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1.
Plasma fibronectin was chemically modified by 4-maleimido-2,2,6,6-tetramethylpiperidinooxyl (maleimide spin label). Only the free sulfhydryl groups of plasma fibronectin were modified by the label under the experimental conditions. The ESR spectrum of spin-labeled fibronectin showed that the sites of labeling were highly immobilized, suggesting that the sulfhydryl groups of the protein are in small, confined environments. The conversion of the strongly immobilized ESR spectrum into a weakly immobilized one was observed when the spin-labeled protein was heated from 30 to 60 degrees C, indicating the thermal unfolding of the protein molecules. The midpoint temperature for the thermal unfolding of plasma fibronectin is about 50 degrees C. The results suggest that plasma fibronectin is stable to about 40 degrees C and starts unfolding above this temperature. The rotational correlation time estimated from the ESR spectrum of spin-labeled fibronectin at 21 degrees C was about 2.0 X 10(-8) s. The rotational correlation time calculated from the Stokes-Einstein equation, assuming a rigid globular configuration for fibronectin with a Stokes radius of 10 nm, was about 7.8 X 10(-7) s. The differences in rotational correlation time by a factor of 39 between experimental and calculated values do not support a globular configuration for plasma fibronectin. 相似文献
2.
Evidence that the two free sulfhydryl groups of plasma fibronectin are in different local environments. Saturation-recovery electron spin resonance study.
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Human plasma fibronectin is a dimer consisting of two subunits; each contains two cryptic thiol groups that were selectively labeled with an 15N,2H-maleimide spin label. Previous studies using conventional X-band electron spin resonance (ESR) methods showed that the spectrum of the labeled protein displays a single strongly immobilized component with an effective rotational correlation time of approximately 17 ns, suggesting that the physical environments of the two labeled sites per chain are indistinguishable. Here we have used saturation-recovery ESR to measure directly electron spin-lattice relaxation time (T1) of the labeled protein in solution at 27 degrees C. Interestingly, the time evolution of the signal was found to be biphasic, which was deconvoluted into two T1 values of 1.37 and 4.53 microseconds. Thus, the two spin-labeled sulfhydryl sites of plasma fibronectin (Fn), being similar in rates of rotational diffusion, differ by a factor of 3.2 in T1. Parallel experiments using various fibronectin fragments showed that the 1.37-microseconds component is associated with the label attached onto the thiol located in between the DNA-binding and the cell-binding domains, and the 4.53-microseconds component is associated with the label attached onto the thiol located within the carboxyl-terminal fibrin-binding domain. The data suggest that the saturation-recovery ESR is a useful method for differentiating multiple spin-labeled sites on macromolecules in which the labels undergo similar rates of rotational motion. 相似文献
3.
We report here a novel approach to label specifically one of the two cryptic, free sulfhydryl groups per subunit of human plasma fibronectin with either an 15N,2H-maleimide spin label or a coumarinylphenyl maleimide fluorescent label. This permits the use of electron spin resonance (ESR) or fluorescence techniques to study molecular dynamics of fibronectin with the label attached to a single site per chain on the protein molecule. The method is based on our observation that upon adsorption of fibronectin to a gelatin-coated surface, the SH1 site, located between the DNA-binding and the cell-binding domains, is partially exposed, while the SH2 site, located within the carboxyl-terminal fibrin-binding domain, remains buried and unreactive. The procedures for the preparation of the selectively labeled fibronectins are described in detail. The physicochemical properties of these single-site labeled fibronectins, particularly as affected by high salt, heparin, surface binding, and temperature, were characterized by ESR spin-label and steady-state fluorescence techniques. The steady-state fluorescence measurement indicates that both local environments of SH1 and SH2 sites are relatively hydrophobic, and that the SH2 site is more hydrophobic than the SH1 site. The ESR results show that heparin or high salt induces an increase in the domainal flexibility in both SH1 and SH2 regions, perhaps through the disruption of domain-domain interactions in the fibronectin molecule, and that the former is more effective than the latter in producing such an effect. The observed heparin effect is reversible by addition of calcium ions in the SH2 regions but not in the SH1 regions. In addition, at temperatures above 44 degrees C, both type III homologous regions containing the free sulfhydryl groups are shown to undergo denaturation and aggregation processes. The data presented here suggest that the newly developed method for differential labeling of the free sulfhydryl groups in fibronectin should be useful for mapping the spatial arrangement of structural domains in the protein molecule using spin-label-spin-probe and fluorescence energy transfer techniques. 相似文献
4.
