Hydrophilic Reduction-Resistant Spin Labels of Pyrrolidine and Pyrroline Series from 3,4-Bis-hydroxymethyl-2,2,5,5-tetraethylpyrrolidine-1-oxyl.

Highly resistant to reduction nitroxides open new opportunities for structural studies of biological macromolecules in their native environment inside living cells and for functional imaging of pH and thiols, enzymatic activity and redox status in living animals. 3,4-Disubstituted nitroxides of 2,2,5,5-tetraethylpyrrolidine and pyrroline series with a functional group for binding to biomolecules and a polar moiety for higher solubility in water and for more rigid attachment via additional coordination to polar sites were designed and synthesized. The EPR spectra, lipophilicities, kinetics of the reduction in ascorbate-containing systems and the decay rates in liver homogenates were measured. The EPR spectra of all 3,4-disubstituted pyrrolidine nitroxides showed additional large splitting on methylene hydrogens of the ethyl groups, while the spectra of similar pyrroline nitroxides were represented with a simple triplet with narrow lines and hyperfine structure of the nitrogen manifolds resolved in oxygen-free conditions. Both pyrrolidine and pyrroline nitroxides demonstrated low rates of reduction with ascorbate, pyrrolidines being a bit more stable than similar pyrrolines. The decay of positively charged nitroxides in the rat liver homogenate was faster than that of neutral and negatively charged radicals, with lipophilicity, rate of reduction with ascorbate and the ring type playing minor role. The EPR spectra of N,N-dimethyl-3,4-bis-(aminomethyl)-2,2,5,5-tetraethylpyrrolidine-1-oxyl showed dependence on pH with pKa = 3, ΔaN = 0.055 mT and ΔaH = 0.075 mT.

Interaction of DWORF with SERCA and PLB as determined by EPR spectroscopy.

Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca2+], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies.

Insights into the Conformational Plasticity of the Protein Kinase Akt1 by Multi-Lateral Dipolar Spectroscopy.

The serine/threonine kinase Akt1 is part of the PI3 K/Akt pathway and plays a key role in the regulation of various cellular processes such as cell growth, proliferation, and apoptosis. Here, we analyzed the elasticity between the two domains of the kinase Akt1, connected by a flexible linker, recording a wide variety of distance restraints by electron paramagnetic resonance (EPR) spectroscopy. We studied full length Akt1 and the influence of the cancer-associated mutation E17K. The conformational landscape in the presence of different modulators, like different types of inhibitors and membranes was presented, revealing a tuned flexibility between the two domains, dependent on the bound molecule.

Combining TEMPO and methyl undecenoate to produce an effective anti-mosquito compound with convenient spin-labeling.

A general method to spin-label a fatty acid was demonstrated as well as an assay of the effectiveness of methyl 10-undecenoate and the spin-labeled version, against the larvae of Aedes aegypti. The LC50s were 66 and 58 µL/120 mL (55 and 48 ppm) respectively, and the LC90s were 108 and 90 µL/120 mL (113 and 90) ppm. This shows that the spin-label has very little effect on the larvicidal activity of the compound. This opens the possibility of the use of spin-labeling as a tool to determine mechanisms of larvicidal effectiveness, as it can be employed without altering the system under study.

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. Additionally, we quantitatively follow the interconversion from the outward- to the inward-facing state in the conformational ensemble under ATP turnover conditions. We finally show that the specificity of the nanobodies for the target protein allows the direct attainment of structural information on the wild-type TM287/288 expressed in cellular membranes without the need to purify or label the investigated membrane protein.

Spin-Label Electron Paramagnetic Resonance Spectroscopy Reveals Effects of Wastewater Filter Membrane Coated with Titanium Dioxide Nanoparticles on Bovine Serum Albumin.

