Nicolai Tidemand Johansen

Ex vivo structure determination of macromolecules from native source is gaining increasing attention from the scientific community, as the method can be employed to dissect the function of important, multi-component molecular machines. However, the existing ex vivo procedures often require genome manipulation or availability high-affinity binders, limiting the general applicability. Here, we report simple yet robust principles for isolation of protein complexes from enriched native biological material, enabling cryoEM-facilitated high-resolution structure determination. We report the structures of ten separate membrane and soluble protein complexes determined from spinach leaves. Moreover, the developed pipeline is likely adaptable to essentially any biological system. As such, the approach may represent an attractive avenue for future structural proteomics efforts.

Because of their ability to promote growth, act as biopesticides, and improve abiotic stress tolerance, Trichoderma spp. have been used for plant seed coating. However, the mechanism for the promotion of plant growth remains unknown. In this study, we investigate the effect of fungal extracts on the plant plasma membrane (PM) H+-ATPase, which is essential for plant growth and often a target of plant-associated microbes. We show that Trichoderma harzianum extract increases H+-ATPase activity, and by fractionation and high-resolution mass spectrometry (MS), we identify the activating components trichorzin PA (tPA) II and tPA VI that belong to the class of peptaibols. Peptaibols are nonribosomal peptides that can integrate into membranes and form indiscriminate ion channels, which causes pesticidal activity. To further investigate peptaibol-mediated H+-ATPase activation, we compare the effect of tPA II and VI to that of the model peptaibol alamethicin (AlaM). We show that AlaM increases H+-ATPase turnover rates in a concentration-dependent manner, with a peak in activity measured at 31.25 mu M, above which activity decreases. Using fluorescent probes and light scattering, we find that the AlaM-mediated increase in activity is not correlated to increased membrane fluidity or vesicle integrity, whereas the activity decrease at high AlaM concentrations is likely due to PM overloading of AlaM pores. Overall, our results suggest that the symbiosis of fungi and plants, specifically related to peptaibols, is a concentration-dependent balance, where peptaibols do not act only as biocontrol agents but also as plant growth stimulants.

The combination of online size-exclusion chromatography and small-angle X-ray scattering (SEC–SAXS) is rapidly becoming a key technique for structural investigations of elaborate biophysical samples in solution. Here, a novel model-refinement strategy centred around the technique is outlined and its utility is demonstrated by analysing data series from several SEC–SAXS experiments on phospholipid bilayer nanodiscs. Using this method, a single model was globally refined against many frames from the same data series, thereby capturing the frame-to-frame tendencies of the irradiated sample. These are compared with models refined in the traditional manner, in which refinement is based on the average profile of a set of consecutive frames from the same data series without an in-depth comparison of individual frames. This is considered to be an attractive model-refinement scheme as it considerably lowers the total number of parameters refined from the data series, produces tendencies that are automatically consistent between frames, and utilizes a considerably larger portion of the recorded data than is often performed in such experiments. Additionally, a method is outlined for correcting a measured UV absorption signal by accounting for potential peak broadening by the experimental setup.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg²⁺ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg²⁺-bound closed structure nor the Mg²⁺-free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg²⁺. We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg²⁺ but that conducting states become more populated in Mg²⁺-free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Isothermal titration calorimetry (ITC) is a widely used method to determine binding affinities and thermodynamics in ligand–receptor interactions, but it also has the capability of providing detailed information on much more complex events. However, the lack of available methods to analyze ITC data is limiting the use of the technique in such multifaceted cases. Here, we present the software ANISPROU. Through a semi-empirical approach that allows for extraction of quantitative information from complex ITC data, ANISPROU solves an inverse problem where three parameters describing a set of predefined functions must be found. In analogy to strategies adopted in other scientific fields, such as geophysics, imaging, and many others, it employs an optimization algorithm which minimizes the difference between calculated and experimental data. In contrast to the existing methods, ANISPROU provides automated and objective analysis of ITC data on sodium dodecyl sulfate (SDS)-induced protein unfolding, and in addition, more information can be extracted from the data. Here, data series on SDS-mediated protein unfolding is analyzed, and binding isotherms and thermodynamic information on the unfolding events are extracted. The obtained binding isotherms as well as the enthalpy of different events are similar to those obtained using the existing manual methods, but our methodology ensures a more robust result, as the entire data set is used instead of single data points. We foresee that ANISPROU will be useful in other cases with complex enthalpograms, for example, in cases with coupled interactions in biomolecular, polymeric, and amphiphilic systems including cases where both structural changes and interactions occur simultaneously.

