Spin-Label EPR for Determining Polarity and Proticity in Biomolecular Assemblies: Transmembrane Profiles

Hyperfine couplings and g-values of nitroxyl spin labels are sensitive to polarity and hydrogen bonding in the environment probed. The dependences of these electronic paramagnetic resonance (EPR) properties on environmental dielectric permittivity and proticity are reviewed. Calibrations are given, in terms of the Block–Walker reaction field and local proton donor concentration, for the nitroxides that are commonly used in spin labeling of lipids and proteins. Applications to studies of the transverse polarity profiles in lipid bilayers, which constitute the permeability barrier of biological membranes, are reviewed. Emphasis is given to parallels with the permeation profiles of oxygen and nitric oxide that are determined from spin-label relaxation enhancements by using nonlinear continuous-wave EPR and saturation recovery EPR, and with permeation profiles of D2O that are determined by using 2H electron spin echo envelope modulation spectroscopy

Spin-echo EPR of Na,K-ATPase unfolding by urea

AbstractDenaturant-perturbation and pulsed EPR spectroscopy are combined to probe the folding of the membrane-bound Na,K-ATPase active transport system. The Na,K-ATPase enzymes from shark salt gland and pig kidney are covalently spin labelled on cysteine residues that either do not perturb or are essential to hydrolytic activity (Class I and Class II –SH groups, respectively). Urea increases the accessibility of water to the spin-labelled groups and increases their mutual separations, as recorded by D2O interactions from ESEEM spectroscopy and instantaneous spin diffusion from echo-detected EPR spectra, respectively. The greater effects of urea are experienced by Class I groups, which indicates preferential unfolding of the extramembrane domains. Conformational heterogeneity induced by urea causes dispersion in spin-echo phase-memory times to persist to higher temperatures. Analysis of lineshapes from partially relaxed echo-detected EPR spectra indicates that perturbation by urea enhances the amplitude and rate of fluctuations between conformational substates, in the higher temperature regime, and also depresses the glasslike transition in the protein. These non-native substates that are promoted by urea lie off the enzymatic pathway and contribute to the loss of function

Differential effects of energy stress on AMPK phosphorylation and apoptosis in experimental brain tumor and normal brain

<p>Abstract</p> <p>Background</p> <p>AMP-activated protein kinase (AMPK) is a known physiological cellular energy sensor and becomes phosphorylated at Thr-172 in response to changes in cellular ATP levels. Activated AMPK acts as either an inducer or suppressor of apoptosis depending on the severity of energy stress and the presence or absence of certain functional tumor suppressor genes.</p> <p>Results</p> <p>Here we show that energy stress differentially affects AMPK phosphorylation and cell-death in brain tumor tissue and in tissue from contra-lateral normal brain. We compared TSC2 deficient CT-2A mouse astrocytoma cells with syngeneic normal astrocytes that were grown under identical condition <it>in vitro</it>. Energy stress induced by glucose withdrawal or addition of 2-deoxyglucose caused more ATP depletion, AMPK phosphorylation and apoptosis in CT-2A cells than in the normal astrocytes. Under normal energy conditions pharmacological stimulation of AMPK caused apoptosis in CT-2A cells but not in astrocytes. TSC2 siRNA treated astrocytes are hypersensitive to apoptosis induced by energy stress compared to control cells. AMPK phosphorylation and apoptosis were also greater in the CT-2A tumor tissue than in the normal brain tissue following implementation of dietary energy restriction. Inefficient mTOR and TSC2 signaling, downstream of AMPK, is responsible for CT-2A cell-death, while functional LKB1 may protect normal brain cells under energy stress.</p> <p>Conclusion</p> <p>Together these data demonstrates that AMPK phosphorylation induces apoptosis in mouse astrocytoma but may protect normal brain cells from apoptosis under similar energy stress condition. Therefore, using activator of AMPK along with glycolysis inhibitor could be a potential therapeutic approach for TSC2 deficient human malignant astrocytoma.</p

Systematically searching for and assessing the literature for self-management of chronic pain: a lay users’ perspective

