The Effect of Reactive Oxygen Species on Respiratory Complex I Activity in Liposomes.

Respiratory complex I (R-CI) is an essential enzyme in the mitochondrial electron transport chain but also a major source of reactive oxygen species (ROS), which are implicated in neurodegenerative diseases and ageing. While the mechanism of ROS production by R-CI is well-established, the feedback of ROS on R-CI activity is poorly understood. Here, we perform EPR spectroscopy on R-CI incorporated in artificial membrane vesicles to reveal that ROS (particularly hydroxyl radicals) reduce R-CI activity by making the membrane more polar and by increasing its hydrogen bonding capability. Moreover, the mechanism that we have uncovered reveals that the feedback of ROS on R-CI activity via the membrane is transient and not permanent; lipid peroxidation is negligible for the levels of ROS generated under these conditions. Our successful use of modular proteoliposome systems in conjunction with EPR spectroscopy and other biophysical techniques is a powerful approach for investigating ROS effects on other membrane proteins.

Ligand-Binding Cooperativity Effects in Polymer-Protein Conjugation.

We present an electron paramagnetic resonance (EPR) spectroscopic characterization of structural and dynamic effects that stem from post-translational modifications of bovine serum albumin (BSA), an established model system for polymer-protein conjugation. Beyond the typical drug delivery and biocompatibility aspect of such systems, we illustrate the causes that alter internal dynamics and therefore functionality in terms of ligand-binding to the BSA protein core. Uptake of the paramagnetic fatty acid derivative 16-doxyl stearic acid by several BSA-based squaric acid macroinitiators and polymer-protein conjugates was studied by EPR spectroscopy, aided by dynamic light scattering (DLS) and zeta potential measurements. The conjugates were grafted from oligo(ethylene glycol) methyl ether methacrylate (OEGMA), forming an overall core-shell-like structure. It is found that ligand-binding and associated parameters such as binding affinity, cooperativity, and the number of binding sites of BSA change drastically with the extent of surface modification. In the course of processing BSA, the ligands also change their preference for individual binding sites, as observed from a comparative view of their spatial alignments in double electron electron resonance (DEER) experiments. The protein-attached polymers constitute a diffusion barrier that significantly hamper ligand uptake. Moreover, zeta potentials (ζ) decrease linearly with the degree of surface modification in protein macroinitiators and an effective dielectric constant can be estimated for the polymer layer in the conjugates. All this suggests that ligand uptake characteristics in BSA can be fine-tuned by the extent and nature of such post-translational modifications (PTMs). We show that EPR spectroscopy is suitable for quantifying these subtle PTM-based functional effects from self-assembly of substrate and ligand.

Tuning the shape anisotropy of loosely bound colloid-like ionic clusters in solution.

We characterize the influence of the ionic ratio on the dynamic self-assembly process involving a macrocyclic tetraimidazolium molecular box and small dianionic salts into highly defined, colloid-like ionic clusters in solution, called ionoids. Based on our studies utilizing dynamic light scattering (DLS) and continuous wave electron paramagnetic resonance (CW EPR) spectroscopy, we determine a region of privileged ionic ratios, which allow the formation of monodisperse, spheroidal structures of loosely bound ions in solution with adjustable (i) hydrodynamic radii between 6 nm and 12 nm and (ii) shape anisotropy. Inspired by Hertzsprung-Russell diagrams (HRDs) used in astrophysics to describe the fate of stars, we construct ionoid evolution diagrams (IEDs). IEDs are essential for grasping and describing the highly complex temporal development of these dynamically self-assembled structures in solution from the level of the individual ionic building blocks to stable clusters with a minimum lifetime of months, and thus aid in crafting future globular ionoids and anisotropic ionic clusters.

Solvent and concentration effects on highly defined, colloid-like ionic clusters in solution.

We characterize the process of ionic self-assembly involving a macrocyclic tetraimidazolium molecular box and small dianionic salts into highly defined, colloid-like ionic clusters called ionoids. Using dynamic light scattering (DLS) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we determine the influence of solvent composition and bulk concentration on the size, shape and durability of the ionoids. Minor aberrations from the established optimum solvent mixture DMSO : glycerol : water 50 : 43 : 7 (v/v/v) in volume stoichiometry still lead to formation of ionoids, but on longer timescales they evolve into separated smaller and bigger entities. The same effect occurs when decreasing the bulk concentration from approximately 1 mM : 3 mM to 10-fold and higher dilutions. This transition at the 0.1 mM : 0.3 mM ratio represents the 'critical ionoid concentration' for the investigated system. We can thus define the solvent and concentration range, in which these soft, yet durable and long lived colloid-like ionic clusters form.

