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PubMed2000

The yeast mitochondrial citrate transport protein: determination of secondary structure and solvent accessibility of transmembrane domain IV using site-directed spin labeling.

To explore the spatial organization and functional dynamics of the citrate transport protein (CTP), a nitroxide scan was carried out along 22 consecutive residues within the fourth transmembrane domain (TMDIV). This domain has been implicated as being of unique importance to the CTP mechanism due to (i) the presence of two intramembranous positive charges that are essential for CTP function and (ii) the existence of a transmembrane aqueous surface within this domain which likely corresponds to a portion of the citrate translocation pathway. The sequence-specific variation in the mobilities of the introduced nitroxides and their accessibilities to molecular O(2) reveal an alpha-helical conformation along the sequence. The accessibilities to NiEDDA are out of phase with accessibilites to O(2), indicating that one face of the helix is solvated by the lipid bilayer while the other is solvated by an aqueous environment. A gradient of NiEDDA accessibility is observed along the helix surface facing the aqueous phase, and the EPR spectral line shapes at these sites indicate considerable motional restriction. In the context of the model where TMDIV lines the translocation pathway, these data suggest a barrier to passive diffusion through the pathway. This paper reports the first use of site-directed spin labeling to study mitochondrial transporter structure.

Amino Acid Sequence↗
PubMed1978

Spin-label studies on the sulfhydryl environment in bovine plasma albumin. 2. The neutral transition and the A isomer.

Since we were able to demonstrate that the sulfhydryl group is located in the crevice which opens during the N--F transition (Cornell, C. N., & Kaplan, L. J. (1978) Biochemistry 17 (preceding paper in this issue), the investigation was extended by characterizing the environment during the N--B transition and in the A isomer. The results indicate that the N--B and N--F transitions are very similar in that the sulfhydryl group moves from a restricted to unhindered environment during both. The use of molecular dipstick technique further demonstrated the similarity between the F and A forms. However, since A is a covalently stabilized form of albumin after a pH dependent transition, it retains its properties during subsequent pH changes rather than reverting to the N form. We were thus able to titrate spin-labeled A through the pH range of the acidic transitions without detecting the N--F transition. Isoelectric focusing analysis of A generated during alkaline aging and purified by SP-Sephadex chromatography indicates that it is a mixture of a small number of albumin forms rather than the large number of components once thought to be formed during aging.

Chemical Phenomena↗
PubMed1995

An NMR and spin label study of the effects of binding calcium and troponin I inhibitory peptide to cardiac troponin C.

The paramagnetic relaxation reagent, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (HyTEMPO), was used to probe the surface exposure of methionine residues of recombinant cardiac troponin C (cTnC) in the absence and presence of Ca2+ at the regulatory site (site II), as well as in the presence of the troponin I inhibitory peptide (cTnIp). Methyl resonances of the 10 Met residues of cTnC were chosen as spectral probes because they are thought to play a role in both formation of the N-terminal hydrophobic pocket and in the binding of cTnIp. Proton longitudinal relaxation rates (R1's) of the [13C-methyl] groups in [13C-methyl]Met-labeled cTnC(C35S) were determined using a T1 two-dimensional heteronuclear single- and multiple-quantum coherence pulse sequence. Solvent-exposed Met residues exhibit increased relaxation rates from the paramagnetic effect of HyTEMPO. Relaxation rates in 2Ca(2+)-loaded and Ca(2+)-saturated cTnC, both in the presence and absence of HyTEMPO, permitted the topological mapping of the conformational changes induced by the binding of Ca2+ to site II, the site responsible for triggering muscle contraction. Calcium binding at site II resulted in an increased exposure of Met residues 45 and 81 to the soluble spin label HyTEMPO. This result is consistent with an opening of the hydrophobic pocket in the N-terminal domain of cTnC upon binding Ca2+ at site II. The binding of the inhibitory peptide cTnIp, corresponding to Asn 129 through Ile 149 of cTnI, to both 2Ca(2+)-loaded and Ca(2+)-saturated cTnC was shown to protect Met residues 120 and 157 from HyTEMPO as determined by a decrease in their measured R1 values. These results suggest that in both the 2Ca(2+)-loaded and Ca(2+)-saturated forms of cTnC, cTnIp binds primarily to the C-terminal domain of cTnC.