The accumulation of proteins in filter membranes limits the efficiency of filtering technologies for cleaning wastewater. Efforts are ongoing to coat commercial filters with different materials (such as titanium dioxide, TiO2) to reduce the fouling of the membrane. Beyond monitoring the desired effect of the retention of biomolecules, it is necessary to understand what the biophysical changes are in water-soluble proteins caused by their interaction with the new coated filter membranes, an aspect that has received little attention so far. Using spin-label electron paramagnetic resonance (EPR), aided with native fluorescence spectroscopy and dynamic light scattering (DLS), here, we report the changes in the structure and dynamics of bovine serum albumin (BSA) exposed to TiO2 (P25) nanoparticles or passing through commercial polyvinylidene fluoride (PVDF) membranes coated with the same nanoparticles. We have found that the filtering process and prolonged exposure to TiO2 nanoparticles had significant effects on different regions of BSA, and denaturation of the protein was not observed, neither with the TiO2 nanoparticles nor when passing through the TiO2-coated filter membranes.

GdIII -19 F Distance Measurements for Proteins in Cells by Electron-Nuclear Double Resonance.

Studies of protein structure and dynamics are usually carried out in dilute buffer solutions, conditions that differ significantly from the crowded environment in the cell. The double electron-electron resonance (DEER) technique can track proteins' conformations in the cell by providing distance distributions between two attached spin labels. This technique, however, cannot access distances below 1.8 nm. Here, we show that GdIII -19 F Mims electron-nuclear double resonance (ENDOR) measurements can cover part of this short range. Low temperature solution and in-cell ENDOR measurements, complemented with room temperature solution and in-cell GdIII -19 F PRE (paramagnetic relaxation enhancement) NMR measurements, were performed on fluorinated GB1 and ubiquitin (Ub), spin-labeled with rigid GdIII tags. The proteins were delivered into human cells via electroporation. The solution and in-cell derived GdIII -19 F distances were essentially identical and lie in the 1-1.5 nm range revealing that both, GB1 and Ub, retained their overall structure in the GdIII and 19 F regions in the cell.

The molecular basis of OH-PCB estrogen receptor activation.

Polychlorinated bisphenols (PCBs) continue to contaminate food chains globally where they concentrate in tissues and disrupt the endocrine systems of species throughout the ecosphere. Hydroxylated PCBs (OH-PCBs) are major PCB metabolites and high-affinity inhibitors of human estrogen sulfotransferase (SULT1E1), which sulfonates estrogens and thus prevents them from binding to and activating their receptors. OH-PCB inhibition of SULT1E1 is believed to contribute significantly to PCB-based endocrine disruption. Here, for the first time, the molecular basis of OH-PCB inhibition of SULT1E1 is revealed in a structure of SULT1E1 in complex with OH-PCB1 (4'-OH-2,6-dichlorobiphenol) and its substrates, estradiol (E2), and PAP (3'-phosphoadenosine-5-phosphosulfate). OH-PCB1 prevents catalysis by intercalating between E2 and catalytic residues and establishes a new E2-binding site whose E2 affinity and positioning are greater than and competitive with those of the reactive-binding pocket. Such complexes have not been observed previously and offer a novel template for the design of high-affinity inhibitors. Mutating residues in direct contact with OH-PCB weaken its affinity without compromising the enzyme's catalytic parameters. These OH-PCB resistant mutants were used in stable transfectant studies to demonstrate that OH-PCBs regulate estrogen receptors in cultured human cell lines by binding the OH-PCB binding pocket of SULT1E1.

Arterial Spin Labeling for the Etiological Workup of Intracerebral Hemorrhage in Children.

