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1.
We have used spin labels and electron paramagnetic resonance (EPR) to study the correlation between the rotational dynamics of protein and lipid in sarcoplasmic reticulum (SR) membranes. A short-chain maleimide spin label was used to monitor the submillisecond rotational mobility of the Ca-ATPase enzyme (using saturation transfer EPR); a free fatty acid spin label was used to monitor the submicrosecond rotational mobility of the bulk lipid hydrocarbon chains (using conventional EPR); and a fatty acid spin label derivative (long-chain maleimide) attached to the enzyme was used to monitor the mobility of hydrocarbon chains adjacent to the protein (i.e., boundary lipid). In the native SR membranes, the protein was highly mobile (effective correlation time 50 microseconds). The spectra of the hydrocarbon probes both contained at least two components. For the unattached probe, the major component indicated nearly as much mobility as in the absence of protein (effective rotational correlation time 3 ns), while a minor component, corresponding to 25-30% of the total signal, indicated strong immobilization (effective correlation time greater than or equal to 10 ns). For the attached hydrocarbon probe, the major component (approximately 70% of the total) was strongly immobilized, and the mobile component was less mobile than that of the unattached probe. When the lipid-to-protein ratio was reduced 55% by treatment with deoxycholate, protein mobility decreased considerably, suggesting protein aggregation. A concomitant increase was observed in the fraction of immobilized spin labels for both the free and attached hydrocarbon probes. The observed hydrocarbon immobilization probably arises in part from immobilization at the protein-lipid boundary, but protein-protein interactions that trap hydrocarbon chains may also contribute. When protein aggregation was induced by glutaraldehyde crosslinking, submillisecond protein mobility was eliminated, but there was no effect on either hydrocarbon probe. Thus protein aggregation does not necessarily cause hydrocarbon chain immobilization.  相似文献   

2.
The interaction of quinone with luciferase from Photobacterium leiognathi was studied based on the fluorescence decay measurements of the endogenous flavin bound to the enzyme. Homologous 1,4-quinones, 1,4-benzoquinone, methyl-1,4-benzoquinone, 2-methyl-5-isopropyl-1,4-benzoquine and 1,4-naphthoquinone, were investigated. In the absence of quinone, the fluorescence intensity and anisotropy decays of the endogenous flavin exhibited two intensity decay lifetimes (~ 1 and 5 ns) and two anisotropy decay lifetimes (~ 0.2 and 20 ns), suggesting a heterogeneous quenching and a rotational mobility microenvironment of the active site of the luciferase, respectively. In the presence of quinone, the intensity decay heterogeneity was largely maintained, whereas the fraction of the short anisotropy decay component and the averaged rotational rate of FMN increased with the increasing hydrophobicity of the quinone. We hypothesize that the hydrophobicity of the quinone plays a role in the non-specific inhibition mechanism of xenobiotic molecules in the bacterial bioluminescence system via altering the rotational mobility of the endogenous flavin in the luciferase.  相似文献   

3.
We have developed a saturation transfer EPR (ST-EPR) method to measure selectively the rotational dynamics of those lipids that are motionally restricted by integral membrane proteins and have applied this methodology to measure lipid-protein interactions in native sarcoplasmic reticulum (SR) membranes. This analysis involves the measurement of spectral saturation using a series of six stearic acid spin labels that are labeled with a nitroxide at different carbon atom positions. A large amount of spectral saturation is observed for spin labels in native SR membranes, but not for spin labels in dispersions of extracted SR lipids, implying that the motional properties of those lipids interacting with the Ca-ATPase, i.e., the boundary or annular lipid, can be directly measured without the need for spectral subtraction procedures. A comparison of the motional properties of the restricted lipid, measured by ST-EPR, with those measured by digital subtraction of conventional EPR spectra qualitatively agree, for in both cases the Ca-ATPase restricts the rotational mobility of a population of lipids, whose rotational mobility increases as the nitroxide is positioned toward the center of the bilayer. However, the ability of ST-EPR to directly measure the motionally restricted lipid in a model-independent means provides the greater precision necessary to measure small changes in the rotational dynamics of the lipid at the protein-lipid interface, providing a valuable tool in clarifying the relationship between the physical nature of the protein-lipid interface and membrane function.  相似文献   