Amino Acid Sequence↗
PubMed2003

The reduction of a nitroxide spin label as a probe of human blood antioxidant properties.

The kinetics of reduction of the radical R*, 5-dimethylaminonaphthalene-1-sulfonyl-4-amino-2,2,6,6-tetramethyl-1-piperidine-oxyl by blood and its components were studied using the EPR technique. The results demonstrate that R* is adsorbed to the outer surface of the membrane and does not penetrate into the erythrocytes. A series of control experiments in PBS demonstrate that ascorbate is the only natural reducing agent that reacts with R*. The observed first order rate of disappearance of the nitroxide radical k, is: k(blood) > k(eryth) > k(plasma) and k(blood) approximately = k(eryth) + k(plasma). The results demonstrate that: a. The erythrocytes catalyze the reduction of R* by ascorbate. b. The rate of reduction of the radical is high though it does not penetrate the cells. c. In human erythrocytes there is an efficient electron transfer route through the cell membrane. d. The study points out that R* is a suitable spin label for measuring the reduction kinetics and antioxidant capacity in blood as expressed by reduction by ascorbate.

Antioxidants↗
PubMed1982

Orientation of spin labels attached to cross-bridges in contracting muscle fibres.

Electron micrographs showing different cross-bridge orientations in different states of muscle fibres, and X-ray diffraction patterns indicating axial cross-bridge disorder in contracting muscle first suggested that force generation in the contracting muscle involved a change in orientation of the myosin heads that form cross-bridges between thick and thin filaments. This has been supported by subsequent work; the myosin molecule has the required flexibility for changes in orientation. The orientation of muscle tryptophans and of probes attached to the myosin heads of permeable muscle fibres depends on the state of the muscle. Recently, fluorescence polarization fluctuations and time-resolved X-ray diffraction patterns have suggested that cross-bridges of a contracting muscle can rotate. We have used electron paramagnetic resonance (EPR) spectroscopy to monitor the orientation of spin labels attached specifically to a reactive sulphydryl on the myosin heads in glycerinated rabbit psoas skeletal muscle. Previously, it has been shown that the paramagnetic probes are highly ordered in rigor muscle, with a nearly random angular distribution in relaxed muscle. We show here that during the generation of isometric tension, approximately 80% of the probes display a random angular distribution as in relaxed muscle while the remaining 20% are highly oriented at the same angle as found in rigor muscle. These findings indicate that a domain of the myosin head does not change orientation during the power stroke of the contractile interaction.

Actins↗
PubMed2002

Overexpression, purification, and site-directed spin labeling of the Nramp metal transporter from Mycobacterium leprae.

It has long been recognized that the pathogenicity of a broad range of intracellular parasites depends on the availability of transition metal ions, especially iron. Nramp1 (natural resistance-associated macrophage protein 1), a proton-coupled divalent metal ion transporter, has been identified as a controlling factor in the resistance or susceptibility to infection with a diverse range of intracellular pathogens such as Toxoplasma, Salmonella, Mycobacterium, and Leishmania. The role of divalent metal ion transport is even more compelling given the existence of Nramp homologs in several intracellular parasites, such as mycobacteria. We have confirmed the functional homology of the Nramp homologue from Mycobacterium leprae by using a yeast complementation assay for divalent cation uptake. To facilitate a concerted biochemical and structural analysis of this important class of transporters, the M. leprae Nramp was expressed in Escherichia coli. Dual affinity tags were engineered at the N and C termini to allow for isolation of full-length protein at >95% purity. Site-directed spin labeling of Cys-299 reveals a flexible hinge-like domain. A weak dipolar interaction is detected between the nitroxide and paramagnetic transition ions, indicating this position is approximately 19 A from the nearest high affinity binding site.

Base Sequence↗
PubMed1995

Distance determination in proteins using designed metal ion binding sites and site-directed spin labeling: application to the lactose permease of Escherichia coli.