BACKGROUND AND PURPOSE: Pediatric nontraumatic intracerebral hemorrhage accounts for half of stroke in children. Early diagnostic of the causative underlying lesion is the first step toward prevention of hemorrhagic recurrence. We aimed to investigate the performance of arterial spin labeling sequence (ASL) in the acute phase etiological workup for the detection of an arteriovenous shunt (AVS: including malformation and fistula), the most frequent cause of pediatric nontraumatic intracerebral hemorrhage. METHODS: Children with a pediatric nontraumatic intracerebral hemorrhage between 2011 and 2019 enrolled in a prospective registry were retrospectively included if they had undergone ASL-magnetic resonance imaging before any etiological treatment. ASL sequences were reviewed using cerebral blood flow maps by 2 raters for the presence of an AVS. The diagnostic performance of ASL was compared with admission computed tomography angiography, other magnetic resonance imaging sequences including contrast-enhanced sequences and subsequent digital subtraction angiography. RESULTS: A total of 121 patients with pediatric nontraumatic intracerebral hemorrhage were included (median age, 9.9 [interquartile range, 5.8-13]; male sex 48.8%) of whom 76 (63%) had a final diagnosis of AVS. Using digital subtraction angiography as an intermediate reference, visual ASL inspection had a sensitivity and a specificity of, respectively, 95.9% (95% CI, 88.5%-99.1%) and 79.0% (95% CI, 54.4%-94.0%). ASL had a sensitivity, specificity, and accuracy of 90.2%, 97.2%, and 92.5%, respectively for the detection of the presence of an AVS, with near perfect interrater agreement (κ=0.963 [95% CI, 0.912-1.0]). The performance of ASL alone was higher than that of other magnetic resonance imaging sequences, individually or combined, and higher than that of computed tomography angiography. CONCLUSIONS: ASL has strong diagnostic performance for the detection of AVS in the initial workup of intracerebral hemorrhage in children. If our findings are confirmed in other settings, ASL may be a helpful diagnostic imaging modality for patients with pediatric nontraumatic intracerebral hemorrhage. REGISTRATION: URL: https://www.clinicaltrials.gov; Unique identifiers: 3618210420, 2217698.

Spin-Labeled Riboswitch Synthesized from a Protected TPA Phosphoramidite Building Block.

The nitroxide TPA (2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene) is an excellent spin label for EPR studies of RNA. Previous synthetic methods, however, are complicated and require special equipment. Herein, we describe a uridine derived phosphoramidite with a photocaged TPA unit attached. The light sensitive 2-nitrobenzyloxymethyl group can be removed in high yield by short irradiation at 365 nm. Based on this approach, a doubly spin-labeled 27mer neomycin sensing riboswitch was synthesized and studied by PELDOR. The overall thermal stability of the fold is not much reduced by TPA. In-line probing nevertheless detected changes in local mobility.

Cerebral blood flow alterations specific to freezing of gait in Parkinson's disease.

BACKGROUND: Freezing of gait (FOG) have been associated with deficits in the cortico-basal ganglia-thalamic network. However, the resting-state cerebral blood flow (CBF) alterations specific to FOG in Parkinson's disease (PD) remain unknown. METHODS: In total, sixty PD individuals, including 30 PD with FOG (PD-FOG) and 30 PD without FOG (PD-NFOG), and 30 healthy controls (HC) underwent arterial spin labeling magnetic resonance image. The CBF were voxel-wise compared among the three groups and validated in a different cohort of PD-FOG and PD-NFOG. RESULTS: The results revealed that patients with PD-FOG had increased CBF in bilateral thalamus and the left caudate nucleus and decreased CBF in the left inferior parietal cortex compared to patients with PD-NFOG. The inter-group differences of CBF between PD-FOG and PD-NFOG was confirmed in a different cohort in the validation analysis. Moreover, the CBF in left caudate nucleus was positively correlated with severity of FOG in PD-FOG patients. CONCLUSIONS: Perfusion alterations in both cortical and subcortical regions in the cortico-basal ganglia-thalamic network are related to the development of FOG in PD patients.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy.

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Differences in the properties of porcine cortical and nuclear fiber cell plasma membranes revealed by saturation recovery EPR spin labeling measurements.

