In vivo EPR on spin labeled colicin A reveals an oligomeric assembly of the pore-forming domain in E. coli membranes.

We report on the application of site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) spectroscopy to study possible oligomerization of the bacterial toxin colicin A (ColA) upon membrane insertion in vitro and in vivo. We applied SDSL-EPR protocols and optimized experimental conditions to perform continuous wave EPR experiments and double electron-electron resonance distance measurements on intact Escherichia coli cells interacting with nitroxide spin-labeled ColA. Our data suggest that ColA forms dimers upon membrane insertion, thus explaining previously reported pore diameters of about 1 nm, which are unlikely to be formed by a single colicin A monomer.

Hydrogen bonding of nitroxide spin labels in membrane proteins.

On the basis of experiments at 275 GHz, we reconsider the dependence of the continuous-wave EPR spectra of nitroxide spin-labeled protein sites in sensory- and bacteriorhodopsin on the micro-environment. The high magnetic field provides the resolution necessary to disentangle the effects of hydrogen bonding and polarity. In the gxx region of the 275 GHz EPR spectrum, bands are resolved that derive from spin-label populations carrying no, one or two hydrogen bonds. The gxx value of each population varies hardly from site to site, significantly less than deduced previously from studies at lower microwave frequencies. The fractions of the populations vary strongly, which provides a consistent description of the variation of the average gxx and the average nitrogen-hyperfine interaction Azz from site to site. These variations reflect the difference in the proticity of the micro-environment, and differences in polarity contribute marginally. Concomitant W-band ELDOR-detected NMR experiments on the corresponding nitroxide in perdeuterated water resolve population-specific nitrogen-hyperfine bands, which underlies the interpretation for the proteins.

Prevention of peroxidation of cardiolipin liposomes by quinol-based antioxidants.

In mammalian mitochondria, cardiolipin molecules are the primary targets of oxidation by reactive oxygen species. The interaction of oxidized cardiolipin molecules with the constituents of the apoptotic cascade may lead to cell death. In the present study, we compared the effects of quinol-containing synthetic and natural amphiphilic antioxidants on cardiolipin peroxidation in a model system (liposomes of bovine cardiolipin). We found that both natural ubiquinol and synthetic antioxidants, even being introduced in micro- and submicromolar concentrations, fully protected the liposomal cardiolipin from peroxidation. The duration of their action, however, varied; it increased with the presence of either methoxy groups of ubiquinol or additional reduced redox groups (in the cases of rhodamine and berberine derivates). The concentration of ubiquinol in the mitochondrial membrane substantially exceeds the concentrations of antioxidants we used and would seem to fully prevent peroxidation of membrane cardiolipin. In fact, this does not happen: cardiolipin in mitochondria is oxidized, and this process can be blocked by amphiphilic cationic antioxidants (Y. N. Antonenko et al. (2008) Biochemistry (Moscow), 73, 1273-1287). We suppose that a fraction of mitochondrial cardiolipin could not be protected by natural ubiquinol; in vivo, peroxidation most likely threatens those cardiolipin molecules that, being bound within complexes of membrane proteins, are inaccessible to the bulky hydrophobic ubiquinol molecules diffusing in the lipid bilayer of the inner mitochondrial membrane. The ability to protect these occluded cardiolipin molecules from peroxidation may explain the beneficial therapeutic action of cationic antioxidants, which accumulate electrophoretically within mitochondria under the action of membrane potential.

Conformational changes of the betaine transporter BetP from Corynebacterium glutamicum studied by pulse EPR spectroscopy.

The betaine transporter BetP from Corynebacterium glutamicum is activated by hyperosmotic stress critically depending on the presence and integrity of its sensory C-terminal domain. The conformational properties of the trimeric BetP reconstituted in liposomes in the inactive state and during osmotic activation were investigated by site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy. Comparison of intra- and intermolecular inter spin distance distributions obtained by double electron-electron resonance (DEER) EPR with the crystal structure of BetP by means of a rotamer library analysis suggest a rotation of BetP protomers within the trimer by about 15° as compared to the X-ray structure. Furthermore, we observed conformational changes upon activation of BetP, which are reflected in changes of the distances between positions 545 and 589 of different protomers in the trimer. Introduction of proline at positions 550 and 572, both leading to BetP variants with a permanent (low level) transport activity, caused changes of the DEER data similar to those observed for the activated and inactivated state, respectively. This indicates that not only displacements of the C-terminal domain in general but also concomitant interactions of its primary structure with surrounding protein domains and/or lipids are crucial for the activity regulation of BetP.