4.
In order to investigate the roles of the physical states of phospholipid and protein in the enzymatic behavior of the Ca2+ -ATPase from sarcoplasmic reticulum, we have modified the lipid phase of the enzyme, observed the effects on the enzymatic activity at low temperatures, and correlated these effects with spectroscopic measurements of the rotational motions of both the lipid and protein components. Replacement of the native lipids with dipalmitoyl phosphatidylcholine inhibits ATPase activity and decreases both lipid fluidity, as monitored by EPR spectroscopy on a stearic acid spin label, and protein rotational mobility, as monitored by saturation transfer EPR spectroscopy on the covalently spin-labeled enzyme. Solubilization of the lipid-replaced enzyme with Triton X-100 reverses all three of these effects. Ten millimolar CaCl2 added either to the enzyme associated with the endogenous lipids or to the Triton X-100 soulbilized enzyme inhibits both ATPase activity and protein rotational mobility but has no detectable effect on the lipid mobility. These results are consistent with the proposal that both lipid fluidity and protein rotational mobility are essential for enzymatic activity.  相似文献   

5.
Colicin A (ColA) is a water-soluble toxin that forms a voltage-gated channel in the cytoplasmic membrane of Escherichia coli. Until now, two models were proposed for the closed channel state: the umbrella model and the penknife model. Mutants of ColA, each containing a single cysteine, were labeled with a nitroxide spin label, reconstituted into liposomes, and studied by electron paramagnetic resonance (EPR) spectroscopy to study the membrane-bound closed channel state. The spin-labeled ColA variants in solution and in liposomes of native E. coli lipid composition were analyzed in terms of the mobility of the nitroxide, its accessibility to paramagnetic reagents, and the polarity of its microenvironment. The EPR data determined for the soluble ColA pore-forming domain are in agreement with its crystal structure. Moreover, the EPR results show that ColA has a conformation in liposomes different from its water-soluble conformation. Residues that belong to helices H8 and H9 are significantly accessible for O2 but not for nickel-ethylene diamine diacetic acid, indicating their location inside the membrane. In addition, the polarity values determined from the hyperfine tensor component Azz of residues 176, 181, and 183 (H9) indicate the location of these residues close to the center of the lipid bilayer, supporting a transmembrane orientation of the hydrophobic hairpin. Furthermore, the accessibility and polarity data suggest that the spin-labeled side chains of the amphipathic helices (H1-H7 and H10) are located at the membrane-water interface. Evidence that the conformation of the closed channel state in artificial liposomes depends on lipid composition is given. The EPR results for ColA reconstituted into liposomes of E. coli lipids support the umbrella model for the closed channel state.  相似文献   

6.
The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin-label rigidly bound at Cys-374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin. Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F-actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F-actin, suggesting that the slow formation of interfilamental cross-links in concentrated F-actin solutions contributes to this process. We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F-actin in its rotational mobility but differs from the complex of F-actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub-millisecond internal motion in actin polymer and for a non-cooperative change in the intramonomer conformation around Cys-374 on the binding of HMM.  相似文献   

7.
S M Lewis  D D Thomas 《Biochemistry》1986,25(16):4615-4621
We have studied the effects of vanadate on the rotational motion of the calcium adenosine-triphosphatase (Ca-ATPase) from sarcoplasmic reticulum (SR), using saturation-transfer electron paramagnetic resonance (ST-EPR). Vanadate has been proposed to act as a phosphate analogue and produce a stable intermediate state similar to the phosphoenzyme. This study provides evidence about the physical state of this intermediate. In particular, since ST-EPR provides a sensitive measure of microsecond protein rotational mobility, and hence of protein-protein association, these studies allowed us to ask (a) whether the vanadate-induced protein association observed in electron micrographs of SR vesicles also occurs under physiological (as opposed to fixed, stained, or frozen) conditions and (b) whether vanadate-induced changes in protein association also occur under conditions sufficient for enzyme inhibition but not for the production of large arrays detectable by electron microscopy (EM). At 5 mM decavanadate, a concentration sufficient to crystallize the ATPase on greater than 90% of the membrane surface area in EM, ST-EPR showed substantial immobilization of the spin-labeled protein, indicating protein-protein association in the unstained vesicles. Conventional EPR spectra of lipid probes showed that lipid hydrocarbon chain mobility is unaffected by decavanadate-induced protein crystallization in SR, suggesting that changes in protein-protein contacts do not involve the lipid hydrocarbon region. At 5 mM monovanadate, a concentration sufficient to inhibit the ATPase but not to form crystals detectable by EM, no changes were observed in ST-EPR or conventional EPR spectra of either protein or lipid.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