Profound Changes in Functional Structure and Dynamics of Serum Albumin in Children with Nephrotic Syndrome: An Exploratory Research Study.

Patients with nephrotic syndrome (NS) suffer from urinary loss of albumin. As a cause, previous studies focused on the glomerular filter rather than analyzing the molecular properties of albumin itself. Later one was initiated by clinical observations indicating unexplained molecular alterations of human serum albumin (HSA) in an NS pediatric patient. Therefore, we examined serum from eight pediatric patients with steroid-sensitive and -resistant NS and compared it with serum from healthy subjects as well as commercial HSA. We used dynamic and electrophoretic light scattering to characterize the protein size and effective surface charge and electron paramagnetic resonance spectroscopy to measure the local environment and binding dynamics of up to seven fatty acids associated with HSA. Our findings suggest that pronounced differences in binding behavior and surface charge of HSA could enhance their filtration through the GBM, leading to direct toxicity of HSA to podocytes.

Using light scattering to assess how phospholipid-protein interactions affect complex I functionality in liposomes.

Complex I is an essential membrane protein in respiration, oxidising NADH and reducing ubiquinone to contribute to the proton-motive force that powers ATP synthesis. Liposomes provide an attractive platform to investigate complex I in a phospholipid membrane with the native hydrophobic ubiquinone substrate and proton transport across the membrane, but without convoluting contributions from other proteins present in the native mitochondrial inner membrane. Here, we use dynamic and electrophoretic light scattering techniques (DLS and ELS) to show how physical parameters, in particular the zeta potential (ζ-potential), correlate strongly with the biochemical functionality of complex I-containing proteoliposomes. We find that cardiolipin plays a crucial role in the reconstitution and functioning of complex I and that, as a highly charged lipid, it acts as a sensitive reporter on the biochemical competence of proteoliposomes in ELS measurements. We show that the change in ζ-potential between liposomes and proteoliposomes correlates linearly with protein retention and catalytic oxidoreduction activity of complex I. These correlations are dependent on the presence of cardiolipin, but are otherwise independent of the liposome lipid composition. Moreover, changes in the ζ-potential are sensitive to the proton motive force established upon proton pumping by complex I, thereby constituting a complementary technique to established biochemical assays. ELS measurements may thus serve as a more widely useful tool to investigate membrane proteins in lipid systems, especially those that contain charged lipids.

Insights into metalloproteins and metallodrugs from electron paramagnetic resonance spectroscopy.

Metal ions play an important role in diverse biological processes, and much of the basic knowledge derived from studying native bioinorganic systems are applied in the synthesis of new molecules with the aim of diagnosing and treating diseases. At first glance, metalloproteins and metallodrugs are very different systems, but metal ion coordination, redox chemistry and substrate binding play essential roles in advancing both of these research fields. In this article, we discuss recent metalloprotein and metallodrug studies where electron paramagnetic resonance spectroscopy served as a major tool to gain a better understanding of metal-based structures and their function.

Influence of different polymer belts on lipid properties in nanodiscs characterized by CW EPR spectroscopy.

With this study we aim at comparing the well-known lipid membrane model system of liposomes and polymer-encapsulated nanodiscs regarding their lipid properties. Using differential scanning calorimetry (DSC) and continuous-wave electron paramagnetic resonance (CW EPR) spectroscopy, we characterize the temperature-dependent lipid behavior within 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes and nanodiscs made from such liposomes by application of various polymers based on styrene-co-maleic acid (SMA), diisobutylene-alt-maleic acid (DIBMA), and styrene-co-maleic amide sulfobetaine (SMA-SB), a new SMA-derived copolymer containing sulfobetaine side chains. By incorporating a spin label doxyl moiety into the lipid bilayer in position 16 or 5 we were able to study the micropolarity as well as rotational restrictions onto the lipids in the apolar bilayer center and the chain region adjacent to the carbonyl groups, respectively. Our results suggest that all polymers broaden the main melting transition of DMPC, change the water accessibility within the lipid bilayer, and exhibit additional constraints onto the lipids. Independent of the used polymer, the rotational mobility of both spin-labeled lipids decreased with DIBMA exerting less restraints probably due to its aliphatic side chains. Our findings imply that the choice of the solubilizing polymer has to be considered an important step to form lipid nanodiscs which should be included into research of lipid membranes and membrane proteins in the future.

