EquipSent - Enabling Sustainable Education, Everywhere.

EquipSent is a volunteer-based non-profit organization aiming at creating conditions for sustainable teaching, study, and academic research worldwide. Used, functional equipment is collected by its members, who are responsible for matching the donations with the receivers in need. After starting in 2017, nine big transfers were accomplished that significantly impacted the quality of local scientific and educational life.

Entropy Tug-of-War Determines Solvent Effects in the Liquid-Liquid Phase Separation of a Globular Protein.

Liquid-liquid phase separation (LLPS) plays a key role in the compartmentalization of cells via the formation of biomolecular condensates. Here, we combined atomistic molecular dynamics (MD) simulations and terahertz (THz) spectroscopy to determine the solvent entropy contribution to the formation of condensates of the human eye lens protein γD-Crystallin. The MD simulations reveal an entropy tug-of-war between water molecules that are released from the protein droplets and those that are retained within the condensates, two categories of water molecules that were also assigned spectroscopically.

Dissecting the Molecular Origin of -Tensor Heterogeneity and Strain in Nitroxide Radicals in Water: Electron Paramagnetic Resonance Experiment versus Theory.

Nitroxides are common EPR sensors of microenvironmental properties such as polarity, numbers of H-bonds, pH, and so forth. Their solvation in an aqueous environment is facilitated by their high propensity to form H-bonds with the surrounding water molecules. Their - and -tensor elements are key parameters to extracting the properties of their microenvironment.

Electron paramagnetic resonance spectroscopy in structural-dynamic studies of large protein complexes.

Macromolecular protein assemblies are of fundamental importance for many processes inside the cell, as they perform complex functions and constitute central hubs where reactions occur. Generally, these assemblies undergo large conformational changes and cycle through different states that ultimately are connected to specific functions further regulated by additional small ligands or proteins. Unveiling the 3D structural details of these assemblies at atomic resolution, identifying the flexible parts of the complexes, and monitoring with high temporal resolution the dynamic interplay between different protein regions under physiological conditions is key to fully understanding their properties and to fostering biomedical applications.

A Potent Auto-Umpolung Ligand for Conjugative Radical Stabilization.

Carbenes with conjugatively connected redox system act as "auto-umpolung" ligands. Due to their electronic flexibility, they should also be particularly suitable to stabilize open-shell species. Herein, the first neutral radical of such sort is described in form of a dialkylamino-substituted bis(dicyanomethylene)cyclopropanide.

The ABC transporter MsbA adopts the wide inward-open conformation in cells.

Membrane proteins are currently investigated after detergent extraction from native cellular membranes and reconstitution into artificial liposomes or nanodiscs, thereby removing them from their physiological environment. However, to truly understand the biophysical properties of membrane proteins in a physiological environment, they must be investigated within living cells. Here, we used a spin-labeled nanobody to interrogate the conformational cycle of the ABC transporter MsbA by double electron-electron resonance.

Orthogonal spin labeling and pulsed dipolar spectroscopy for protein studies.

Different types of spin labels are currently available for structural studies of biomolecules both in vitro and in cells using Electron Paramagnetic Resonance (EPR) and pulse dipolar spectroscopy (PDS). Each type of label has its own advantages and disadvantages, that will be addressed in this chapter. The spectroscopically distinct properties of the labels have fostered new applications of PDS aimed to simultaneously extract multiple inter-label distances on the same sample.

Neural networks in pulsed dipolar spectroscopy: A practical guide.

This is a methodological guide to the use of deep neural networks in the processing of pulsed dipolar spectroscopy (PDS) data encountered in structural biology, organic photovoltaics, photosynthesis research, and other domains featuring long-lived radical pairs and paramagnetic metal ions. PDS uses distance dependence of magnetic dipolar interactions; measuring a single well-defined distance is straightforward, but extracting distance distributions is a hard and mathematically ill-posed problem requiring careful regularisation and background fitting. Neural networks do this exceptionally well, but their "robust black box" reputation hides the complexity of their design and training - particularly when the training dataset is effectively infinite.

Benchmark Test and Guidelines for DEER/PELDOR Experiments on Nitroxide-Labeled Biomolecules.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results.

A Joint Venture of Ab Initio Molecular Dynamics, Coupled Cluster Electronic Structure Methods, and Liquid-State Theory to Compute Accurate Isotropic Hyperfine Constants of Nitroxide Probes in Water.

The isotropic hyperfine coupling constant (HFCC, ) of a pH-sensitive spin probe in a solution, HMI (2,2,3,4,5,5-hexamethylimidazolidin-1-oxyl, CHNO) in water, is computed using an ensemble of state-of-the-art computational techniques and is gauged against X-band continuous wave electron paramagnetic resonance (EPR) measurement spectra at room temperature. Fundamentally, the investigation aims to delineate the cutting edge of current first-principles-based calculations of EPR parameters in aqueous solutions based on using rigorous statistical mechanics combined with correlated electronic structure techniques. In particular, the impact of solvation is described by exploiting fully atomistic, RISM integral equation, and implicit solvation approaches as offered by ab initio molecular dynamics (AIMD) of the periodic bulk solution (using the spin-polarized revPBE0-D3 hybrid functional), embedded cluster reference interaction site model integral equation theory (EC-RISM), and polarizable continuum embedding (using CPCM) of microsolvated complexes, respectively.

