Insights into Folding and Molecular Environment of Lyophilized Proteins Using Pulsed Electron Paramagnetic Resonance Spectroscopy.

There is still an insufficient understanding of how the characteristics of protein drugs are maintained in the solid state of lyophilizates, including aspects such as protein distances, local environment, and structural preservation. To this end, we evaluated protein folding and the molecules' nearest environment by electron paramagnetic resonance (EPR) spectroscopy. Double electron-electron resonance (DEER) probe distances of up to approximately 200 Å and is suitable to investigate protein folding, local concentration, and aggregation, whereas electron spin echo envelope modulation (ESEEM) allows the study of the near environment within approximately 10 Å of the spin label.

Protein and solutes freeze-concentration in water/glycerol mixtures revealed by pulse EPR.

Lyophilization can extend protein drugs stability and shelf life, but it also can lead to protein degradation in some cases. The development of safe freeze-drying approaches for sensitive proteins requires a better understanding of lyophilization on the molecular level. The evaluation of the freezing process and its impact on the protein environment in the nm scale is challenging because feasible experimental methods are scarce.

Membrane-Sugar Interactions Probed by Pulsed Electron Paramagnetic Resonance of Spin Labels.

Sugars can stabilize biological systems under extreme desiccation and freezing conditions. Hypothetical molecular mechanisms suggest that the stabilization effect may be determined either by specific interactions of sugars with biological molecules or by the influence of sugars on the solvating shell of the biomolecule. To explore membrane-sugar interactions, we applied electron spin echo envelope modulation (ESEEM) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR), to phospholipid bilayers with spin-labeled lipids added and solvated by aqueous deuterated sucrose and trehalose solutions.

Heterogeneities in Cholesterol-Containing Model Membranes Observed by Pulsed Electron Paramagnetic Resonance of Spin Labels.

Biological membranes are supposed to have heterogeneous structure containing lipid rafts-lateral micro- and nanodomains enriched in cholesterol (chol) and sphingolipids. In this work, lipid bilayers containing a small amount of the spin-labeled chol analogue 3β-doxyl-5α-cholestane (chlstn) were studied using electron spin echo (ESE) spectroscopy, which is a pulsed version of electron paramagnetic resonance (EPR). Bilayers were prepared from an equimolecular mixture of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) with chol added at different concentrations.

Low-temperature molecular motions in lipid bilayers in the presence of sugars: insights into cryoprotective mechanisms.

Sugars and sugar alcohols can stabilize biological systems under extreme conditions of desiccation and freezing. Phospholipid bilayers solvated by aqueous solutions of sucrose, trehalose, and sorbitol at concentrations of 0.2 and 1 M and containing incorporated spin-labeled stearic acids were studied by electron spin echo (ESE) spectroscopy, a pulsed version of electron paramagnetic resonance (EPR).

Mitochondria-targeted plastoquinone antioxidant SkQ1 prevents amyloid-β-induced impairment of long-term potentiation in rat hippocampal slices.

Bath application of 200 nM amyloid-β1-42 (Aβ) to rat hippocampal slices impairs induction of long-term potentiation (LTP) of the population spike in pyramidal layer of the CA1 field of the hippocampus. Intraperitoneal injection of mitochondria-targeted plastoquinone derivative SkQ1 at very low concentrations (250 nmol/kg body weight) given 24 h before the slice preparation or 1 h treatment of hippocampal slices with 250 nM SkQ1 prevents the deleterious effect of Aβ on LTP. To elucidate which part of the molecule is responsible for this type of neuroprotective activity, the effect of the analog of SkQ1 lacking plastoquinone (C12TPP) was studied.

The mitochondria-targeted antioxidants and remote kidney preconditioning ameliorate brain damage through kidney-to-brain cross-talk.

Background: Many ischemia-induced neurological pathologies including stroke are associated with high oxidative stress. Mitochondria-targeted antioxidants could rescue the ischemic organ by providing specific delivery of antioxidant molecules to the mitochondrion, which potentially suffers from oxidative stress more than non-mitochondrial cellular compartments. Besides direct antioxidative activity, these compounds are believed to activate numerous protective pathways.

Small-amplitude backbone motions of the spin-labeled lipopeptide trichogin GA IV in a lipid membrane as revealed by electron spin echo.

Trichogin GA IV is a lipopeptide antibiotic of fungal origin, which is known to be able to modify the membrane permeability. TOAC nitroxide spin-labeled analogues of this membrane active peptide were investigated in hydrated bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) by electron spin echo (ESE) spectroscopy. Because the TOAC nitroxide spin label is rigidly attached to the peptide backbone, it may report on the backbone orientational dynamics.

Small-angle orientational motions of spin-labeled lipids in cholesterol-containing bilayers studied at low temperatures by electron spin echo spectroscopy.

Electron spin echo (ESE) study was performed for a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer containing an admixture of lipids (of 1:100 molar ratio) spin-labeled at the polar head or at different positions along the acyl chain and optionally containing additionally cholesterol that substituted POPC lipids in a 50:50 molar ratio. ESE signal was observed below 150-115 K, depending on the label position. Three-pulse stimulated ESE is sensitive to two types of orientational motion of spin labels at these temperatures.

Acidosis-induced zinc-dependent death of cultured cerebellar granule neurons.

Severe acidosis caused death of cultured cerebellar granule neurons (CGNs). Acidosis was accompanied by a progressive increase of the intracellular zinc ions ([Zn(2+)](i)) and decrease of [Ca(2+)](i). Zn(2+) chelator, N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), prevented the increase of [Zn(2+)](i) and acidosis-induced neuronal death.

Effect of transitory glucose deprivation on mitochondrial structure and functions in cultured cerebellar granule neurons.

We found that 60-min glucose deprivation leads to progressive decrease in the mitochondrial membrane potential and increase in [Ca(2+)](i) in cultured cerebellar granule neurons. The latter effect was fully reversible, returning to the basal level 60 min after restoration of normal glucose level in the incubation medium, whereas mitochondrial membrane potential remained at 10.0+/-1.

Fast stochastic librations and slow rotations of spin labeled stearic acids in a model phospholipid bilayer at cryogenic temperatures.

The spin label DOXYL (4,4-dimethyl-oxazolidine-1-oxyl) is a nitroxyl ring that can be attached rigidly at specific C-atom positions in the stearic acid. 5-DOXYL-stearic acid and 16-DOXYL-stearic acid in 1-Palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine (POPC) lipid bilayers were studied using electron spin echo (ESE) spectroscopy at low temperatures. The anisotropy of ESE decay across the electron paramagnetic resonance (EPR) spectrum evidence that these spin labels participate in orientational motions at temperatures down to approximately 120 K for 5-DOXYL-stearic acid and down to approximately 80 K for 16-DOXYL-stearic acid.

Desferrioxamine induces delayed tolerance against cerebral ischemia in vivo and in vitro.

The widely prescribed drug desferrioxamine is a known activator of the hypoxia-inducible transcription factor 1 (HIF-1) and the subsequent transcription of erythropoietin. In the brain, HIF-1 is a master switch of the transcriptional response to hypoxia, whereas erythropoietin is a potent neuroprotectant. The authors show that desferrioxamine dose-dependently and time-dependently induces tolerance against focal cerebral ischemia in rats and mice, and against oxygen-glucose deprivation in purified cortical neurons.