How far in-silico computing meets real experiments. A study on the structure and dynamics of spin labeled vinculin tail protein by molecular dynamics simulations and EPR spectroscopy.

BACKGROUND: Investigation of conformational changes in a protein is a prerequisite to understand its biological function. To explore these conformational changes in proteins we developed a strategy with the combination of molecular dynamics (MD) simulations and electron paramagnetic resonance (EPR) spectroscopy. The major goal of this work is to investigate how far computer simulations can meet the experiments. METHODS: Vinculin tail protein is chosen as a model system as conformational changes within the vinculin protein are believed to be important for its biological function at the sites of cell adhesion. MD simulations were performed on vinculin tail protein both in water and in vacuo environments. EPR experimental data is compared with those of the simulated data for corresponding spin label positions. RESULTS: The calculated EPR spectra from MD simulations trajectories of selected spin labelled positions are comparable to experimental EPR spectra. The results show that the information contained in the spin label mobility provides a powerful means of mapping protein folds and their conformational changes. CONCLUSIONS: The results suggest the localization of dynamic and flexible regions of the vinculin tail protein. This study shows MD simulations can be used as a complementary tool to interpret experimental EPR data.

Structure and dynamics of spin-labeled insulin entrapped in a silica matrix by the sol-gel method.

The structure and conformational dynamics of insulin entrapped into a silica matrix was monitored during the sol to maturated-gel transition by electron paramagnetic resonance (EPR) spectroscopy. Insulin was successfully spin-labeled with iodoacetamide and the bifunctional nitroxide reagent HO-1944. Room temperature continuous wave (cw) EPR spectra of insulin were recorded to assess the mobility of the attached spin labels. Insulin conformation and its distribution within the silica matrix were studied using double electron-electron resonance (DEER) and low-temperature cw-EPR. A porous oxide matrix seems to form around insulin molecules with pore diameters in the order of a few nanometers. Secondary structure of the encapsulated insulin investigated by Fourier transform infrared spectroscopy proved a high structural integrity of insulin even in the dried silica matrix. The results show that silica encapsulation can be used as a powerful tool to effectively isolate and functionally preserve biomolecules during preparation, storage, and release.

Time-resolved detection of structural changes during the photocycle of spin-labeled bacteriorhodopsin.

Bacteriorhodopsin was selectively spin labeled at residues 72, 101, or 105 after replacement of the native amino acids by cysteine. Only the electron paramagnetic resonance spectrum of the label at 101 was time-dependent during the photocycle. The spectral change rose with the decay of the M intermediate and fell with recovery of the ground state. The transient signal is interpreted as the result of movement in the C-D or E-F interhelical loop, or in both, coincident with protonation changes at the key aspartate 96 residue. These results link the optically characterized intermediates with localized conformational changes in bacteriorhodopsin during the photocycle.

Detergent-free solubilization of human Kv channels expressed in mammalian cells.

Styrene-maleic acid (SMA) copolymers are used to extract lipid-encased membrane proteins from lipid bilayers in a detergent-free manner, yielding SMA lipid particles (SMALPs). SMALPs can serve as stable water-soluble nanocontainers for structural and functional studies of membrane proteins. Here, we used SMA copolymers to study full-length pore-forming α-subunits hKCNH5 and hKCNQ1 of human neuronal and cardiac voltage-gated potassium (Kv) channels, as well as the fusion construct comprising of an α-subunit hKCNQ1 and its regulatory transmembrane KCNE1 ß-subunit (hKCNE1-hKCNQ1) with added affinity tags, expressed in mammalian COS-1 cells. All these recombinant proteins were shown to be functionally active. Treatment with the SMA copolymer, followed by purification on the affinity column, enabled extraction of all three channels. A DLS experiment demonstrated that negative stain electron microscopy and single particle image analysis revealed a four-fold symmetry within channel-containing SMALPs, which indicates that purified hKCNH5 and hKCNQ1 channels, as well as the hKCNE1-hKCNQ1 fusion construct, retained their structural integrity as tetramers.

Temperature-jump studies and polarized absorption spectroscopy of methemoglobin-thiocyanate single crystals.

