Rational design of19F NMR labelling sites to probe protein structure and interactions DOI Creative Commons
Julian O. Streit, Sammy H. S. Chan,

Saifu Daya

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Dec. 12, 2024

Abstract Proteins are investigated in increasingly more complex biological systems, where 19 F NMR is proving highly advantageous due to its high gyromagnetic ratio and background-free spectra. Its application has, however, been hindered by limited chemical shift dispersions an incomprehensive relationship between shifts protein structure. We exploit the sensitivity of ring currents designing labels with direct contact a native or engineered aromatic ring. Fifty variants predicted AlphaFold molecular dynamics simulations show 80-90% success rates correlations their experimental magnitude current. Our method consequently improves dispersion through simple 1D experiments enables structural analyses alternative conformational states, including ribosome-bound folding intermediates, in-cell measurements thermodynamics protein-protein interactions. strategy thus provides sensitive tool extract residue restraints from for previously intractable systems.

Language: Английский

On the distance to the transition state of protein folding in optical tweezers experiments DOI

Carlos Correa,

Christian A.M. Wilson

Biophysical Reviews, Journal Year: 2025, Volume and Issue: 17(1), P. 45 - 54

Published: Jan. 9, 2025

Language: Английский

Citations

2
Single-molecule orientation-localization microscopy: Applications and approaches DOI
Oumeng Zhang, Matthew D. Lew

Quarterly Reviews of Biophysics, Journal Year: 2024, Volume and Issue: 57

Published: Jan. 1, 2024

Abstract Single-molecule orientation-localization microscopy (SMOLM) builds upon super-resolved localization by imaging orientations and rotational dynamics of individual molecules in addition to their positions. This added dimensionality provides unparalleled insights into nanoscale biophysical biochemical processes, including the organization actin networks, movement molecular motors, conformations DNA strands, growth remodeling amyloid aggregates, composition changes within lipid membranes. In this review, we discuss recent innovations SMOLM cover three key aspects: (1) enabled labeling strategies that endow fluorescent probes bind targets with orientation specificity; (2) advanced techniques leverage physics light-matter interactions estimation theory encode information high fidelity microscope images; (3) computational methods ensure accurate precise data analysis interpretation, even presence severe shot noise. Additionally, compare approaches, hardware, publicly available software aid community choosing best implementation for specific application. Finally, highlight future directions SMOLM, such as development improved photostability specificity, design “smart” adaptive use approaches handle large, complex datasets. review underscores significant current potential impact deepening our understanding dynamics, paving way breakthroughs fields biophysics, biochemistry, materials science.

Language: Английский

Citations

4
Keeping Cells Alive in Microscopy DOI Creative Commons
Herbert Schneckenburger, Christoph Cremer

Biophysica, Journal Year: 2025, Volume and Issue: 5(1), P. 1 - 1

Published: Jan. 6, 2025

Light microscopy has emerged as one of the fundamental methods to analyze biological systems; novel techniques 3D and super-resolution (SRM) with an optical resolution down sub-nanometer range have recently been realized. However, most these achievements made fixed specimens, i.e., direct information about dynamics biosystem studied was not possible. This stimulated development live cell imaging approaches, including Low Illumination Fluorescence Microscopy, Sheet (Fluorescence) Microscopy (LSFM), or Structured (SIM). Here, we discuss perspectives, methods, relevant light doses advanced fluorescence keep cells alive at low levels phototoxicity.

Language: Английский

Citations

0
Mechanism of sensor kinase CitA transmembrane signaling DOI Creative Commons

Xizhou Cecily Zhang,

Kai Xue,

Michele Salvi

et al.

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: Jan. 22, 2025

Abstract Membrane bound histidine kinases (HKs) are ubiquitous sensors of extracellular stimuli in bacteria. However, a uniform structural model is still missing for their transmembrane signaling mechanism. Here, we used solid-state NMR conjunction with crystallography, solution and distance measurements to investigate the mechanism paradigmatic citrate sensing membrane embedded HK, CitA. Citrate binding sensory extracytoplasmic PAS domain (PASp) causes linker helix 2 (TM2) adopt helical conformation. This triggers piston-like pulling TM2 quaternary structure rearrangement cytosolic (PASc). Crystal structures PASc reveal both anti-parallel parallel dimer conformations. An transition upon agrees interdimer distances measured lipid protein using site-specific 19 F label PASc. These data show how Angstrom scale changes sensor transmitted across be converted amplified into nm shift phosphorylation subdomain kinase.

