RESUMO
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.
Assuntos
Colicinas/análise , Escherichia coli/química , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Escherichia coli/citologia , Modelos Moleculares , Multimerização Proteica , Marcadores de SpinRESUMO
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.
Assuntos
Ligação de Hidrogênio , Proteínas de Membrana/química , Óxidos de Nitrogênio/química , Marcadores de Spin , Espectroscopia de Ressonância de Spin EletrônicaRESUMO
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.
Assuntos
Antioxidantes/química , Antioxidantes/farmacologia , Cardiolipinas/metabolismo , Hidroquinonas/química , Peroxidação de Lipídeos/efeitos dos fármacos , Lipossomos/metabolismo , Animais , Bovinos , Estrutura Molecular , Ubiquinona/análogos & derivados , Ubiquinona/farmacologiaRESUMO
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.
Assuntos
Proteínas de Bactérias/química , Proteínas de Transporte/química , Corynebacterium glutamicum/química , Cristalografia por Raios X , Espectroscopia de Ressonância de Spin Eletrônica , Estrutura Terciária de Proteína , SimportadoresRESUMO
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.
Assuntos
Simulação por Computador , Espectroscopia de Ressonância de Spin Eletrônica , Simulação de Dinâmica Molecular , Vinculina/química , Conformação Proteica , Marcadores de SpinRESUMO
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.
Assuntos
Portadores de Fármacos/química , Hipoglicemiantes/química , Insulina/química , Sílica Gel/química , Animais , Bovinos , Composição de Medicamentos , Espectroscopia de Ressonância de Spin Eletrônica , Humanos , Modelos Moleculares , Nanopartículas/química , Tamanho da Partícula , Transição de Fase , Porosidade , Estrutura Secundária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Marcadores de SpinRESUMO
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.
Assuntos
Bacteriorodopsinas/química , Conformação Proteica , Bacteriorodopsinas/genética , Espectroscopia de Ressonância de Spin Eletrônica , Luz , Modelos Moleculares , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Marcadores de SpinRESUMO
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.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Animais , Células CHO , Células COS , Chlorocebus aethiops , Cricetinae , Cricetulus , Detergentes/química , Humanos , Microscopia Eletrônica , Técnicas de Patch-Clamp , Poliestirenos/química , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
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.
Assuntos
Metemoglobina/química , Tiocianatos/química , Animais , Cristalização , Cavalos , Cinética , Soluções , Análise Espectral , TemperaturaRESUMO
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.
Assuntos
Metemoglobina/ultraestrutura , Animais , Espectroscopia de Ressonância de Spin Eletrônica , Heme , Histidina , Cavalos , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Movimento (Física) , Conformação Proteica , Temperatura , Termodinâmica , ÁguaRESUMO
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.
Assuntos
Metemoglobina/química , Conformação Proteica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Calorimetria/instrumentação , Calorimetria/métodos , Heme/metabolismo , Cavalos , Cinética , Substâncias Macromoleculares , Matemática , Metemoglobina/isolamento & purificação , Modelos Moleculares , Modelos Teóricos , TermodinâmicaRESUMO
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.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Carotenoides , Halorrodopsinas , Transdução de Sinal Luminoso , Movimento (Física) , Natronobacterium/química , Rodopsinas Sensoriais , Marcadores de Spin , Sequência de Aminoácidos , Substituição de Aminoácidos , Proteínas Arqueais/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteriorodopsinas/genética , Cisteína/genética , Cisteína/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Cinética , Luz , Transdução de Sinal Luminoso/efeitos da radiação , Óxidos de Nitrogênio/metabolismo , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Estrutura Secundária de Proteína/efeitos da radiação , Deleção de Sequência , Relação Estrutura-Atividade , Fatores de TempoRESUMO
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.
Assuntos
Aminoácidos/química , Bacteriorodopsinas/química , Bombas de Próton/química , Bacteriorodopsinas/genética , Bacteriorodopsinas/metabolismo , Transporte Biológico , Óxidos N-Cíclicos , Cisteína/genética , Espectroscopia de Ressonância de Spin Eletrônica , Halobacterium salinarum , Cinética , Mesilatos , Modelos Moleculares , Mutação , Oxalatos , Oxigênio , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Bombas de Próton/genética , Bombas de Próton/metabolismo , Espectrofotometria , Marcadores de SpinRESUMO
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.
Assuntos
Transcriptase Reversa do HIV/química , HIV-1/enzimologia , Marcadores de Spin , Cristalografia por Raios X , Cisteína/genética , Cisteína/metabolismo , DNA/química , DNA/metabolismo , Espectroscopia de Ressonância de Spin Eletrônica , Congelamento , Transcriptase Reversa do HIV/antagonistas & inibidores , Transcriptase Reversa do HIV/genética , Transcriptase Reversa do HIV/metabolismo , Ligantes , Modelos Moleculares , Mutagênese Sítio-Dirigida , Conformação de Ácido Nucleico , Conformação Proteica , RNA/química , RNA/metabolismo , Inibidores da Transcriptase Reversa/metabolismo , Soluções , Temperatura , TermodinâmicaRESUMO
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.
