Biology subjects

Wendler, N.

Publications and source records attributed to Wendler, N..

5 recordsLinked to original sources

bioRxiv2025

A systematic approach for the purification of fluorophore-labelled proteins via anion exchange chromatography

Forster resonance energy transfer (FRET), an optical distance ruler, provides the unique ability to monitor molecular interactions and conformational changes in single biomacromolecules and multi-subunit complexes. It has proven to be a powerful tool to study enzymatic reactions, membrane transport, protein folding, but also the properties of nucleic acids, proteins, molecular motors and many other biological systems and processes. A prerequisite for FRET is targeted (covalent) labelling of macromolecules with two distinct fluorophores. Here, we present a strategy for stochastic labeling of protein residues with the required donor and acceptor dyes via anion exchange chromatography. While this technique has been used before for this purpose, we provide a conceptual basis to systematically design a purification protocol for an arbitrary choice of fluorophores. By characterizing the interaction fluorophore-maleimides with the column material, we are able to select (and predict) which pairs of fluorophores allow successful purification of donor-acceptor-labelled protein with yields up to 98%. We demonstrate the capabilities of the method for bulk and single-molecule FRET assays of various bacterial substrate-binding proteins.

biophysics↗

bioRxiv2024

Determination of Absolute Intramolecular Distances in Proteins by Anomalous X-ray Scattering Interferometry

Biomolecular structures are typically determined using frozen or crystalline samples. Measurement of intramolecular distances in solution can provide additional insights into conformational heterogeneity and dynamics of biological macromolecules and their complexes. The established molecular ruler techniques used for this (NMR, FRET, and EPR) are, however, limited in their dynamic range and require model assumptions to determine absolute distance (distributions). Here, we introduce anomalous X-ray scattering interferometry (AXSI) for intramolecular distance measurements in proteins, which are labeled at two sites with small gold nanoparticles of 0.7 nm radius. We apply AXSI to two different cysteine-variants of maltose binding protein in the presence and absence of its ligand maltose and find distances in quantitative agreement with single-molecule FRET experiments. Our study shows that AXSI enables determination of absolute intramolecular distance distributions under virtually arbitrary solution conditions and we anticipate its broad use to characterize protein conformational ensembles and dynamics.

biophysics↗

bioRxiv2023

High-resolution mapping of Ryd4Hb, a major resistance gene to Barley yellow dwarf virus from Hordeum bulbosum

Virus diseases are causing high yield losses in crops worldwide. The Barley yellow dwarf virus (BYDV) complex is responsible for one of the most widespread and economically important viral diseases of cereals. While no complete resistance gene has been uncovered in the primary genepool of barley, sources of resistance were identified in the wild relative Hordeum bulbosum, representing the secondary genepool of barley. One such locus, Ryd4Hb, has been previously introgressed into barley, and was allocated to chromosome 3H, but is tightly linked to a sublethality factor that prevents the incorporation and utilization of Ryd4Hb in barley varieties. To solve this problem, we fine-mapped Ryd4Hb and separated it from this negative factor. We narrowed the Ryd4Hb locus to a 66.5 kbp physical interval in the barley Morex reference genome. The region comprises one complete and one partial gene from the nucleotide-binding and leucine-rich repeat immune receptor family, typical of dominant virus resistance genes. The closest homolog to these two Ryd4Hb candidate genes is the wheat Sr35 stem rust resistance gene. In addition to the fine mapping, we reduced the sublethality factor interval to 600 kbp in barley. Aphid feeding experiments demonstrated that Ryd4Hb provides a direct resistance to BYDV rather than a resistance to its vector. The presented results, including the high-throughput molecular markers, will permit a more targeted selection of the resistance in breeding, enabling the use of Ryd4Hb in barley varieties. Key messageWe mapped Ryd4Hb in a 66.5 kpb interval in barley and dissociated it from a sublethality factor. These results will enable a targeted selection of the resistance in barley breeding.

plant biology↗

bioRxiv2022

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins

Single-molecule FRET (smFRET) has become an established tool to study biomolecular structure and dynamics in vitro and in live cells. We performed a worldwide blind study involving 19 labs to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems that undergo distinct conformational changes, we obtained an uncertainty of the FRET efficiency of less than {+/-} 0.06, corresponding to an interdye distance precision of [≤] 0.2 nm and accuracy of [≤] 0.5 nm. We further discuss the limits for detecting distance fluctuations with sensitivity down to [lsim] 10% of the Forster distance and provide guidelines on how to detect potential dye perturbations. The ability of smFRET experiments to simultaneously measure distances and avoid averaging of conformational dynamics slower than the fluorescence lifetime is unique for dynamic structural biology.

biophysics↗

bioRxiv2020

High-resolution mapping of Rym14Hb, a wild relative resistance gene to barley yellow mosaic disease

Barley yellow mosaic disease is caused by Barley yellow mosaic virus and Barley mild mosaic virus, and leads to severe yield losses in barley (Hordeum vulgare) in Central Europe and East-Asia. Several resistance loci are used in barley breeding. However, cases of resistance-breaking viral strains are known, raising concerns about the durability of those genes. Rym14Hb is a dominant major resistance gene on chromosome 6HS, originating from barleys secondary genepool wild relative Hordeum bulbosum. As such, the resistance mechanism may represent a case of non-host resistance, which could enhance its durability. A susceptible barley variety and a resistant H. bulbosum introgression line were crossed to produce a large F2 mapping population (n=7,500), to compensate for a ten-fold reduction in recombination rate compared to intraspecific barley crosses. After high-throughput genotyping, the Rym14Hb locus was assigned to a 2Mbp telomeric interval on chromosome 6HS. The co-segregating markers developed in this study can be used for marker-assisted introgression of this locus into barley elite germplasm with a minimum of linkage drag.

plant biology↗