Language: Английский

Citations

0
Matchmaking at the cell surface using bispecifics to put cells on their best behavior DOI Creative Commons
C. Driscoll, Mark Howarth

Current Opinion in Biotechnology, Journal Year: 2025, Volume and Issue: 92, P. 103267 - 103267

Published: Feb. 5, 2025

Language: Английский

Citations

0
MINFLUX achieves molecular resolution with minimal photons DOI
Lukas Scheiderer, Zach Marin, Jonas Ries

et al.

Nature Photonics, Journal Year: 2025, Volume and Issue: 19(3), P. 238 - 247

Published: March 1, 2025

Language: Английский

Citations

0
Microtubules as a versatile reference standard for expansion microscopy DOI Creative Commons
Rajdeep Chowdhury,

Tiago Mimoso,

Abed Alrahman Chouaib

et al.

Communications Biology, Journal Year: 2025, Volume and Issue: 8(1)

Published: March 26, 2025

Abstract Expansion microscopy (ExM) is continually improving, and new ExM variants need to be validated on well-defined biological structures. There no consensus validation structures for ExM, especially as nuclear pore complexes or DNA nanorulers are not popular studies. Here we propose that microtubules should used validation. The diameter of immunostained using primary secondary antibodies sufficiently large the techniques with resolutions better than 50 nm. For higher precision (up ~10 nm), can assembled imaged in vitro, a protocol introduce here. Alternatively, cellular extraction procedure employed, followed by labeling peptide chains tubulin molecules NHS-ester fluorophores. Finally, nanometer-scale techniques, single analyzed. We conclude valuable related technologies.

Language: Английский

Citations

0
NanoDriftGuard: Open-source isotropic ångström-scale active stabilization for super-resolution microscopy DOI

Xiaofan Sun,

Zhengyi Zhan, Chenying He

et al.

Optics and Lasers in Engineering, Journal Year: 2025, Volume and Issue: 190, P. 108957 - 108957

Published: March 26, 2025

Language: Английский

Citations

0
Nanoscale mechano-adaption of integrin-based cell adhesions: New tools and techniques lead the way DOI Creative Commons
R. Nishimura, Pakorn Kanchanawong

Current Opinion in Cell Biology, Journal Year: 2025, Volume and Issue: 94, P. 102509 - 102509

Published: April 8, 2025

Force generation and transmission in biological systems are driven by protein-based machinery organized at the nanoscale. Thus, technological advances that allow for measurement or manipulation of molecular-scale features key to new mechanobiological insights. Integrins, a superfamily adhesion receptors, function forming supramolecular complexes mediate processes such as migration matrix remodeling. This review highlights recent findings harness advanced techniques microscopy, nanotechnology, biosensors uncover nanoscale transformations accompany integrin responses stimuli. Recent discoveries sharpening our understanding diverse functions structural organization different heterodimers their molecular partners, highlighting critical roles cellular processes.

Language: Английский

Citations

0
Penta‐ALFA‐Tagged Substrates for Self‐Labelling Tags Allow Signal Enhancement in Microscopy DOI Creative Commons
Souvik Ghosh,

Ramona Birke,

Ashwin Karthick Natarajan

et al.

Journal of Peptide Science, Journal Year: 2025, Volume and Issue: 31(5)

Published: April 13, 2025

Self-labelling proteins like SNAP- and HaloTag have advanced imaging in life sciences by enabling live-cell labeling with fluorophore-conjugated substrates. However, the typical one-fluorophore-per-protein system limits signal intensity. To address this, we developed a strategy using ALFA-tag system, 13-amino acid peptide recognized bio-orthogonal fluorescently labelled nanobody, for amplification. We synthesized pentavalent ALFA5 used an azidolysine conjugation Cy5-modified or ligand through strain-promoted click chemistry. In vitro measurements on SDS-PAGE showed labelling, peptides covalently reacted their respective tag. HEK293 cells expressing HaloTag-mGluR2 fusion were labeled ALFA5-Cy5 substrates, confocal microscopy revealed significant enhancement far-red intensity upon nanobody addition, as quantified integrated density ratios. Comparisons between substrates superior performance latter, achieving better signal-to-noise signal-to-background ratios, well overall plasma membrane-localized regions. Our results demonstrate potential of ALFA-tag-based systems to amplify SLP fluorescent signals. This combines photostability synthetic fluorophores multivalent labeling, providing powerful tool applications including super-resolution cells. Its versatility is expandable across diverse protein colors.

Language: Английский

Citations

0