Biology subjects

Gunkel, M.

Publications and source records attributed to Gunkel, M..

5 recordsLinked to original sources

bioRxiv2024

A versatile protocol for purifying recombinant proteins from Nicotiana benthamiana for structural studies

Structural biology is an essential tool for understanding the molecular basis of biological processes. Although predicting protein structures by fold recognition algorithms has become increasingly powerful, especially with the integration of deep-learning approaches, experimentally resolved structures are indispensable for guiding structure-function studies and for improving modelling. However, experimental structural studies of protein complexes are still challenging, owing to, for example, the necessity for high protein concentrations and purity for downstream analyses such as cryogenic electron microscopy (cryo-EM). The use of Nicotiana benthamiana leaves as a transient expression system for recombinant proteins has become an increasingly attractive approach as the plant is inexpensive to cultivate, grows rapidly, allows fast experimental turnaround and is easily scalable compared to other established systems such as insect cell cultures. Using N. benthamiana as an expression system, we present here a robust and versatile protocol for the purification of five heterocomplexes with sizes ranging from [~]140 kDa to [~]660 kDa consisting of immunoreceptors and their associated pathogen effectors, followed by electron microscopy. The plant-based protocol was applied to verify the structure of the insect cell-derived wheat Sr35 resistosome and to co-purify and co-resolve a [~]140 kDa homodimer of the AvrSr35 effector from the fungus Puccinia graminis f sp tritici (Pgt). In several cases, only a single epitope tag is needed for complex purification, reducing complications that come with multiple epitope tags and two-step affinity purifications. We identify codon usage, signal peptide fusion, epitope tag choice and detergents as critical factors for expression and purification of recombinant protein from N. benthamiana leaves.

biochemistry↗

bioRxiv2024

Revisiting Sodium Phosphotungstate and Ammonium Molybdate as non-radioactive negative staining agents for single particle analysis

This study reports the successful replacement of uranyl-based stains by either sodium phosphotungstate or ammonium molybdate for negative staining electron microscopy. Using apoferritin as a test specimen, it is demonstrated that in combination with a facile on-grid fixation step both stains yield comparable images to uranyl formate. Subsequently, using {beta}-galactosidase, it is shown that both stains can also successfully be employed for single particle analysis, yielding virtually indistinguishable results from uranyl formate. As both replacement stains are non-radioactive, they are not subjected to the same handling restrictions as uranyl-based stains. Therefore they are not only cheaper to use, but also make decentralized sample grid preparation - thus directly after purification - accessible to a broader range of scientists.

biochemistry↗

bioRxiv2024

The barley MLA13-AVRA13 heterodimer reveals principles for immunoreceptor recognition of RNase-like powdery mildew effectors

Co-evolution between cereals and pathogenic grass powdery mildew fungi is exemplified by sequence diversification of an allelic series of barley resistance genes encoding Mildew Locus A (MLA) nucleotide-binding leucine-rich repeat (NLR) immunoreceptors with a N-terminal coiled-coil domain (CNLs). Each immunoreceptor recognises a matching, strain-specific powdery mildew effector encoded by an avirulence gene (AVRa). We present here the cryo-EM structure of barley MLA13 in complex with its cognate effector AVRA13-1. The effector adopts an RNase-like fold when bound to MLA13 in planta, similar to crystal structures of other RNase-like AVRA effectors purified from E. coli. AVRA13-1 interacts via its basal loops with MLA13 C-terminal leucine rich repeats (LRRs) and the central winged helix domain (WHD). Co-expression of structure-guided MLA13 and AVRA13-1 substitution variants show that the receptor-effector interface plays an essential role in mediating immunity-associated plant cell death. Furthermore, by combining structural information from the MLA13-AVRA13-1 heterocomplex with sequence alignments of other MLA receptors, we designed a single amino acid substitution in MLA7 that enables expanded effector detection of AVRA13-1 and the virulent variant AVRA13-V2. In contrast to the pentameric conformation of previously reported effector-activated CNL resistosomes, MLA13 was purified and resolved as a stable heterodimer from an in planta expression system. Our study suggests that the MLA13-AVRA13-1 heterodimer might represent a CNL output distinct from CNL resistosomes and highlights opportunities for the development of designer gain-of-function NLRs.

plant biology↗

bioRxiv2023

A marine cryptochrome with an inverse photo-oligomerization mechanism

Cryptochromes (CRYs) are a structurally conserved but functionally diverse family of proteins that can confer unique sensory properties to organisms. In the marine bristle worm Platynereis dumerilii, its light receptive cryptochrome L-CRY (PdLCry) allows the animal to discriminate between sunlight and moonlight, an important requirement for synchronizing its lunar cycle-dependent mass spawning. Using cryo-electron microscopy, we show that in the dark, PdLCry adopts a dimer arrangement observed neither in plant nor insect CRYs. Intense illumination disassembles the dimer into monomers. Structural and functional data suggest a mechanistic coupling between the light-sensing flavin adenine dinucleotide chromophore, the dimer interface, and the C-terminal tail helix, with a likely involvement of the phosphate binding loop. Taken together, our work establishes PdLCry as a CRY protein with inverse photo-oligomerization with respect to plant CRYs, and provides molecular insights into how this protein might help discriminating the different light intensities associated with sunlight and moonlight.

biochemistry↗

bioRxiv2023

Targeted volume Correlative Light and Electron Microscopy of an environmental marine microorganism

Photosynthetic microalgae are responsible for an important fraction of CO2 fixation and O2 production on Earth. Three-dimensional ultrastructural characterization of these organisms in their natural environment can contribute to a deeper understanding of their cell biology. However, the low throughput of volume electron microscopy (vEM) methods, along with the complexity and heterogeneity of environmental samples, pose great technical challenges. In the present study, we used a workflow based on a specific EM sample preparation, compatible with both light and vEM imaging in order to target one cell among a complex natural community. This method revealed the 3D subcellular landscape of a photosynthetic dinoflagellate with quantitative characterization of multiple organelles. We could show that this cell contains a single convoluted chloroplast and the arrangement of the flagellar apparatus with its associated photosensitive elements. Moreover, we observed chromatin features that could shed light on how transcriptional activity takes place in organisms where chromosomes are permanently condensed. Together with providing insights in dinoflagellates biology, this proof-of-principle study illustrates an efficient tool for the targeted ultrastructural analysis of environmental microorganisms in heterogeneous mixes.

cell biology↗