Two electron spin resonance (ESR) spin labels were used to monitor the physical state of bacterial and animal cell membranes: 5N10, a nitroxide derivative of decane, and 12NS-GA, a glucosamine derivative of 12-nitroxide stearic acid. Spectra were recorded at 1 degrees C intervals from approximately 5 to 45 degrees C. Arrhenius plots of log hH/hP vs. 1/K were obtained by measuring the amplitudes of the hydrocarbon and water signals, hH and hP, respectively. Two discontinuities in the Arrhenius plot (at characteristic temperatures t1 and th) were observed with bacterial cell membranes independent of the spin label employed. Analysis of sealed animal cell membrane samples revealed four characteristic temperatures when the hydrophobic spin lable 5N10 was used, but only two when the amphiphilic spin label 12NS-GA was used. The specific set of characteristic temperatures revealed with 12NS-GA depended on whether the membrane preparation was inside out (ISO) or right side out (RSO). Analysis of Newcastle disease virus, a source of RSO plasma membrane derived from host, revealed two characteristic temperatures at approximately 14 and 33 degrees C. Analysis of phagosomes, a source of ISO plasma membrane derived from LM cells, revealed two characteristic temperatures at approximately 23 and 38 degrees C. When unsealed or disrupted membrane preparations were spin labeled with 12NS-GA, both sets (RSO and ISO) of characteristic temperatures were revealed. The results indicate that the inner and outer monolayers of animal cell membranes are physically distinct and that the glycosylated spin label, 12NS-GA, is apparently restricted in its ability to flip across the membrane bilayer. In this study, characteristic temperatures were pinpointed by computer analysis of the ESR spectral data. 相似文献
5.
The interaction of human alpha 1-acid glycoprotein (AAG) with a corticosteroid was studied using nitroxide labeled deoxycorticosterone and electron spin resonance (ESR) spectroscopy. The ESR spectra of the spin labeled steroid in the presence of AAG could be used to characterize the ligand-protein interaction at equilibrium without the need of a separation between bound and free species. An association constant Ka of 6.10(5) M-1 at 20 degrees C and a binding capacity of one site per mole protein were found. ESR spectra recorded at equilibrium at various temperatures allowed the calculation of enthalpy and entropy variations for the steroid-protein interaction; these thermodynamic parameters exhibited a rapid change above 45 degrees C which may be related to a protein conformational modification above this temperature, as detected by circular dichroism study. The ESR spectra width could be used to define a polar character for the spin label environment in the steroid binding site of AAG and to calculate an apparent rotational correlation time of 2.8 x 10(-8) sec for the steroid-protein complex in aqueous solution at 20 degrees C. It can be concluded that spin labeling and ESR methodology is of value in the study of steroid-protein interactions of biological significance above all because it can provide direct physico-chemical information concerning the local environment of the ligand in its binding site at equilibrium. 相似文献
6.
Structure and flexibility of plasma fibronectin in solution: electron spin resonance spin-label, circular dichroism, and sedimentation studies 总被引:2,自引:0,他引:2
Human plasma fibronectin has been investigated by electron spin resonance (ESR) spin-label methods in conjunction with circular dichroism (CD) and sedimentation techniques to investigate its structure and flexibility in solution. The buried sulfhydryl groups of fibronectin were modified with a maleimide spin-label [Lai, C.-S., & Tooney, N. M. (1984) Arch. Biochem. Biophys. 228, 465-473]. Both conventional and saturation transfer ESR spectra give a rotational correlation time of about (2-3) X 10(-8) s for plasma fibronectin, a value that is at least 40 times faster than the rotational correlation time calculated from the minimal molecular dimensions. This argues that plasma fibronectin is not a compact, globular protein and suggests that the regions of ordered structural domains have a relatively high degree of independent mobility. ESR, CD, and sedimentation measurements showed that many structural features of plasma fibronectin remain unchanged when the pH is decreased from 7.4 to 3.0. On the other hand, ESR results indicate an unfolding of the protein molecule either at pH 11 or in 4 M urea solution. Similarly, the sedimentation coefficient decreases from about 13 to 8.4 S when the pH is raised to 10.8. At pH values above 11, the CD spectrum resembles a random coil; however, some ordered structure is retained either at pH 11 or in 4 M urea. It is likely that the sulfhydryl-containing regions of the molecule are more sensitive to urea or alkali than are portions of the molecule stabilized by intrachain disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
Spin label electron spin resonance (ESR) was used to characterize the response of lipid regions of the plasma membrane of yeast to photodynamic attack. Following photodynamic attack, the structure of these lipid regions changed resulting in the disappearance of an apparent order—disorder phase transition as well as impeding the diffusion of the steric acid based spin label 12NS into and across the plasma membrane. We propose that singslet molecular oxygen reacting with unsaturated carbon bonds in the fatty acyl chains of lipid surrounding channel proteins leads to an increase in the order of the lipid array and/or a change in the channel protein's conformation and is the cause of the lethal effect of externally sensitized photodynamic action. 相似文献
8.