8.
We have investigated the relationship between function and molecular dynamics of both the lipid and the Ca-ATPase protein in sarcoplasmic reticulum (SR), using temperature as a means of altering both activity and rotational dynamics. Conventional and saturation-transfer electron paramagnetic resonance (EPR) was used to probe rotational motions of spin-labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase sulfhydryl groups in SR. EPR studies were also performed on aqueous dispersions of extracted SR lipids, in order to study intrinsic lipid properties independent of the protein. While an Arrhenius plot of the Ca-ATPase activity exhibits a clear change in slope at 20 degrees C, Arrhenius plots of lipid hydrocarbon chain mobility are linear, indicating that an abrupt thermotropic change in the lipid hydrocarbon phase is not responsible for the Arrhenius break in enzymatic activity. The presence of protein was found to decrease the average hydrocarbon chain mobility, but linear Arrhenius plots were observed both in the intact SR and in extracted lipids. Lipid EPR spectra were analyzed by procedures that prevent the production of artifactual breaks in the Arrhenius plots. Similarly, using sample preparations and spectral analysis methods that minimize the temperature-dependent contribution of local probe mobility to the spectra of spin-labeled Ca-ATPase, we find that Arrhenius plots of overall protein rotational mobility also exhibit no change in slope. The activation energy for protein mobility is the same as that of ATPase activity above 20 degrees C; we discuss the possibility that overall protein mobility may be essential to the rate-limiting step above 20 degrees C.  相似文献   

9.
Thermal stability and internal dynamics of myosin heads in fiber bundles from rabbit psoas muscle has been studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC). Using ADP, ATP and orthovanadate (V(i)), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibers. DSC transitions contained three overlapping endotherms in each state. Deconvolution showed that the transition temperature of 58.4 degrees C was almost independent of the intermediate state of myosin, while nucleotide binding shifted the melting temperatures of 54.0 and 62.3 degrees C, and changed the enthalpies. These changes suggest global rearrangements of the internal structure in myosin head. In the presence of ADP and ADP plus V(i), the conventional EPR spectra showed changes in the ordering of the probe molecules, suggesting local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus orthovanadate corresponding to a weakly binding state of myosin to actin.  相似文献   

10.
DNA photolyase repairs pyrimidine dimer lesions in DNA through light-induced electron donation to the dimer. During isolation of the enzyme, the flavin cofactor necessary for catalytic activity becomes one-electron-oxidized to a semiquinone radical. In the absence of external reducing agents, the flavin can be cycled through the semiquinone radical to the fully reduced state with light-induced electron transfer from a nearby tryptophan residue. This cycle provides a convenient means of studying the process of electron transfer within the protein by using transient EPR. By studying the excitation wavelength dependence of the time-resolved EPR signals we observe, we show that the spin-polarized EPR signal reported earlier from this laboratory as being initiated by semiquinone photochemistry actually originates from the fully oxidized form of the flavin cofactor. Exciting the semiquinone form of the flavin produces two transient EPR signals: a fast signal that is limited by the time response of the instrument and a slower signal with a lifetime of approximately 6 ms. The fast component appears to correlate with a dismutation reaction occurring with the flavin. The longer lifetime process occurs on a time scale that agrees with transient absorption data published earlier; the magnetic field dependence of the amplitude of this kinetic component is consistent with redox chemistry that involves electron transfer between flavin and tryptophan. We also report a new procedure for the rapid isolation of DNA photolyase.  相似文献   

11.
We measured EPR spectra from a spin label on the Cys133 residue of troponin I (TnI) to identify Ca(2+)-induced structural states, based on sensitivity of spin-label mobility to flexibility and tertiary contact of a polypeptide. Spectrum from Tn complexes in the -Ca(2+) state showed that Cys133 was located at a flexible polypeptide segment (rotational correlation time tau=1.9ns) that was free from TnC. Spectra of both Tn complexes alone and those reconstituted into the thin filaments in the +Ca(2+) state showed that Cys133 existed on a stable segment (tau=4.8ns) held by TnC. Spectra of reconstituted thin filaments (-Ca(2+) state) revealed that slow mobility (tau=45ns) was due to tertiary contact of Cys133 with actin, because the same slow mobility was found for TnI-actin and TnI-tropomyosin-actin filaments lacking TnC, T or tropomyosin. We propose that the Cys133 region dissociates from TnC and attaches to the actin surface on the thin filaments, causing muscle relaxation at low Ca(2+) concentrations.  相似文献   