Association equilibria and association kinetics of the thiocyanate binding reaction to methemoglobin in single crystals and solution are studied using temperature-jump technique and polarized absorption spectroscopy. Different kinetic constants are found for the reaction in solution and crystal phase for the alpha- and beta-subunits of the methemoglobin tetramer. The reduction of the reactivity of the alpha- and beta-subunits in crystalline phase is 6-fold and 2.4-fold, respectively, compared to the values found in solution. The intramolecular binding reaction of the N epsilon of the distal histidine E7 which is observed in methemoglobin in solution cannot be detected in single crystals. Our results suggest that crystallization of hemoglobin has little influence on small-scale structural fluctuations which are necessary for ligands to get to the binding sites and large-scale structural motions are suppressed.

Conformational transition of aquomethemoglobin: intramolecular histidine E7 binding reaction to the heme iron in the temperature range between 220 K and 295 K as seen by EPR and temperature-jump measurements.

Temperature-dependent EPR and temperature-jump measurements have been carried out, in order to examine the high-spin to low-spin transition of aquomethemogobin (pH 6.0). Relaxation rates and equilibrium constants could be determined as a function of temperature. As a reaction mechanism for the high-spin to low-spin transition, the binding of N epsilon of His E7 to the heme iron had been proposed; the same mechanism had been suggested for the ms-effect, found in temperature-jump experiments on aquomethemoglobin. A comparison of the thermodynamic quantities, deduced form the measurements in this paper, gives evidence that indeed the same reaction is investigated in both cases. Our results and most of the findings of earlier studies on the spin-state transitions of aquomethemoglobin, using susceptibility, optical, or EPR measurements, can be explained by the transition of methemoglobin with H2O as ligand (with high-spin state at all temperatures) and methemoglobin with ligand N epsilon of His E7 (with a low-spin ground state). Thermal fluctuations of large amplitude have to be postulated for the reaction to take place, so this reaction may be understood as a probe for the study of protein dynamics.

Structural fluctuations and conformational entropy in proteins: entropy balance in an intramolecular reaction in methemoglobin.

The reversible intramolecular binding of the distal histidine side chain to the heme iron in methemoglobin is of special interest due to the very large negative reaction entropy which overcompensates the large reaction enthalpy. It may be considered as a prominent example of the ability of proteins (including enzymes) to provide global entropy in a local process. In this work new experiments and model calculations are reported which aim at finding the structural elements contributing to the reaction entropy. Geometrical studies prove the implication of the 20 residue E-helix being shifted by more than 2 A. Vibrational entropies are calculated by a procedure derived from the method of Karplus and Kushik. It turns out that neither the histidine alone nor the complete E-helix contribute more than 15 per cent of the required entropy.

High-field EPR studies of the structure and conformational changes of site-directed spin labeled bacteriorhodopsin.

Cw and pulsed high-field EPR (95 GHz, 3.4 T) are performed on site-directed spin labeled bacteriorhodopsin (BR) mutants. The enhanced Zeeman splitting leads to spectra with resolved g-tensor components of the nitroxide spin label. The g(xx) component shift determined for 10 spin labels located in the cytoplasmic loop region and in the protein interior along the BR proton channel reveals a maximum close to position 46 between the proton donor D96 and the retinal. A plot of g(xx) versus A(zz) of the nitrogen discloses grouping of 12 spin labeled sites in protic and aprotic sites. Spin labels at positions 46, 167 and 171 show the aprotic character of the cytoplasmic moiety of the proton channel whereas nitroxides at positions 53, 194 and 129 reveal the protic environment in the extracellular channel. The enhanced sensitivity of high-field EPR with respect to anisotropic reorientational motion of nitroxides allows the characterization of different motional modes for spin labels bound to positions 167 and 170. The motional restriction of the nitroxide at position 167 of the double mutant V167C/D96N is decreased in the M(N) photo-intermediate. An outward shift of the cytoplasmic moiety of helix F in the M(N) intermediate would account for the high-field EPR results and is in agreement with diffraction and recent X-band EPR data.

Time-resolved detection of transient movement of helix F in spin-labelled pharaonis sensory rhodopsin II.