Acridine spin labels as probes for nucleic acids. 总被引:2,自引:0,他引:2
Adridine spin labels, 4-[9-(6-chloro-2-methoxy)-acridylamino]- 2,2,6,6-tetramethyl-1-piperidinyloxy (I) and 4-(9-acridylamino)- 2,2,6,6-tetramethyl-1-piperidinyloxy (II), have been synthesized and their interaction with nucleic acids studied by means of electron spin resonance (ESR). The ESR spectra of labels I and II in the presence of calf thymus DNA were characteristic of highly immobilized nitroxide radicals with maximum hyperfine splittings (2Tˌˌ) of 58.7 and 55.5 G, respectively. The melting temperature (Tm) of DNA, determined in the presence of labels I and II by the ESR technique, were closely similar to those obtained by spectrophotometric methods. The ESR spectrum of label I bound to calf liver RNA and yeast RNA indicated that the nitroxide group of this label was highly mobile. These results suggest that spin labels I and II are suitable noncovalent probes for nucleic acids. 相似文献
9.
The topography of the active sites of native horseradish peroxidase and manganic horseradish peroxidase has been studied with the aid of a spin-labeled analog of benzhydroxamic acid (N-(1-oxyl-2,2,5,5-tetramethylpyrroline-3-carboxy)-p-aminobenzhydroxamic acid). The optical spectra of complexes between the spin-labeled analog of benzhydroxamic acid and Fe3+ or Mn3+ horseradish peroxidase resembled the spectra of the corresponding enzyme complexes with benzhydroxamic acid. Electron spin resonance (ESR) measurement indicated that at pH 7 the nitroxide moiety of the spin-labeled analog of benzhydroxamic acid became strongly immobilized when this label bound to either ferric or manganic horseradish peroxidase. The titration of horseradish peroxidase with the spin-labeled analog of benzhydroxamic acid revealed a single binding site with association constant Ka approximately 4.7 . 10(5) M-1. Since the interaction of ligands (e.g. F-, CN-) and H2O2 with horseradish peroxidase was found to displace the spin label, it was concluded that the spin label did not indeed bind to the active site of horseradish peroxidase. At alkaline pH values, the high spin iron of native horseradish peroxidase is converted to the low spin form and the binding of the spin-labeled analog of benzhydroxamic acid to horseradish peroxidase is completely inhibited. From the changes in the concentration of both bound and free spin label with pH, the pK value of the acid-alkali transition of horseradish peroxidase was found to be 10.5. The 2Tm value of the bound spin label varied inversely with temperature, reaching a value of 68.25 G at 0 degree C and 46.5 G at 52 degrees C. The dipolar interaction between the iron atom and the free radical accounted for a 12% decrease in the ESR signal intensity of the spin label bound to horseradish peroxidase. From this finding, the minimum distance between the iron atom and nitroxide group and hence a lower limit to the depth of the heme pocket of horseradish peroxidase was estimated to be 22 A. 相似文献
10.
The applicability of a new steroidal spin label, 3-oxo-androstan-17 beta-yl-(2",2",6",6"-tetramethyl-N-oxyl) piperidyl butan-1',4'-dioate, in studying the phase transition properties of model membrane L-alpha-dipalmitoyl phosphatidyl choline (DPPC) in the presence and absence of drugs has been explored. Its synthesis and characterization has been described herein. Besides, the localization of this spin label in lipid liposomes has been studied using electron spin resonance (ESR), differential scanning calorimetry (DSC) and 1H and 31P NMR spectroscopic techniques. The label has also been used to study the permeability of epinephrine into membrane. The results show that the spin label has a good potential as a spin probe in the study of biomembranes. 相似文献
11.