12.
The spin trapping ability of the nitrones 2,4-disulphophenyl-N-tert-butyl nitrone (NXY-059), 2-sulphophenyl-N-tert-butyl nitrone (S-PBN) and alpha-phenyl-N-tert-butyl nitrone (PBN) for both hydroxyl and methanol radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy. The radicals of interest were generated in situ in the spectrometer under constant flow conditions in the presence of each nitrone. The spin adducts formed were detected by EPR spectroscopy. This approach allowed for quantitative comparison of the EPR spectra of the spin adducts of each nitrone. The results obtained showed that NXY-059 trapped a greater number of hydroxyl and methanol radicals than the other two nitrones, under the conditions studied.  相似文献   

13.
Genistein (5,7,4′-trihydroxyisoflavone) the common soy beans isoflavone has attracted scientific interest due to its antioxidant, estrogenic, antiangiogenic and aniticancer activities. The aim of the present study was to investigate the interaction of genistein with biological (erythrocyte) and model membranes (dimyristoyl- and dipalmitoylphosphatidylcholine). Using Laurdan and Prodan as fluorescent probes, we demonstrated phase behavior and membrane fluidity changes induced by genistein. ESR spectroscopy revealed alterations caused by genistein in membrane domains structure and mobility of spin probes with free radicals located at different depths of membrane. The method of ESR spectra decomposition and computer simulation of the recorded spectra were used in order to visualize domain coexistence by GHOST condensation method. Fluorescence and ESR spectroscopy experiments performed at different temperatures enabled us to observe the effect of isoflavone on phospholipid bilayers in either gel or liquid crystalline phase. It was concluded that genistein preferentially intercalated into lipid headgroup region, to some extent into polar–apolar interface and only in minimal degree into hydrophobic core of the membrane. According to our best knowledge this is the first study on modification of domain structure of membranes by genistein.  相似文献   

14.
Di-tert-butylnitroxide dissolved in an aqueous suspension of egg yolk lecithin vesicles is distributed between the two phases. Partition coefficients of the nitroxide between the lipid and the water, calculated from the nitroxide electron paramagnetic resonance (EPR) spectra, decrease with decreasing temperature until approximately the freezing point of the solvent. Below this temperature the nitroxide is detected only in the lecithin. The rotational correlation times of the spin label present in the lecithin were calculated for the temperature range from +45 to -60 degrees C. At low temperatures, the EPR spectra are characteristic of a superposition of two spectra resulting from the nitroxide dissolved in the lipid in two environments with different rotational correlation times.  相似文献   

15.
Dunford AJ  Rigby SE  Hay S  Munro AW  Scrutton NS 《Biochemistry》2007,46(17):5018-5029
Multiple solution-state techniques have been employed in investigating the nature and control of electron transfer in the context of the proposed "domain shuffle hypothesis" for intraprotein electron transfer inferred from the crystal structure of the nitric oxide synthase reductase domain. NADPH analogues and fragments have been used to map those regions of this substrate that are important in eliciting a conformational change, observed in both the fluorescence emission of the flavin cofactors of the enzyme and the EPR spectra of the FMN flavosemiquinone state. EPR and UV-visible potentiometric methods have demonstrated a substantial calmodulin-dependent perturbation in the midpoint reduction potentials of the redox couples of both flavin cofactors, in contrast to a previous report [Noble, M. A., et al. (1999) Biochemistry 38, 16413-16418]. These studies support a model in which FMN domain mobility, triggered by Ca2+-calmodulin binding and antagonized by substrate binding, facilitates electron transfer in nitric oxide synthase through conformational change and effects a major change in the midpoint reduction potentials of the flavin redox couples. These results are discussed in light of the recent crystal structure of the NADPH-locked reductase domain.  相似文献   

16.
The trend of evidence suggests that general anesthetics act directly on proteins in the neural membrane. However, the fact that the functions of nicotinic acetylcholine receptor (sodium permeability, desensitization rate) are modulated by the composition of the membrane in which it is reconstituted has been thought to be a result of the variation of interactions between acetylcholine receptor and membrane. In this study, protein-lipid interaction at the level of the lipid headgroup was investigated using electron paramagnetic resonance (EPR) and headgroup spin label. Lipid headgroup mobility was evaluated with rotational correlation time from the EPR spectrum. Protein-lipid interaction at headgroup depth was demonstrated from the motionally restricted component of the spectrum. Rotational correlation time increased to 13 ns from 7 ns due to protein-lipid interaction. The effect of anesthetic (ethanol, 1-hexanol, and isoflurane) on protein-lipid interaction was investigated, and the correlation time was 13 ns. It is concluded that the anesthetics used in this study did not alter protein-lipid interaction at the level of the lipid headgroup, so far as observed by rotational correlation time, without excluding the possibility that anesthetics that perturb protein-lipid interactions modulate receptor functions via this mechanism.  相似文献   