Sensory rhodopsin II (also called phoborhodopsin) from the archaeal Natronobacterium pharaonis (pSRII) functions as a repellent phototaxis receptor. The excitation of the receptor by light triggers the activation of a transducer molecule (pHtrII) which has close resemblance to the cytoplasmic domain of bacterial chemotaxis receptors. In order to elucidate the first step of the signal transduction chain, the accessibility as well as static and transient mobility of cytoplasmic residues in helices F and G were analysed by electron paramagnetic resonance spectroscopy. The results indicate an outward tilting of helix F during the early steps of the photocycle which is sustained until the reformation of the initial ground state. Co-expression of pSRII with a truncated fragment of pHtrII affects the accessibility and/or the mobility of certain spin-labelled residues on helices F and G. The results suggest that these sites are located within the binding surface of the photoreceptor with its transducer.

Site-directed spin-labeling reveals the orientation of the amino acid side-chains in the E-F loop of bacteriorhodopsin.

Due to high temperature factors and the lack of considerable electron density, electron microscopy and X-ray experiments on the cytoplasmic E-F loop of bacteriorhodopsin result in a variety of structural models. As the experimental conditions regarding ionic strength, temperature and the presence of detergents may affect the structure of the E-F loop, we employ electron paramagnetic resonance and site-directed spin-labeling to study the structure of this loop under physiological conditions. The amino acid residues at positions 154 to 171 were replaced by cysteine residues and derivatized with a sulfhydryl-specific nitroxide spin label one by one. The conventional and power saturation electron paramagnetic spectroscopy provide the mobility of the nitroxide and its accessibility to dissolved molecular oxygen and membrane-impermeable chromium oxalate in the respective site. The results show that K159 and A168 are located at the water-lipid interface of helices E and F, respectively. The orientation of the amino acid side-chains in the helical regions from positions 154 to 159 and 166 to 171 were found to agree with published structural data for bacteriorhodopsin. In the residue sequence from positions 160 to 165 the EPR data yield evidence for a turned loop structure with the side-chains of M163 and S162 oriented towards the proton channel and the water phase, respectively.

Temperature-dependent equilibrium between the open and closed conformation of the p66 subunit of HIV-1 reverse transcriptase revealed by site-directed spin labelling.

X-ray crystallographic studies of human immunodeficiency virus type 1 reverse transcriptase complexed with or without substrates or inhibitors show that the heterodimeric enzyme adopts distinct conformations that differ in the orientation of the so-called thumb subdomain in the large subunit. Site-directed spin labelling of mutated residue positions W24C and K287C is applied here to determine the distances between the fingers and thumb subdomains of liganded and unliganded RT in solution. The inter-spin distances of a DNA/DNA and a pseudoknot RNA complexed reverse transcriptase in solution was found to agree with the respective crystal data of the open and closed conformations. For the unliganded reverse transcriptase a temperature-dependent equilibrium between these two states was observed. The fraction of the closed conformation decreased from 95% at 313 K to 65% at 273 K. The spectral separation between the two structures was facilitated by the use of a perdeuterated ([15)N]nitroxide methane-thiosulfonate spin label.

Domain motions accompanying Tet repressor induction defined by changes of interspin distances at selectively labeled sites.

To investigate internal movements in Tet repressor (TetR) during induction by tetracycline (tc) we determined the interspin distances between pairs of nitroxide spin labels attached to specific sites by electron paramagnetic resonance (EPR) spectroscopy. For this purpose, we constructed six TetR variants with engineered cysteine pairs located in regions with presumed conformational changes. These are I22C and N47C in the DNA reading head, T152C/Q175C, A161C/Q175C and R128C/D180C near the tc-binding pocket, and T202C in the dimerization surface. All TetR mutants show wild-type activities in vivo and in vitro. The binding of tc results in a considerable decrease of the distance between the nitroxide groups attached to both I22C residues in the TetR dimer and an increase of the distance between the N47C residues. These opposite effects are consistent with a twisting motion of the DNA reading heads. Changes of the spin-spin interactions between nitroxide groups attached to residues near the tc-binding pocket demonstrate that the C-terminal end of alpha-helix 9 moves away from the protein core upon DNA binding. Alterations of the dipolar interaction between nitroxide groups at T202C indicate different conformations for tc and DNA-bound repressor also in the dimerization area. These results are used to model structural changes of TetR upon induction.

Serum nitrite sensitively reflects endothelial NO formation in human forearm vasculature: evidence for biochemical assessment of the endothelial L-arginine-NO pathway.