Eye lens membranes are complex biological samples. They consist of a variety of lipids that form the lipid bilayer matrix, integral proteins embedded into the lipid bilayer, and peripheral proteins. This molecular diversity in membrane composition induces formation of lipid domains with particular physical properties that are responsible for the maintenance of proper membrane functions. These domains can be, and have been, effectively described in terms of the rotational diffusion of lipid spin labels and oxygen collision with spin labels using the saturation recovery (SR) electron paramagnetic resonance method and, now, using stretched exponential function for the analysis of SR signals. Here, we report the application of the stretched exponential function analysis of SR electron paramagnetic resonance signals coming from cholesterol analog, androstane spin label (ASL) in the lipid bilayer portion of intact fiber cell plasma membranes (IMs) isolated from the cortex and nucleus of porcine eye lenses. Further, we compare the properties of these IMs with model lens lipid membranes (LLMs) derived from the total lipids extracted from cortical and nuclear IMs. With this approach, the IM can be characterized by the continuous probability density distribution of the spin-lattice relaxation rates associated with the rotational diffusion of a spin label, and by the distribution of the oxygen transport parameter within the IM (i.e., the collision rate of molecular oxygen with the spin label). We found that the cortical and nuclear LLMs possess very different, albeit homogenous, spin lattice relaxation rates due to the rotational diffusion of ASL, indicating that the local rigidity around the spin label in nuclear LLMs is considerably greater than that in cortical LLMs. However, the oxygen transport parameter around the spin label is very similar and slightly heterogenous for LLMs from both sources. This heterogeneity was previously missed when distinct exponential analysis was used. The spin lattice relaxation rates due to either the rotational diffusion of ASL or the oxygen collision with the spin label in nuclear IMs have slower values and wider distributions compared with those of cortical IMs. From this evidence, we conclude that lipids in nuclear IMs are less fluid and more heterogeneous than those in cortical membranes. Additionally, a comparison of properties of IMs with corresponding LLMs, and lipid and protein composition analysis, allow us to conclude that the decreased lipid-to-protein ratio not only induces greater rigidity of nuclear IMs, but also creates domains with the considerably decreased and variable oxygen accessibility. The advantages and disadvantages of this method, as well as its use for the cluster analysis, are discussed.

Interaction of alpha-crystallin with four major phospholipids of eye lens membranes.

It is well-studied that the significant factor in cataract formation is the association of α-crystallin, a major eye lens protein, with the fiber cell plasma membrane of the eye lens. The fiber cell plasma membrane of the eye lens consists of four major phospholipids (PLs), i.e., phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), and sphingomyelin (SM). Despite several attempts to study the interaction of α-crystallin with PLs of the eye lens membrane, the role of individual PL for the binding with α-crystallin is still unclear. We recently developed the electron paramagnetic resonance (EPR) spin-labeling method to study the binding of α-crystallin to the PC membrane (Mainali et al., 2020a). Here, we use the recently developed EPR method to explicitly measure the binding affinity (Ka) of α-crystallin to the individual (PE*, PS, and SM) and two-component mixtures (SM/PE, SM/PS, and SM/PC in 70:30 and 50:50 mol%) of PL membranes as well as the physical properties (mobility parameter and maximum splitting) of these membranes upon binding with α-crystallin. One of the key findings of this study was that the Ka of α-crystallin binding to individual PL membranes followed the trends: Ka(PC) > Ka(SM) > Ka(PS) > Ka(PE*), indicating PE* inhibits binding the most whereas PC inhibits binding the least. Also, the Ka of α-crystallin binding to two-component mixtures of PL membranes followed the trends: Ka(SM/PE) > Ka(SM/PS) > Ka(SM/PC), indicating SM/PC inhibits binding the most whereas SM/PE inhibits binding the least. Except for the PE* membrane, for which there was no binding of α-crystallin, the mobility parameter for all other membranes decreased with an increase in α-crystallin concentration. It represents that the membranes become more immobilized near the headgroup regions of the PLs when more and more α-crystallin binds to them. The maximum splitting increased only for the SM and the SM/PE (70:30 mol%) membranes, with an increase in the binding of α-crystallin. It represents that the PL headgroup regions of these membranes become more ordered after binding of α-crystallin to these membranes. Our results showed that α-crystallin binds to PL membranes in a saturable manner. Also, our data suggest that the binding of α-crystallin to PL membranes likely occurs through hydrophobic interaction between α-crystallin and the hydrophobic fatty acid core of the membranes, and such interaction is modulated by the PL headgroup's size and charge, hydrogen bonding between headgroups, and PL curvature. Thus, this study provides an in-depth understanding of α-crystallin interaction with the PL membranes made of individual and two-component mixtures of the four major PLs of the eye lens membranes.

Studies of transmembrane peptides by pulse dipolar spectroscopy with semi-rigid TOPP spin labels.