The CorA family of proteins regulates the homeostasis of divalent metal ions in many bacteria, archaea, and eukaryotic mitochondria, making it an important target in the investigation of the mechanisms of transport and its functional regulation. Although numerous structures of open and closed channels are now available for the CorA family, the mechanism of the transport regulation remains elusive. Here, we investigated the conformational distribution and associated dynamic behaviour of the pentameric Mg ²⁺ channel CorA at room temperature using small-angle neutron scattering (SANS) in combination with molecular dynamics (MD) simulations and solid-state nuclear magnetic resonance spectroscopy (NMR). We find that neither the Mg ²⁺ -bound closed structure nor the Mg ²⁺ -free open forms are sufficient to explain the average conformation of CorA. Our data support the presence of conformational equilibria between multiple states, and we further find a variation in the behaviour of the backbone dynamics with and without Mg ²⁺ . We propose that CorA must be in a dynamic equilibrium between different non-conducting states, both symmetric and asymmetric, regardless of bound Mg ²⁺ but that conducting states become more populated in Mg ²⁺ -free conditions. These properties are regulated by backbone dynamics and are key to understanding the functional regulation of CorA.

Nanodiscs based on membrane scaffold proteins (MSPs) and phospholipids are used as membrane mimics to stabilize membrane proteins in solution for structural and functional studies. Combining small-angle X-ray scattering (SAXS), differential scanning calorimetry (DSC), and time-resolved small-angle neutron scattering (TR-SANS), we characterized the structure and lipid bilayer properties of five different nanodiscs made with dimyristoylphosphatidylcholine and different MSPs varying in size, charge, and circularization. Our SAXS modeling showed that the structural parameters of the embedded lipids are all similar, irrespective of the MSP properties. DSC showed that the lipid packing is not homogeneous in the nanodiscs and that a 20 Å wide boundary layer of lipids with perturbed packing is located close to the MSP, while the packing of central lipids is tighter than in large unilamellar vesicles. Finally, TR-SANS showed that lipid exchange rates in nanodiscs decrease with increasing nanodisc size and are lower for the nanodiscs made with supercharged MSPs compared to conventional nanodiscs. Altogether, the results provide a thorough biophysical understanding of the nanodisc as a model membrane system, which is important in order to carry out and interpret experiments on membrane proteins embedded in such systems.

Pentameric ligand-gated ion channels undergo subtle conformational cycling to control electrochemical signal transduction in many kingdoms of life. Several crystal structures have now been reported in this family, but the functional relevance of such models remains unclear. Here, we used small-angle neutron scattering (SANS) to probe ambient solution-phase properties of the pH-gated bacterial ion channel GLIC under resting and activating conditions. Data collection was optimized by inline paused-flow size-exclusion chromatography, and exchanging into deuterated detergent to hide the micelle contribution. Resting-state GLIC was the best-fit crystal structure to SANS curves, with no evidence for divergent mechanisms. Moreover, enhanced-sampling molecular dynamics simulations enabled differential modeling in resting versus activating conditions, with the latter corresponding to an intermediate ensemble of both the extracellular and transmembrane domains. This work demonstrates state-dependent changes in a pentameric ion channel by SANS, an increasingly accessible method for macromolecular characterization with the coming generation of neutron sources.

Apolipoproteins are vital to lipid metabolism and cholesterol transport in the human body. Here we present a structural study of the lipid-bound particles formed by ApoE3 in a full-length and a truncated version. The particles are formed with, respectively, POPC and DMPC and investigated by small-angle X-ray scattering and negative stain electron microscopy. We find that lipid-bound ApoE3 particles are elliptical, disc-shaped particles composed of a central lipid bilayer encircled by two amphipathic ApoE3 proteins. We went on to investigate a truncated form of ApoE3 containing only residue 80 to 255 (ApoE3 ^80-255 ), which is the central helical repeat segment of ApoE3. The lipid-bound ApoE3 ^80-255 particles are found to have the same morphology as the particles with full-length ApoE3. However, they are larger, and form more heterogeneous discoidal structures with four proteins per particle. This behavior is in contrast to ApoA1 where the highly similar helical repeat domain determines the size and stoichiometry of the formed particles both in the case of full-length and truncated ApoA1. Our data hence points towards different mechanisms for lipid bilayer structural modulation by ApoA1 and ApoE3 due to different roles of the non-repeat segments.