OBJECTIVE: A reduced bioactivity of endothelial nitric oxide (NO) has been implicated in the pathogenesis of atherosclerosis. In humans, the endothelial L-arginine-NO pathway has been indirectly assessed via the flow response to endothelium-dependent vasodilators locally administered into the coronary, pulmonary or forearm circulation. However, biochemical quantification of endothelial NO formation in these organ circulations has been hampered so far because of the rapid metabolism of NO. Therefore, we aimed to work out a reliable biochemical index to assess endothelial NO formation in human circulation. METHODS: In 33 healthy volunteers, forearm blood flow (FBF) was measured by standard techniques of venous occlusion plethysmography at rest, after local application of the endothelium-dependent vasodilator acetylcholine (ACH), the endothelium-independent vasodilator papaverine (PAP), the stereospecific inhibitor of endothelial NO synthase (eNOS) L-NMMA, and L-arginine (ARG), the natural substrate of eNOS. In parallel, nitrite and nitrate concentrations in blood samples taken from the antecubital vein were measured by HPLC using anion-exchange chromatography in combination with electrochemical and ultraviolet detection following a specific sample preparation method. RESULTS: ACH dose-dependently increased resting FBF (from 3.0 +/- 0.3 to 10.4 +/- 0.9 ml/min per 100 ml tissue) and serum nitrite concentration (from 402 +/- 59 to 977 +/- 82 nmol/l, both p < 0.05, n = 12). A significant correlation was observed between the changes in FBF and the serum nitrite concentration (r = 0.61, p < 0.0001). L-NMMA reduced resting FBF and endothelium-dependent vasodilation by 30% and this was paralleled by a significant reduction in serum nitrite concentration at the highest dose of ACH (n = 9, p < 0.001). PAP increased FBF more than fourfold, but did not affect serum nitrite concentration (n = 11), whereas ARG significantly increased both FBF and nitrite. Basal serum nitrate amounted to 25 +/- 4 mumol/l and remained constant during the application of ACH, PAP and L-NMMA. CONCLUSIONS: The concentration of serum nitrite sensitively reflects changes in endothelial NO formation in human forearm circulation. This biochemical measure may help to characterize the L-arginine-NO pathway in disease states associated with endothelial dysfunction and to further elucidate its pathophysiological significance for the development of atherosclerosis in humans.

Computerized transaxial tomography and cerebral serial scintigraphy in intracranial tumors--rates of detection and tumor-type identification: concise communication.

In 215 cases of intracranial tumors with subsequent histological identification, computerized transaxial tomography (CTT), and cerebral serial scintigraphy (CSS) with [99mTc] pertechnetate were carried out to compare the efficiency of each method in detecting and classifying such tumors. With a tumor detection rate of 99%, CTT turned out to be superior to CSS (91%). On the other hand, CSS findings enhanced the CTT rate of correctly identified tumor types in meningiomas from 85 to 92% and in high-grade gliomas from 82 to 89%. In metastases, low-grade gliomas, and various other tumors, CSS supported CTT by confirming a number of these tumor types. Therefore, the most important use of CSS in intracranial tumors today is its role as a supplement to CTT in order to establish and confirm type-specific diagnoses.

Enhanced renal function associated with intermittent mandatory ventilation in acute respiratory failure.

In ten patients suffering from acute respiratory failure (ARF) renal function was evaluated during 2-h periods of intermittent mandatory ventilation (IMV) or controlled mechanical ventilation (CMV). Urine flow, osmolal and creatinine clearances were significantly lower during CMV in comparison to both IMV phases and the free water clearance was less negative. Potassium excretion declined with CMV but remained reduced during the second IMV phase. There was no change in sodium excretion. This study suggests that in order to maintain renal function and prevent water retention the use of IMV should be considered whenever a sufficient mechanical reserve for partial spontaneous ventilation is present.

Facilitation of renal function by intermittent mandatory ventilation.

The effects of intermittent mandatory ventilation (IMV) and controlled mechanical ventilation (CMV) on excretory function and the hemodynamics of the kidneys were studied in two groups of anaesthetized dogs during periods of 3 and 4 h. IMV was associated with statistically significant improved urinary output and renal plasma flow of approximately 50 and 35%, respectively. Graphical and statistical analysis revealed certain cross-over effects indicating that the beneficial effect of IMV was more pronounced if it was used following CMV. The improvements in renal function were interpreted as consequences of decreased mean intrathoracic pressures during IMV as compared to CMV. A correlation to global hemodynamic changes could not be established. IMV does facilitate kidney function and hence may successfully counteract the retention of water and salt which occurs during prolonged mechanical ventilation.