bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.11.23.394080

Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET

Peter, M. F.·Gebhardt, C.·Maechtel, R.·Glaenzer, J.·Thomas, G. H.·Cordes, T.·Hageluecken, G.
View original source ↗Export metadata ↓

Abstract

Pulsed electron-electron double resonance spectroscopy (PELDOR or DEER) and single molecule Forster resonance energy transfer spectroscopy (smFRET) are recent additions to the toolbox of integrative structural biology. Both methods are frequently used to visualize conformational changes and to determine nanometer-scale distances in biomacromolecules including proteins and nucleic acids. A prerequisite for the application of PELDOR/DEER and smFRET is the presence of suitable spin centers or fluorophores in the target molecule, which are usually introduced via chemical biology methods. The application portfolio of the two methods is overlapping: each allows determination of distances, to monitor distance changes and to visualize conformational heterogeneity and -dynamics. Both methods can provide qualitative information that facilitates mechanistic understanding, for instance on conformational changes, as well as quantitative data for structural modelling. Despite their broad application, a comprehensive comparison of the accuracy of PELDOR/DEER and smFRET is still missing and we set out here to fill this gap. For this purpose, we prepared a library of double cysteine mutants of three well-studied substrate binding proteins that undergo large-scale conformational changes upon ligand binding. The distances between the introduced spin- or fluorescence labels were determined via PELDOR/DEER and smFRET, using established standard experimental protocols and data analysis routines. The experiments were conducted in the presence and absence of the natural ligands to investigate how well the ligand-induced conformational changes could be detected by the two methods. Overall, we found good agreement for the determined distances, yet some surprising inconsistencies occurred. In our set of experiments, we identified the source of discrepancies as the use of cryoprotectants for PELDOR/DEER and label-protein interactions for smFRET. Our study highlights strength and weaknesses of both methods and paves the way for a higher confidence in quantitative comparison of PELDOR/DEER and smFRET results in the future.

Source connections

Explore related subjects

biophysicsPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Peter, M. F., Gebhardt, C., Maechtel, R., Glaenzer, J., Thomas, G. H., Cordes, T., Hageluecken, G.. 2020-11-23. Cross-validation of distance measurements in proteins by PELDOR/DEER and single-molecule FRET. https://doi.org/10.1101/2020.11.23.394080

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Frequency-Dependent Structural Responses of Amyloid-β Fibrils to Terahertz Pulses: A Nonequilibrium Molecular Dynamics Study

The abnormal aggregation of amyloid proteins into fibrils is closely associated with several neurodegenerative diseases, highlighting the importance of developing effective strategies to modulate fibril structural stability. Here, we employed nonequilibrium molecular dynamics(NEMD) simulations to examine the effects of intense terahertz-to-mid-infrared pulses on three amyloid fibrils (2LMP, 2MXU, and 5OQV), focusing on the combined effects of pulse frequency and electric-field strength. Frequency scans from 0.5 to 60 THz in 0.5 THz steps revealed frequency-dependent losses of {beta}-sheet content, with several common response patterns in these fibrils. Comparisons at 2, 23, 33, and 50 THz showed that pulse-induced changes in secondary structure differed from those in high-temperature controls. Hydrogen-bond analysis showed that total hydrogen-bond counts could partially recover even as interchain hydrogen bonds continued to decrease, with distinct responses among representative chain pairs. We therefore propose a two-threshold electric-field framework for fibril disruption: Eth1 describes the onset of structural perturbation, whereas Eth2 indicates persistent structural loss after the pulse. This framework offers a testable approach for characterizing amyloid structural responses to external fields, although the underlying energy-coupling mechanisms, quantitative thresholds, and long-term recovery require further validation. These findings may provide a theoretical basis for exploring external-field-based strategies to modulate protein aggregation.

biophysics↗
bioRxiv2026

Organised streaming of cerebrospinal fluid in the human third ventricle

Quasi-periodic pressure pulses associated with cardiac and respiratory activity generate reciprocating cerebrospinal fluid (CSF) motion in the human ventricles. Regular waves dominated by the cardiac cycle are overlaid with cycles of low-frequency respiratory waves, the latter to become stronger in forced breathing or even dominated by large successive flushes in NREM sleep. The present study investigates how such waves contribute to residual streaming within the human ventricles. Transient CFD simulations were performed in the third ventricle at varying parameters of frequency, stroke volume and wave-form asymmetry. Increasing the aqueduct peak Reynolds-number, either by small stroke volume and high frequency or large stroke volume at low frequency, transfers the flow from a reversible flow pattern to the formation of clear preferential pathways, alternating between the in- and outflow cycle. The resulting permanent streaming flux across the aqueduct relative to the mean oscillatory flow-rate, i.e. the dimensionless streaming ratio SR follows a rational saturation relation, SR [~] ReAq,peak/(C + ReAq,peak), demonstrating that increasing the peak aqueduct Reynolds number yields diminishing returns and bounds the relative contribution of residual inter-ventricular streaming. These findings demonstrate differences in hydrodynamic organisation in the third ventricle and highlight the importance of this organisation for intraventricular fluid exchange.

biophysics↗
bioRxiv2026

Amyloid stability and condensate material properties independently govern the hnRNPA2 condensate to fibril transition

hnRNPA2 is a component of cellular membraneless organelles many of which contain RNA and RNA-binding proteins (RBPs). RBPs frequently contain low-complexity domains (LCDs) that can also assemble into amyloid fibrils. Fibrils isolated from patients with frontotemporal lobar degeneration contain ordered segments of these LCDs and disease-associated mutations can enhance the homotypic interactions that promote aggregation. Although previous studies have largely examined the condensate-to-fibril transition by altering condensate stability, how amyloid structure governs this process remains poorly understood. Here, we show that the hnRNPA2 LCD forms a reproducible fibril fold and by combining structure-guided thermodynamic profiling, peptide design, mutagenesis and in vitro characterization under conditions with (two-phase) and without (one-phase) condensates, we define the molecular determinants of its assembly. We identify an aromatic triad within the fibril core that controls fibril formation kinetics and thermodynamic stability. Mutational analysis further reveals that condensate stability and fibril stability are largely independent. Finally, microrheology linked reduced condensate viscosity to faster fibril formation, supporting a role for condensate material properties in regulating aggregation kinetics. Together, these findings support a model in which the condensate-to-fibril transition is governed by the interplay between a disordered ''fuzzy coat'' and a fibril-forming aromatic triad within hnRNPA2. This work provides a structural framework for understanding how condensates can transition into amyloid assemblies.

biophysics↗