Electron paramagnetic resonance (EPR)-based pulsed dipolar spectroscopy measures the dipolar interaction between paramagnetic centers that are separated by distances in the range of about 1.5-10 nm. Its application to transmembrane (TM) peptides in combination with modern spin labelling techniques provides a valuable tool to study peptide-to-lipid interactions at a molecular level, which permits access to key parameters characterizing the structural adaptation of model peptides incorporated in natural membranes. In this mini-review, we summarize our approach for distance and orientation measurements in lipid environment using novel semi-rigid TOPP [4-(3,3,5,5-tetramethyl-2,6-dioxo-4-oxylpiperazin-1-yl)-L-phenylglycine] labels specifically designed for incorporation in TM peptides. TOPP labels can report single peak distance distributions with sub-angstrom resolution, thus offering new capabilities for a variety of TM peptide investigations, such as monitoring of various helix conformations or measuring of tilt angles in membranes.

Factors determining barrier properties to oxygen transport across model and cell plasma membranes based on EPR spin-label oximetry.

This review is motivated by the exciting new area of radiation therapy using a phenomenon termed FLASH in which oxygen is thought to have a central role. Well-established principles of radiation biology and physics suggest that if oxygen has a strong role, it should be the level at the DNA. The key aspect discussed is the rate of oxygen diffusion. If oxygen freely diffuses into cells and rapidly equilibrates, then measurements in the extracellular compartment would enable FLASH to be investigated using existing methodologies that can readily measure oxygen in the extracellular compartment. EPR spin-label oximetry allows evaluation of the oxygen permeability coefficient across lipid bilayer membranes. It is established that simple fluid phase lipid bilayers are not barriers to oxygen transport. However, further investigations indicate that many physical and chemical (compositional) factor can significantly decrease this permeation. In biological cell plasma membranes, the lipid bilayer forms the matrix in which integral membrane proteins are immersed, changing organization and properties of the lipid matrix. To evaluate oxygen permeability coefficients across these complex membranes, oxygen permeation across all membrane domains and components must be considered. In this review, we consider many of the factors that affect (decrease) oxygen permeation across cell plasma membranes. Finally, we address the question, can the plasma membrane of the cell form a barrier to the free diffusion of oxygen into the cell interior? If there is a barrier then this must be considered in the investigations of the role of oxygen in FLASH.

Atomistic simulations modify interpretation of spin-label oximetry data. Part 1: intensified water-lipid interfacial resistances.

The role of membrane cholesterol in cellular function and dysfunction has been the subject of much inquiry. A few studies have suggested that cholesterol may slow oxygen diffusive transport, altering membrane physical properties and reducing oxygen permeability. The primary experimental technique used in recent years to study membrane oxygen transport is saturation-recovery electron paramagnetic resonance (EPR) oximetry, using spin-label probes targeted to specific regions of a lipid bilayer. The technique has been used, in particular, to assess the influence of cholesterol on oxygen transport and membrane permeability. The reliability of such EPR recordings at the water-lipid interface near the phospholipid headgroups has been challenged by all-atom molecular dynamics (MD) simulation data that show substantive agreement with spin-label probe measurements throughout much of the bilayer. This work uses further MD simulations, with an updated oxygen model, to determine the location of the maximum resistance to permeation and the rate-limiting barrier to oxygen permeation in 1-palmitoyl,2-oleoylphosphatidylcholine (POPC) and POPC/cholesterol bilayers at 25 and 35°C. The current simulations show a spike of resistance to permeation in the headgroup region that was not detected by EPR but was predicted in early theoretical work by Diamond and Katz. Published experimental nuclear magnetic resonance (NMR) oxygen measurements provide key validation of the MD models and indicate that the positions and relative magnitudes of the phosphatidylcholine resistance peaks are accurate. Consideration of the headgroup-region resistances predicts bilayer permeability coefficients lower than estimated in EPR studies, giving permeabilities lower than the permeability of unstirred water layers of the same thickness. Here, the permeability of POPC at 35°C is estimated to be 13 cm/s, compared with 10 cm/s for POPC/cholesterol and 118 cm/s for simulation water layers of similar thickness. The value for POPC is 12 times lower than estimated from EPR measurements, while the value for POPC/cholesterol is 5 times lower. These findings underscore the value of atomic resolution models for guiding the interpretation of experimental probe-based measurements.