The spin-label method was used for structural study of different subclasses of human immunoglobulin A. The spin label was incorporated into the protein part, as well as into carbohydrates of the IgA molecules. Well resolved outer wide extrema were characteristic of the ESR spectra of IgA spin-labeled at the protein moiety. ESR spectra of IgA tagged at carbohydrates reflected moderately immobilized rotation of the spin label. Dependencies of the parameters of ESR spectra of spin-labeled IgA1 and IgA2 upon viscosity at constant temperature have been investigated and a quantitative analysis of the isotherms was carried out. Spin-labeled oligosaccharide chains of IgA2 possessed great freedom of rotation. At least some of IgA1 oligosaccharides were closely attached to the protein moiety. Both proteins under study have shown flexible structure. The Fc fragment of IgA1 molecule appeared to have a rigid structure. 相似文献
12.
Analysis of existing models of spin label movement during spin labeling of biological macromolecules
G N Bondarev V V Isaev-Ivanov L S Isaeva-Ivanova A R Kle?ner V N Krymov 《Molekuliarnaia biologiia》1987,21(6):1655-1663
The spin-labeled bovine serum albumin and IgG were studied in search of an experimental approach for comparison of different models of rotational mobility of spin label. These models are: the model of isotropic motion of spin label together with the macromolecule (IM); the model of highly anisotropic motion of spin label (HAM); and the model of slow isotropic motion of label around the binding site (SIML). The experimental spectra were measured on a common X-band ESR spectrometer and on the unique 140 GHZ (lambda = 2 mm) ESR spectrometer under the same conditions. Theoretical spectra were computer-calculated according to Freed's theory. We have found, that the results of temperature-viscosity experiments in X-band are contradictory to the model of IM both for the BSA and IgG species. The models of HAM and SIML for the BSA give identical X-band spectra. The bovine serum albumin spectra in the 2 mm region strongly contradict to the assumptions of the HAM model. Also, the SIML model fails to describe the experimental spectra in terms of isotropic motion of the spin label around the binding site. X-band spectra of IgG can not be explained by the SIML model, while the same spectra in the 2 mm region can not be explained by the HAM model. 相似文献
13.
Hedin EM Høyrup P Patkar SA Vind J Svendsen A Fransson L Hult K 《Biochemistry》2002,41(48):14185-14196
The binding orientation of the interfacially activated Thermomyces lanuginosa lipase (TLL, EC 3.1.1.3) on phospholipid vesicles was investigated using site-directed spin labeling and electron spin resonance (ESR) relaxation spectroscopy. Eleven TLL single-cysteine mutants, each with the mutation positioned at the surface of the enzyme, were selectively spin labeled with the nitroxide reagent (1-oxyl-2,2,5,5-tetramethyl-Delta(3)-pyrroline-3-methyl) methanethiosulfonate. These were studied together with small unilamellar vesicles (SUV) consisting of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylglycerol (POPG), to which TLL has previously been shown to bind in a catalytically active form [Cajal, Y., et al. (2000) Biochemistry 39, 413-423]. The orientation of TLL with respect to the lipid membrane was investigated using a water-soluble spin relaxation agent, chromium(III) oxalate (Crox), and a recently developed ESR relaxation technique [Lin, Y., et al. (1998) Science 279, 1925-1929], here modified to low microwave amplitude (<0.36 G). The exposure to Crox for the spin label at the different positions on the surface of TLL was determined in the absence and presence of vesicles. The spin label at positions Gly61-Cys and Thr267-Cys, closest to the active site nucleophile Ser146 of the positions analyzed, displayed the lowest exposure factors to the membrane-impermeable spin relaxant, indicating the proximity to the vesicle surface. As an independent technique, fluorescence spectroscopy was employed to measure fluorescence quenching of dansyl-labeled POPG vesicles as exerted by the protein-bound spin labels. The resulting Stern-Volmer quenching constants showed excellent agreement with the ESR exposure factors. An interfacial orientation of TLL is proposed on the basis of the obtained results. 相似文献
14.