Assuntos
Proteínas Repressoras/química , Espectroscopia de Ressonância de Spin Eletrônica , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Marcadores de SpinRESUMO
Site-directed spin labeling of membrane proteins has been used to determine: (1) the topography of the polypeptide chain with respect to the membrane/solution interface, and (2) the identity and orientation of secondary structure in selected regions. These features are deduced from the collision rates of nitroxide side chains with paramagnetic reagents in solution, and the principles of the method are reviewed with reference to bacteriorhodopsin. The dynamics of the nitroxide side chains relative to the backbone reveal tertiary interactions of the labeled site, and provide a promising means of time-resolving conformational changes. This aspect is illustrated by recent studies of structural changes in bacteriorhodopsin during the photocycle. In these experiments, nitroxide side chains were introduced at residues 72, 101 and 105 after replacement of the original residues by cysteine. Upon flash photolysis, the electron paramagnetic resonance spectrum of a nitroxide at 101, but not those at 72 or 105, is time-dependent. The spectral change develops during the decay of the M-intermediate, and reverses upon return to the ground state. The results suggest a movement of the C-D or E-F interhelical loops during the protonation changes of aspartate 96.
Assuntos
Bacteriorodopsinas/química , Bacteriorodopsinas/metabolismo , Conformação Proteica , Sítios de Ligação , Espectroscopia de Ressonância de Spin Eletrônica/métodos , Cinética , Modelos Moleculares , Mutagênese Sítio-Dirigida , Mutação Puntual , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Soluções , Marcadores de Spin , Fatores de TempoRESUMO
A method using nitroxide radical spin labels for determining both the isotropic rotational correlation time tau R and the environmental polarity of the label is described. By means of a least square fitting method, the values of an effective hyperfine tensor A' and of an effective g value tensor g' of randomly oriented spin labels are determined from X-band EPR spectra on the basis of an effective time-independent Hamiltonian. The traces of the tensors deliver the information about the environmental polarity of the label and are not dependent on the rotational correlation time tau R. A new averaging parameter S (tau R), calculated on the basis of the principal values of the tensor A', permits the evaluation of the rotational correlation time tau R in a very wide time range between 10(-10) and 10(-6) s. An application of this method to spin-labeled methemoglobin over a large temperature range and in environments of different polarity is discussed.
Assuntos
Espectroscopia de Ressonância de Spin Eletrônica/métodos , Conformação Molecular , Marcadores de Spin , Matemática , Modelos TeóricosRESUMO
A laser temperature jump apparatus is constructed where the T-jump is achieved by means of the direct absorption of continuous laser radiation of low intensity by a solid sample. The final temperature in the irradiated volume element is reached when the absorbed radiation power equals the dissipation of heat by heat conduction. The time range from the beginning of irradiation to the stationary state depends on the geometry of the irradiated volume element and is less than 10 ms. The heating laser beam is simultaneously used to detect the relaxation to the new chemical equilibrium in the sample. Relaxation processes with relaxation rates between 10(2) s-1 and less than 10(-3) s-1 on samples with volumes less than 10(-3) mm3 may be investigated using this T-jump method. One application of this method is the determination of reaction rates of ligand reactions in hemoglobin single crystals. Rate constants obtained for the reaction of thiocyanate with crystallized horse methemoglobin are presented.
Assuntos
Hemoglobinas , Temperatura Alta , Lasers , Animais , Fenômenos Químicos , Química , Cristalização , Cavalos/sangue , Metemoglobina , Soluções , Termodinâmica , TiocianatosRESUMO
A cavity perturbation method for the absolute determination of the complex permittivity of small samples in the microwave range is developed and tested. Samples with volumes less than 0.4 mm3, for example protein powder or single crystals of macromolecules, may be investigated in a temperature range between 180 and 300 K, using this method. As an application the complex permittivity of hemoglobin single crystals is determined at three frequencies, v = 3.06 GHz, v = 8.76 GHz and v = 17.0 GHz in a temperature range between 180 and 300 K.
Assuntos
Cristalografia/métodos , Hemoglobinas/química , Eletroquímica , Micro-Ondas , Modelos Químicos , Água/análiseRESUMO
The location of cysteines accessible in octopus rhodopsin were characterized by a spin-labeling technique. Two cysteines were found to bind a methanthiosulfonate spin label. One of the spin labels is attached to helix V with the side chain located within the membrane, most probably close to the polar head group region. The second spin label was found to be attached to cysteine 345 in the C terminus. Light-induced reversible electron paramagnetic resonance spectral changes were observed for the spin label attached at position 345. It is concluded that conformational changes occur during the rhodopsin to metarhodopsin transition in the vicinity of the C-terminus position 345.