbert Langer

Abstract Live-cell labelling techniques to visualize proteins with minimal disturbance are important; however, the currently available methods are limited in their labelling efficiency, specificity and cell permeability. We describe high-throughput protein labelling facilitated by minimalistic probes delivered to mammalian cells by microfluidic cell squeezing. High-affinity and target-specific tracing of proteins in various subcellular compartments is demonstrated, culminating in photoinduced labelling within live cells. Both the fine-tuned delivery of subnanomolar concentrations and the minimal size of the probe allow for live-cell super-resolution imaging with very low background and nanometre precision. This method is fast in probe delivery (∼1,000,000 cells per second), versatile across cell types and can be readily transferred to a multitude of proteins. Moreover, the technique succeeds in combination with well-established methods to gain multiplexed labelling and has demonstrated potential to precisely trace target proteins, in live mammalian cells, by super-resolution microscopy.

Live cell imaging

Single-Cell Analysis

10.1038/ncomms10372

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Citations (104)

Lichtstrukturierte Matrizen für die räumlich-zeitliche Proteinanordnung

BIOspektrum (2018)

Ralph Wieneke

Ex vivo

10.1007/s12268-018-0947-0

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Single-molecule FRET reveals the pre-initiation and initiation conformations of influenza virus promoter RNA

Nucleic Acids Research (2016)

Nicole C. Robb Aartjan J. W. te Velthuis Ralph Wieneke Robert Tampé Thorben Cordes

Influenza viruses have a segmented viral RNA (vRNA) genome, which is replicated by the viral RNA-dependent RNA polymerase (RNAP). Replication initiates on the vRNA 3΄ terminus, producing a complementary RNA (cRNA) intermediate, which serves as a template for the synthesis of new vRNA. RNAP structures show the 3΄ terminus of the vRNA template in a pre-initiation state, bound on the surface of the RNAP rather than in the active site; no information is available on 3΄ cRNA binding. Here, we have used single-molecule Förster resonance energy transfer (smFRET) to probe the viral RNA conformations that occur during RNAP binding and initial replication. We show that even in the absence of nucleotides, the RNAP-bound 3΄ termini of both vRNA and cRNA exist in two conformations, corresponding to the pre-initiation state and an initiation conformation in which the 3΄ terminus of the viral RNA is in the RNAP active site. Nucleotide addition stabilises the 3΄ vRNA in the active site and results in unwinding of the duplexed region of the promoter. Our data provide insights into the dynamic motions of RNA that occur during initial influenza replication and has implications for our understanding of the replication mechanisms of similar pathogenic viruses.

10.1093/nar/gkw884

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Citations (38)

Live-cell protein labelling with nanometre precision by cell squeezing

Nature (2016)

Alina Kollmannsperger Armon Sharei Anika Raulf Mike Heilemann Róbert Langer

Live cell imaging

Nanometre

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Force-tuned Avidity of Spike Variant-ACE2 Interactions viewed on the Single-Molecule Level - MD simulations Dataset

Zenodo (CERN European Organization for Nuclear Research) (2022)

Rong Zhou Daniel Canena Mateusz Sikora Miriam Klausberger Hannah Seferovic

Models of SARS-CoV-2 virus spike protein bound to 1-3 of ACE2 receptors embedded in lipid nanodisks. Systems include all files in GROMACS format needed to reproduce simulations performed in the "Force-tuned Avidity of Spike Variant-ACE2 Interactions viewed on the Single-Molecule Level" article. Details system box size (x-y-z) [nm] Number of atoms Spike +
1x ACE2, full length 33.44834 28.96711 57.95573 5,665,217 Spike +
1x ACE2, truncated 28.05757 24.29856 46.74417 3,203,907 Spike +
2x ACE2, truncated 28.30864 21.23141 48.40873 2,936,398 Spike +
3x ACE2, truncated 28.32733 21.24544 48.30436 2,936,588

Avidity

spike protein

10.5281/zenodo.7401939

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