17.
We have developed a quantitative and relatively model-independent measure of lipid fluidity using EPR and have applied this method to compare the temperature dependence of lipid hydrocarbon chain fluidity, overall protein rotational mobility, and the calcium-dependent enzymatic activity of the Ca-ATPase in sarcoplasmic reticulum. We define membrane lipid fluidity to be T/eta, where eta is the viscosity of a long chain hydrocarbon reference solvent in which a fatty acid spin label gives the same EPR spectrum (quantitated by the order parameter S) as observed for the same probe in the membrane. This measure is independent of the reference solvent used as long as the spectral line shapes in the membrane and the solvent match precisely, indicating that the same type of anisotropic probe motion occurs in the two systems. We argue that this empirical measurement of fluidity, defined in analogy to the macroscopic fluidity (T/eta) of a bulk solvent, should be more directly related to protein rotational mobility (and thus to protein function) than are more conventional measures of fluidity, such as the rate or amplitude of rotational motion of the lipid hydrocarbon chains themselves. This new definition thus offers a fluidity measure that is more directly relevant to the protein's behavior. The direct relationship between this measure of membrane fluidity and protein rotational mobility is supported by measurements in sarcoplasmic reticulum. The overall rotational motion of the spin-labeled Ca-ATPase protein was measured by saturation-transfer EPR. The Arrhenius activation energy for protein rotational mobility (11-12 kcal/mol/degree) agrees well with the activation energy for lipid fluidity, if defined as in this study, but not if more conventional definitions of lipid fluidity are used. This agreement, which extends over the entire temperature range from 0 to 40 degrees C, suggests that protein mobility depends directly on lipid fluidity in this system, as predicted from hydrodynamic theory. The same activation energy is observed for the calcium-dependent ATPase activity under physiological conditions, suggesting that protein rotational mobility (dependent on lipid fluidity) is involved in the rate-limiting step of active calcium transport.  相似文献   

18.
Sphagnum mosses sampled in the background and industry-affected regions within an oil deposit situated in the southern zone of the Bolshezemelskaya tundra were analyzed by EPR spectroscopy. The EPR spectra of plants vary in intensity. As shown by heating samples, the intensity of EPR signals of free radicals is an exponential function of temperature. Experiments with preliminary evacuation demonstrated the presence of paramagnetic centers possessing different sensitivity to atmospheric oxygen. This specific oxygen effect is the most profound in plants taken from polluted areas. Therefore, EPR spectroscopy may be used for estimating the state of plants in oil-producing regions.  相似文献   

19.
Di-tert-butylnitroxide dissolved in an aqueous suspension of egg yolk lecithin vesicles is distributed between the two phases. Partition coefficients of the nitroxide between the lipid and the water, calculated from the nitroxide electron paramagnetic resonance (EPR) spectra, decrease with decreasing temperature until approximately the freezing point of the solvent. Below this temperature the nitroxide is detected only in the lecithin. The rotational correlation times of the spin label present in the lecithin were calculated for the temperature range from +45 to ?60 °C. At low temperatures, the EPR spectra are characteristic of a superposition of two spectra resulting from the nitroxide dissolved in the lipid in two environments with different rotational correlation times.  相似文献   

20.
The singlet and triplet excited states properties of lumiflavin (LF), riboflavin (RF), flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in reversed micelles (RM) of sodium docusate (AOT) in n-hexane solutions were evaluated as a function of the water to surfactant molar ratio, w(0) = [H(2)O]/[AOT], by both steady-state and time-resolved absorption and fluorescence spectroscopy. The results indicated that hydrogen-bonding interactions between the isoalloxazine ring of the flavins with the water molecules of the micellar interior play a crucial role on the modulation of the excited state properties of the flavins. Fluorescence dynamic experiments in the RM, allowed the calculation of similar values for both the internal rotational time of the flavins (θ(i)) and the hydrogen-bonding relaxation time (τ(HB)), e.g.≈ 7 and 1.5 ns at w(0) = 1 and 20, respectively. In turn, the triplet state lifetimes of the flavins were also enlarged in RM solutions at low w(0), without modifications of their quantum yields. A hydrogen bonding relaxation model is proposed to explain the singlet excited state properties of the flavins, while the changes of the triplet state decays of the flavins were related with the global composition and strength of the hydrogen bonding network inside of the RM.  相似文献   

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