Background: The Engaging with older adults in the development of strategies for the self management of chronic pain (EOPIC) study aims to design and develop self management strategies to enable older adults to manage their own pain. Involving older adults in research into chronic pain management will better enable the identification and development of strategies that are more appropriate for their use, but how can perspectives really be utilised to the best possible outcomes? Method: Seven older adults were recruited through a local advertising campaign to take part. We also invited participants from the local pain services, individuals who had been involved in earlier phase of the EOPIC study and a previous ESRC funded project. The group undertook library training and research skills training to facilitate searching of the literature and identified sources of material. A grading tool was developed using perceived essential criteria identified by the older adults and material was graded according to the criteria within this scale. Results: Fifty-seven resources from over twenty-eight sources were identified. These materials were identified as being easily accessible, readable and relevant. Many of the web based materials were not always easy to find or readily available so they were excluded by the participants. All but one were UK based. Forty-four items were identified as meeting the key criteria for inclusion in the study. This included five key categories as follows; books, internet, magazines, leaflets, CD’s/Tapes. Conclusion: This project was able to identify a number of exemplars of self management material along with some general rules regarding the categories identified. We must point out that the materials identified were not age specific, were often locally developed and would need to be adapted to older adults with chronic pain. For copyright issues we have not included them in this paper. The key message is really related to the format rather than the content. However, the group acknowledge that these may vary according to the requirements of each individual older adult and therefore recommend the development of a leaflet to help others in their search for resources. This leaflet has been developed as part of Phase IV of the EOPIC study

Prescription and Other Medication Use in Pregnancy

OBJECTIVE: To characterize prescription and other medication use in a geographically and ethnically diverse cohort of women in their first pregnancy. METHODS: In a prospective, longitudinal cohort study of nulliparous women followed through pregnancy from the first trimester, medication use was chronicled longitudinally throughout pregnancy. Structured questions and aids were used to capture all medications taken as well as reasons they were taken. Total counts of all medications taken including number in each category and class were captured. Additionally, reasons the medications were taken were recorded. Trends in medications taken across pregnancy and in the first trimester were determined. RESULTS: Of the 9,546 study participants, 9,272 (97.1%) women took at least one medication during pregnancy with 9,139 (95.7%) taking a medication in the first trimester. Polypharmacy, defined as taking at least five medications, occurred in 2,915 (30.5%) women. Excluding vitamins, supplements, and vaccines, 73.4% of women took a medication during pregnancy with 55.1% taking one in the first trimester. The categories of drugs taken in pregnancy and in the first trimester include the following: gastrointestinal or antiemetic agents (34.3%, 19.5%), antibiotics (25.5%, 12.6%), and analgesics (23.7%, 15.6%, which includes 3.6%; 1.4% taking an opioid pain medication). CONCLUSION: In this geographically and ethnically diverse cohort of nulliparous pregnant women, medication use was nearly universal and polypharmacy was common

Electron spin resonance in membrane research: protein–lipid interactions from challenging beginnings to state of the art

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments

Multiple Binding Sites for Fatty Acids on the Potassium Channel KcsA

Interactions of fatty acids with the potassium channel KcsA were studied using Trp fluorescence quenching and electron paramagnetic resonance (EPR) techniques. The brominated analogue of oleic acid was shown to bind to annular sites on KcsA and to the nonannular sites at each protein-protein interface in the homotetrameric structure with binding constants relative to dioleoylphosphatidylcholine of 0.67 ± 0.04 and 0.87 ± 0.08, respectively. Mutation of the two Arg residues close to the nonannular binding sites had no effect on fatty acid binding. EPR studies with a spin-labeled analogue of stearic acid detected a high-affinity binding site for the fatty acid with strong immobilization. Fluorescence quenching studies with the spin-labeled analogue showed that the binding site detected in the EPR experiments could not be one of the annular or nonannular binding sites. Instead, it is proposed that the EPR studies detect binding to the central hydrophobic cavity of the channel, with a binding constant in the range of ~0.1-1 ?M