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Solid-state NMR (ssNMR) spectroscopy facilitates the non-destructive characterization of structurally heterogeneous biomolecules in their native setting, for example, comprising proteins, lipids and polysaccharides. Here we demonstrate the utility of high and ultra-high field 1 H-detected fast MAS ssNMR spectroscopy, which exhibits increased sensitivity and spectral resolution, to further elucidate the atomic-level composition and structural arrangement of the cell wall of Schizophyllum commune, a mushroom-forming fungus from the Basidiomycota phylum. These advancements allowed us to reveal that Cu(II) ions and the antifungal peptide Cathelicidin-2 mainly bind to cell wall proteins at low concentrations while glucans are targeted at high metal ion concentrations. In addition, our data suggest the presence of polysaccharides containing N-acetyl galactosamine (GalNAc) and proteins, including the hydrophobin proteins SC3, shedding more light on the molecular make-up of cells wall as well as the positioning of the polypeptide layer. Obtaining such information may be of critical relevance for future research into fungi in material science and biomedical contexts.

We thank C. Li from the Max Planck Institute of Colloids and Interfaces in Potsdam, Germany, for help during synchrotron measurements at the μSpot beamline at BESSY at the Helmholtz-Zentrum Berlin für Materialien und Energie in Berlin, Germany. We acknowledge the provision of facilities and technical support by Aalto University at the OtaNano Nanomicroscopy Center (Aalto-NMC). This work was supported by the Academy of Finland project 348628, the Jenny and Antti Wihuri Foundation (Centre for Young Synbio Scientists), and the Academy of Finland Center of Excellence Program (2022–2029) in Life-Inspired Hybrid Materials (LIBER) project number 346106, and by internal funding from the VTT Technical Research Center of Finland Ltd. We also acknowledge the Dutch Research Council (NWO, domain Applied and Engineering Sciences: MYCOAT project number 18425) and the Horizon 2020 programs of the European Union (FUNGAR; project 58132 and iNEXT-Discovery, project 871037) for NMR studies. Furthermore, the high-field NMR experiments were supported by uNMR-NL, the National Roadmap Large-Scale NMR Facility of the Netherlands (NWO grant 184.032.207), and the uNMR-NL grid (NWO grant 184.035.002). High strength, hardness, and fracture toughness are mechanical properties that are not commonly associated with the fleshy body of a fungus. Here, we show with detailed structural, chemical, and mechanical characterization that Fomes fomentarius is an exception, and its architectural design is a source of inspiration for an emerging class of ultralightweight high-performance materials. Our findings reveal that F. fomentarius is a functionally graded material with three distinct layers that undergo multiscale hierarchical self-assembly. Mycelium is the primary component in all layers. However, in each layer, mycelium exhibits a very distinct microstructure with unique preferential orientation, aspect ratio, density, and branch length. We also show that an extracellular matrix acts as a reinforcing adhesive that differs in each layer in terms of quantity, polymeric content, and interconnectivity. These findings demonstrate how the synergistic interplay of the aforementioned features results in distinct mechanical properties for each layer.

Cellular dynamic nuclear polarization (DNP) has been an effective means of overcoming the intrinsic sensitivity limitations of solid-state nuclear magnetic resonance (ssNMR) spectroscopy, thus enabling atomic-level biomolecular characterization in native environments. Achieving DNP signal enhancement relies on doping biological preparations with biradical polarizing agents (PAs). Unfortunately, PA performance within cells is often limited by their sensitivity to the reductive nature of the cellular lumen. Herein, we report the synthesis and characterization of a highly bioresistant and hydrophilic PA (StaPol-1) comprising the trityl radical OX063 ligated to a gem-diethyl pyrroline nitroxide via a rigid piperazine linker. EPR experiments in the presence of reducing agents such as ascorbate and in HeLa cell lysates demonstrate the reduction resistance of StaPol-1. High DNP enhancements seen in small molecules, proteins and cell lysates at 18.8 T confirm that StaPol-1 is an excellent PA for DNP ssNMR investigations of biomolecular systems at high magnetic fields in reductive environments.

For almost five decades, solid-state NMR (ssNMR) has been used to study complex biomolecular systems. This article gives a view on how ssNMR methods and applications have evolved during this time period in a broader structural biology context. It also discusses possible directions for additional developments and the future role of ssNMR in a life science context and beyond.

Regulation of DNA-templated processes such as gene transcription and DNA repair depend on the interaction of a wide range of proteins with the nucleosome, the fundamental building block of chromatin. Both solution and solid-state NMR spectroscopy have become an attractive approach to study the dynamics and interactions of nucleosomes, despite their high molecular weight of ∼200 kDa. For solid-state NMR (ssNMR) studies, dilute solutions of nucleosomes are converted to a dense phase by sedimentation or precipitation. Since nucleosomes are known to self-associate, these dense phases may induce extensive interactions between nucleosomes, which could interfere with protein-binding studies. Here, we characterized the packing of nucleosomes in the dense phase created by sedimentation using NMR and small-angle X-ray scattering (SAXS) experiments. We found that nucleosome sediments are gels with variable degrees of solidity, have nucleosome concentration close to that found in crystals, and are stable for weeks under high-speed magic angle spinning (MAS). Furthermore, SAXS data recorded on recovered sediments indicate that there is no pronounced long-range ordering of nucleosomes in the sediment. Finally, we show that the sedimentation approach can also be used to study low-affinity protein interactions with the nucleosome. Together, our results give new insights into the sample characteristics of nucleosome sediments for ssNMR studies and illustrate the broad applicability of sedimentation-based NMR studies.