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Instruments / Technical Area	Publication Details	Published
B21-High Throughput SAXS	The dynamic nature of netrin-1 and the structural basis for glycosaminoglycan fragment-induced filament formation Markus Meier , Monika Gupta , Serife Akgül , Matthew Mcdougall , Thomas Imhof , Denise Nikodemus , Raphael Reuten , Aniel Moya-Torres , Vu To , Fraser Ferens , Fabian Heide , Gay P. Padilla-Meier , Philipp Kukura , Wenming Huang , Birgit Gerisch , Matthias Mörgelin , Kate Poole , Adam Antebi , Manuel Koch , Jörg Stetefeld Nature Communications 14 DOI: 10.1038/s41467-023-36692-w Open Access Show Abstract ▼ Abstract: Netrin-1 is a bifunctional chemotropic guidance cue that plays key roles in diverse cellular processes including axon pathfinding, cell migration, adhesion, differentiation, and survival. Here, we present a molecular understanding of netrin-1 mediated interactions with glycosaminoglycan chains of diverse heparan sulfate proteoglycans (HSPGs) and short heparin oligosaccharides. Whereas interactions with HSPGs act as platform to co-localise netrin-1 close to the cell surface, heparin oligosaccharides have a significant impact on the highly dynamic behaviour of netrin-1. Remarkably, the monomer-dimer equilibrium of netrin-1 in solution is abolished in the presence of heparin oligosaccharides and replaced with highly hierarchical and distinct super assemblies leading to unique, yet unknown netrin-1 filament formation. In our integrated approach we provide a molecular mechanism for the filament assembly which opens fresh paths towards a molecular understanding of netrin-1 functions.	Mar 2023
B21-High Throughput SAXS	Solution structure and oligomeric state of the E. coli glycerol facilitator Mary Hernando , George Orris , Jacqueline Perodeau , Shixing Lei , Fraser G. Ferens , Trushar R. Patel , Jörg Stetefeld , Andrew J. Nieuwkoop , Joe D. O'Neil Biochimica Et Biophysica Acta (bba) - Biomembranes DOI: 10.1016/j.bbamem.2020.183191 Diamond Proposal Number(s): [16028] Show Abstract ▼ Abstract: Protein dynamics at atomic resolution can provide deep insights into the biological activities of proteins and enzymes but they can also make structure and dynamics studies challenging. Despite their well-known biological and pharmaceutical importance, integral membrane protein structure and dynamics studies lag behind those of water-soluble proteins mainly owing to solubility problems that result upon their removal from the membrane. Escherichia coli glycerol facilitator (GF) is a member of the aquaglyceroporin family that allows for the highly selective passive diffusion of its substrate glycerol across the inner membrane of the bacterium. Previous molecular dynamics simulations and hydrogen-deuterium exchange studies suggested that protein dynamics play an important role in the passage of glycerol through the protein pore. With the aim of studying GF dynamics by solution and solid-state nuclear magnetic resonance (NMR) spectroscopy we optimized the expression of isotope-labelled GF and explored various solubilizing agents including detergents, osmolytes, amphipols, random heteropolymers, lipid nanodiscs, bicelles and other buffer additives to optimize the solubility and polydispersity of the protein. The GF protein is most stable and soluble in lauryl maltose neopentyl glycol (LMNG), where it exists in a tetramer-octamer equilibrium. The solution structures of the GF tetramer and octamer were determined by negative-stain transmission electron microscopy (TEM), size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) and solid-state magic-angle spinning NMR spectroscopy. Although NMR sample preparation still needs optimization for full structure and dynamics studies, negative stain TEM and SEC-SAXS revealed low-resolution structures of the detergent-solubilized tetramer and octamer particles. The non-native octamer appears to form from the association of the cytoplasmic faces of two tetramers, the interaction apparently mediated by their disordered N- and C-termini. This information may be useful in future studies directed at reducing the heterogeneity and self-association of the protein.	Jan 2020
B21-High Throughput SAXS	A cholesterol analogue induces an oligomeric reorganization of VDAC Fraser G. Ferens , Trushar R. Patel , George Orriss , Deborah A. Court , Jörg Stetefeld Biophysical Journal DOI: 10.1016/j.bpj.2019.01.031 Diamond Proposal Number(s): [16028] Show Abstract ▼ Abstract: The oligomeric organization of the voltage-dependent anion-selective channel (VDAC) and its interactions with hexokinase play integral roles in mitochondrially mediated apoptotic signaling. Various small to large assemblies of VDAC are observed in mitochondrial outer membranes but they do not predominate in detergent solubilized VDAC samples. In this study, a cholesterol analogue, cholesteryl-hemisuccinate (CHS), was shown induce the formation of detergent soluble VDAC multimers. The various oligomeric states of VDAC induced by the addition of CHS were deciphered through an integrated biophysics approach using microscale thermophoresis, analytical ultracentrifugation and size exclusion chromatography-small angle X-ray scattering. Furthermore, CHS stabilizes the interaction between VDAC and hexokinase (Kd of 27 ± 6 μM), confirming the biological relevance of oligomers generated. Thus, sterols such as cholesterol in higher eukaryotes or ergosterol in fungi may regulate VDAC oligomeric state and may provide a potential target for the modulation of apoptotic signaling by effecting VDAC-VDAC and VDAC-hexokinase interactions. In addition, the integrated biophysical approach described provides a powerful platform for the study of membrane protein complexes in solution.	Feb 2019

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