Characterization of Aqueous Lower-Polarity Solvation Shells Around Amphiphilic 2,2,6,6-Tetramethylpiperidine-1-oxyl Radicals in Water.

Solvation of the amphiphilic nitroxide radical 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) and hydrophilic 4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPONE) in water and tetrahydrofuran (THF) is studied in detail. The existence of pure water shells enclosing TEMPO in an aqueous solution that leads to significantly reduced local polarity at the nitroxide moiety is shown with multifrequency electron paramagnetic resonance (EPR) spectroscopy at X- and Q-bands as well as spectral simulations. These aqueous lower-polarity solvation shells (ALPSS) offer TEMPO a local polarity that is similar to that in organic solvents like THF. Furthermore, using double electron-electron resonance spectroscopy, local enrichment and inhomogeneous distribution without direct molecular encounters of dissolved TEMPO in water are found that can be correlated with potentially attractive interactions mediated through ALPSS. However, no local enrichment of TEMPO is found in organic solvents such as THF. In contrast to TEMPO, the structurally very similar nitroxide radical TEMPONE shows no ALPSS encapsulation behavior with water molecules in aqueous solutions. Ensemble-averaging methods such as dynamic light scattering and electrospray ionization mass spectrometry substantiate the EPR spectroscopically obtained results of ALPSS-encased TEMPO and attractive interactions between them, leading to a higher local concentration. Furthermore, force field molecular dynamics simulations and metadynamics deliver support for our conclusions.

Dynamic self-assembly of ions with variable size and charge in solution.

Recently it was found that at ambient temperatures and in specific ternary solvents a cationic macrocyclic tetraimidazolium molecular box and small dianionic salts can self-assemble into highly defined, colloid-like ionic clusters, called ionoids. Here, we present evidence that the solution-based ionic self-assembly process leading to ionoids is a general phenomenon by characterizing new ionic building blocks which are capable of generating loosely bound globular and anisotropic structures similar to those in the established system. Using new cationic and anionic molecules, we show that variations in the size ratio between cationic and anionic component mainly affect size, shape and durability of the ionic clusters. Utilizing dynamic light scattering (DLS), continuously monitored phase-analysis light scattering (cmPALS) and continuous wave electron paramagnetic resonance (CW EPR) spectroscopy, we can thus define generalized ionic ratios, in which specific combinations of ionic compounds with certain size and charge densities are able to form these soft yet durable and long-lived ionic clusters. Furthermore, we characterize the temporal development of our dynamically self-assembled structures in solution from the level of the individual ionic building blocks to stable clusters with minimum lifetimes of months through previously established ionoid evolution diagrams (IEDs). The direct comparison of various cluster systems with respect to their shape, size and charges allows correlations of structural changes of the individual building blocks with the fate of self-assembled entities inside the crafted IEDs. This work generalizes the concept of ionoid formation to ions of specific sizes and charge densities, which may broaden the scope of this new type of highly dynamic and soft yet remarkably durable structures in the field of supramolecular chemistry.

Shape, Size, and Internal Dynamics of Loosely Bound Colloidlike Ionic Clusters in Ternary Solvent Systems.

We characterize the influence of preferential solvation on the dynamic self-assembly process between small dianionic salts and a macrocyclic tetraimidazolium molecular box into highly defined, colloidlike ionic clusters in solution, called ionoids. Here, we substitute individual solvents in the established optimal ternary solvent mixture dimethyl sulfoxide (DMSO)/glycerol/water 50:43:7 (v/v/v), namely, DMSO through dimethyl formamide, glycerol through ethylene glycol and water through N-methylpropionamide, and such can characterize the changes in shape and size of the structures of loosely bound ionic clusters induced by the substitution of a specific solvent component. Using dynamic light scattering we associate size, shape, and initial durability of ionic clusters with solvent parameters like dynamic viscosity and relative permittivity to highlight the importance of solvent composition for the build-up of globular ionoids as well as anisotropic ionic clusters. To further analyze the solvation state of our dianionic building unit inside the initial ion cloud state, which later affects the self-assembly process of ionic clusters, we perform continuous wave (CW) electron paramagnetic resonance spectroscopy measurements at X-band (∼9.4 GHz) and Q-band (∼34 GHz) frequencies.