Oxygen Transport Parameter in Plasma Membrane of Eye Lens Fiber Cells by Saturation Recovery EPR.

A probability distribution of rate constants contained within an exponential-like saturation recovery (SR) electron paramagnetic resonance signal can be constructed using stretched exponential function fitting parameters. Previously (Stein et al. Appl. Magn. Reson. 2019.), application of this method was limited to the case where only one relaxation process, namely spin-lattice relaxations due to the rotational diffusion of the spin labels in the intact eye-lens membranes, contributed to an exponential-like SR signal. These conditions were achieved for thoroughly deoxygenated samples. Here, the case is described where the second relaxation process, namely Heisenberg exchange between the spin label and molecular oxygen that occurs during bimolecular collisions, contributes to the decay of SR signals. We have further developed the theory for application of stretched exponential function to analyze SR signals involving these two processes. This new approach allows separation of stretched exponential parameters, namely characteristic stretched rates and heterogeneity parameters for both processes. Knowing these parameters allowed us to separately construct the probability distributions of spin-lattice relaxation rates determined by the rotational diffusion of spin labels and the distribution of relaxations induced strictly by collisions with molecular oxygen. The later distribution is determined by the distribution of oxygen diffusion concentration products within the membrane, which forms a sensitive new way to describe membrane fluidity and heterogeneity. This method was validated in silico and by fitting SR signals from spin-labeled intact nuclear fiber cell plasma membranes extracted from porcine eye lenses equilibrated with different fractions of air.

Lipid Dynamics in Diisobutylene-Maleic Acid (DIBMA) Lipid Particles in Presence of Sensory Rhodopsin II.

Amphiphilic diisobutylene/maleic acid (DIBMA) copolymers extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding nanosized, discoidal DIBMA lipid particles (DIBMALPs). Depending on the DIBMA/lipid ratio, the size of DIBMALPs can be broadly varied which makes them suitable for the incorporation of proteins of different sizes. Here, we examine the influence of the DIBMALP sizes and the presence of protein on the dynamics of encased lipids. As shown by a set of biophysical methods, the stability of DIBMALPs remains unaffected at different DIBMA/lipid ratios. Coarse-grained molecular dynamics simulations confirm the formation of viable DIBMALPs with an overall size of up to 35 nm. Electron paramagnetic resonance spectroscopy of nitroxides located at the 5th, 12th or 16th carbon atom positions in phosphatidylcholine-based spin labels reveals that the dynamics of enclosed lipids are not altered by the DIBMALP size. The presence of the membrane protein sensory rhodopsin II from Natronomonas pharaonis (NpSRII) results in a slight increase in the lipid dynamics compared to empty DIBMALPs. The light-induced photocycle shows full functionality of DIBMALPs-embedded NpSRII and a significant effect of the protein-to-lipid ratio during preparation on the NpSRII dynamics. This study indicates a possible expansion of the applicability of the DIBMALP technology on studies of membrane protein-protein interaction and oligomerization in a constraining environment.

A Simple Method of Synthesis of 3-Carboxy-2,2,5,5-Tetraethylpyrrolidine-1-oxyl and Preparation of Reduction-Resistant Spin Labels and Probes of Pyrrolidine Series.

Stable free radicals are widely used as molecular probes and labels in various biophysical and biomedical research applications of magnetic resonance spectroscopy and imaging. Among these radicals, sterically shielded nitroxides of pyrrolidine series demonstrate the highest stability in biological systems. Here, we suggest new convenient procedure for preparation of 3-carboxy-2,2,5,5-tetraethylpyrrolidine-1-oxyl, a reduction-resistant analog of widely used carboxy-Proxyl, from cheap commercially available reagents with the yield exceeding the most optimistic literature data. Several new spin labels and probes of 2,2,5,5-tetraethylpyrrolidine-1-oxyl series were prepared and reduction of these radicals in ascorbate solutions, mice blood and tissue homogenates was studied.