Site-directed mutagenesis was used to produce 27 single cysteine mutants of bacteriophage M13 major coat protein spanning the whole primary sequence of the protein. Single-cysteine mutants were labeled with nitroxide spin labels and incorporated into phospholipid bilayers with increasing acyl chain length. The SDSL is combined with ESR and CD spectroscopy. CD spectroscopy provided information about the overall protein conformation in different mismatching lipids. The spin label ESR spectra were analyzed in terms of a new spectral simulation approach based on hybrid evolutionary optimization and solution condensation. This method gives the residue-level free rotational space (i.e., the effective space within which the spin label can wobble) and the diffusion constant of the spin label attached to the protein. The results suggest that the coat protein has a large structural flexibility, which facilitates a stable protein-to-membrane association in lipid bilayers with various degrees of hydrophobic mismatch. 相似文献
15.
The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed. 相似文献
16.
Frkanec R Noethig-Laslo V Vranesić B Mirosavljević K Tomasić J 《Biochimica et biophysica acta》2003,1611(1-2):187-196
The interaction of immunostimulating compounds, the peptidoglycan monomer (PGM) and structurally related adamantyltripeptides (AdTP1 and AdTP2), respectively, with phospholipids in liposomal bilayers were investigated by electron paramagnetic resonance spectroscopy. (1). The fatty acids bearing the nitroxide spin label at different positions along the acyl chain were used to investigate the interaction of tested compounds with negatively charged multilamellar liposomes. Electron spin resonance (ESR) spectra were studied at 290 and 310 K. The entrapment of the adamantyltripeptides affected the motional properties of all spin labelled lipids, while the entrapment of PGM had no effect. (2). Spin labelled PGM was prepared and the novel compound bearing the spin label attached via the amino group of diaminopimelic acid was chromatographically purified and chemically characterized. The rotational correlation time of the spin labelled molecule dissolved in buffer at pH 7.4 was studied as a function of temperature. The conformational change was observed above 300 K. The same effect was observed with the spin labelled PGM incorporated into liposomes. Such effect was not observed when the spin labelled PGM was studied at alkaline pH, probably due to the hydrolysis of PGM molecule. The study of possible interaction with liposomal membrane is relevant to the use of tested compounds incorporated into liposomes, as adjuvants in vivo. 相似文献
17.
18.
A quantitative determination of maleimide spin label (MAL) binding in oxi and met hemoglobin (Hb) and bovine serum albumin are investigated using double integration to the ESR signal. This determination permitted the observation that a considerable fraction of MAL is reduced, losing its paramagnetism. Experiments using the same spin label with myoglobin and Hb with blocked-SH groups, where reduction was not observed, indicate the involvement of SH groups in the process. The 4-hydroxy-2,2,6,6-tetramethylpiperidino-1-oxyl spin label (which is not able to bind in the SH group) is reduced too, but the dependence on the molar ratio is different in comparison with the MAL case. In both cases the reduction percentage depends on the molar ratio spin label to protein and to the protein concentration. In order to obtain the total SH groups labeled (two in the Hb case) it is necessary to use an excessive amount of label (around 18:1) in the 0.5 mM Hb concentration. 相似文献
19.
Riske KA Nascimento OR Peric M Bales BL Lamy-Freund MT 《Biochimica et biophysica acta》1999,1418(1):133-146
A small, highly aqueous soluble, deuterated, cationic spin label, 4-trimethylammonium-2,2,6,6-tetramethylpiperidine-d17-1-oxyl iodide (dCAT1), was used to directly monitor the negatively charged DMPG vesicle surface in order to test a recent suggestion (Riske et al., Chem. Phys. Lipids, 89 (1997) 31-44) that alterations in the surface potential accompanied apparent phase transitions observed by light scattering. The temperature dependence of the label partition between the lipid surface and the aqueous medium indicated an increase in the surface potential at the gel to liquid-crystal transition, supporting the previous suggestion. Results at the phase transition occurring at a higher temperature were less definitive. Although some change in the dCAT1 ESR spectra was observed, the interpretation of the phenomena is still rather unclear. DMPG surface potentials were estimated from the dCAT1 partition ratios (surface label moles/total label moles), using a simple two-sites model, where the electrostatic potential is zero everywhere but at the vesicle surface, and the interaction between the spin label and the membrane surface is chiefly electrostatic. The Gouy-Chapman-Stern model predicts surface potentials similar to those observed, although the measured decrease in the surface potential with ionic strength is somewhat steeper than that predicted by the model. 相似文献