From in vitro towards in situ: structure-based investigation of ABC exporters by electron paramagnetic resonance spectroscopy.

ATP-binding cassette (ABC) exporters have been studied now for more than four decades, and recent structural investigation has produced a large number of protein database entries. Yet, important questions about how ABC exporters function at the molecular level remain debated, such as which are the molecular recognition hotspots and the allosteric couplings dynamically regulating the communication between the catalytic cycle and the export of substrates. This conundrum mainly arises from technical limitations confining all research to in vitro analysis of ABC transporters in detergent solutions or embedded in membrane-mimicking environments.

Biophysical Characterization of Pro-apoptotic BimBH3 Peptides Reveals an Unexpected Capacity for Self-Association.

Bcl-2 proteins orchestrate the mitochondrial pathway of apoptosis, pivotal for cell death. Yet, the structural details of the conformational changes of pro- and antiapoptotic proteins and their interactions remain unclear. Pulse dipolar spectroscopy (double electron-electron resonance [DEER], also known as PELDOR) in combination with spin-labeled apoptotic Bcl-2 proteins unveils conformational changes and interactions of each protein player via detection of intra- and inter-protein distances.

Spin-labeled nanobodies as protein conformational reporters for electron paramagnetic resonance in cellular membranes.

Nanobodies are emerging tools in a variety of fields such as structural biology, cell imaging, and drug discovery. Here we pioneer the use of their spin-labeled variants as reporters of conformational dynamics of membrane proteins using DEER spectroscopy. At the example of the bacterial ABC transporter TM287/288, we show that two gadolinium-labeled nanobodies allow us to quantify, via analysis of the modulation depth of DEER traces, the fraction of transporters adopting the outward-facing state under different experimental conditions.

Changes in the fraction of strongly attached cross bridges in mouse atrophic and hypertrophic muscles as revealed by continuous wave electron paramagnetic resonance.

Electron paramagnetic resonance (EPR), coupled with site-directed spin labeling, has been proven to be a particularly suitable technique to extract information on the fraction of myosin heads strongly bound to actin upon muscle contraction. The approach can be used to investigate possible structural changes occurring in myosin of fiber s altered by diseases and aging. In this work, we labeled myosin at position Cys707, located in the SH1-SH2 helix in the myosin head cleft, with iodoacetamide spin label, a spin label that is sensitive to the reorientational motion of this protein during the ATPase cycle and characterized the biochemical states of the labeled myosin head by means of continuous wave EPR.

Atomistic Mechanism of Large-Scale Conformational Transition in a Heterodimeric ABC Exporter.

ATP-binding cassette (ABC) transporters are ATP-driven molecular machines, in which ATP binding and hydrolysis in the nucleotide-binding domains (NBDs) is chemomechanically coupled to large-scale, alternating access conformational changes in the transmembrane domains (TMDs), ultimately leading to the translocation of substrates across biological membranes. The precise nature of the structural dynamics behind the large-scale conformational transition as well as the coupling of NBD and TMD motions is still unresolved. In this work, we combine all-atom molecular dynamics (MD) simulations with electron paramagnetic resonance (EPR) spectroscopy to unravel the atomic-level mechanism of the dynamic conformational transitions underlying the functional working cycle of the heterodimeric ABC exporter TM287/288.

Identifying conformational changes with site-directed spin labeling reveals that the GTPase domain of HydF is a molecular switch.

[FeFe]-hydrogenases catalyse the reduction of protons to hydrogen at a complex 2Fe[4Fe4S] center called H-cluster. The assembly of this active site is a multistep process involving three proteins, HydE, HydF and HydG. According to the current models, HydF has the key double role of scaffold, upon which the final H-cluster precursor is assembled, and carrier to transfer it to the target hydrogenase.

Distance measurements in peridinin-chlorophyll a-protein by light-induced PELDOR spectroscopy. Analysis of triplet state localization.

Triplet-triplet energy transfer from chlorophylls to carotenoids is the mechanism underlying the photoprotective role played by carotenoids in many light harvesting complexes, during photosynthesis. The peridinin-chlorophyll-a protein (PCP) is a water-soluble light harvesting protein of the dinoflagellate Amphidinium carterae, employing peridinin as the main carotenoid to fulfil this function. The dipolar coupling of the triplet state of peridinin, populated under light excitation in isolated PCP, to the MTSSL nitroxide, introduced in the protein by site-directed mutagenesis followed by spin labeling, has been measured by Pulse ELectron-electron DOuble Resonance (PELDOR) spectroscopy.