As shown in the accompanying paper, the magnetic dipolar interaction between site-directed metal-nitroxide pairs can be exploited to measure distances in T4 lysozyme, a protein of known structure. To evaluate this potentially powerful method for general use, particularly with membrane proteins that are difficult to crystallize, both a paramagnetic metal ion binding site and a nitroxide side chain were introduced at selected positions in the lactose permease of Escherichia coli, a paradigm for polytopic membrane proteins. Thus, three individual cysteine residues were introduced into putative helix IV of a lactose permease mutant devoid of native cysteine residues containing a high-affinity divalent metal ion binding site in the form of six contiguous histidine residues in the periplasmic loop between helices III and IV. In addition, the construct contained a biotin acceptor domain in the middle cytoplasmic loop to facilitate purification. After purification and spin labeling, electron paramagnetic resonance spectra were obtained with the purified proteins in the absence and presence of Cu(II). The results demonstrate that positions 103, 111, and 121 are 8, 14, and > 23 A from the metal binding site. These data are consistent with an alpha-helical conformation of transmembrane domain IV of the permease. Application of the technique to determine helix packing in lactose permease is discussed.

Amino Acid Sequence↗
PubMed1999

Structural features and light-dependent changes in the sequence 59-75 connecting helices I and II in rhodopsin: a site-directed spin-labeling study.

Twenty-one single-cysteine substitution mutants were prepared in the sequence 56-75 between transmembrane helices I and II at the cytoplasmic surface of bovine rhodopsin. Each mutant was reacted with a sulfhydryl-specific reagent to produce a nitroxide side chain. The electron paramagnetic resonance of the labeled proteins in dodecyl maltoside solution was analyzed to provide the relative mobility and accessibility of the nitroxide side chain to both polar and nonpolar paramagnetic reagents. The results indicate that the hydrophobic-water interface of the micelle intersects helices I and II near residues 64 and 71, respectively. Thus, the sequence 64-71 is in the aqueous phase, while 56-63 and 72-75 lie in the transmembrane helices I and II, respectively. The lipid-facing surfaces on transmembrane helices I and II near the cytoplasmic surface correspond to approximately 180 degrees and 90 degrees of arc on the helical surfaces, respectively. Photoactivation of rhodopsin produced changes in structure in the region investigated, primarily around helix II. However, these changes are much smaller than those noted by spin labels in helix VI (Altenbach, C., Yang, K., Farrens, D., Farahbakhsh, Z., Khorana, H. G., and Hubbell, W. L. (1996) Biochemistry 35, 12470).

Amino Acid Sequence↗
PubMed1991

Precipitation and 13C-NMR relaxation enhancement measurements of the interactions of bile acids with synthetic cationic bile acid derivatives, and with spin labelled fatty acids.

In an investigation of novel potential bile acid sequestrants, the affinities of the sodium salts of the glycine and taurine conjugates of naturally occurring bile acids (cholate, deoxycholate, chenodeoxycholate and lithocholate) for several cationic ammonium bile acid derivatives have been investigated by measurements of the extent to which the derivatives are able to precipitate the bile acids. This is roughly proportional to the lipophilicity of the interacting species. Thus, amino and ammonium derivatives of cholic acid do not precipitate taurocholate or glycocholate to any great extent, whereas ammonium derivatives of deoxycholate and lithocholate are much more effective. To complement the precipitation measurements, high resolution 13C-NMR has been applied to investigate the weaker interactions between the ammonium cholate derivative and glycocholate, glycodeoxycholate and glycochenodeoxycholate. Addition of either of the latter two bile acids to the cationic ammonium compound results in considerable broadening of the 13C resonances of both species, indicating the formation of relatively rigid structures. In addition, we have used T2 relaxation enhancement induced by spin-labelled fatty acids to examine the mechanism of interaction with bile acids of amphiphilic anions, which might compete with bile acids for sites on bile acid sequestrants. Low concentrations of 16-DOXY L-Stearate dramatically broaden the 13C-NMR resonances of deoxycholate carbons 19, 18 and 7 in particular, while 5-DOXY L-Stearate exerts much less specific effects. These results have been incorporated into a snapshot model of bile acid-fatty acid interactions.

Bile Acids and Salts↗
PubMedDate not supplied

Spin labeling and kinetic studies of a membrane-immobilized proteolytic enzyme.

Membrane-based bioreactors can greatly influence the rate and extent of chemical reactions and consequently lower the costs associated with the corresponding engineering processes. However, in order to progress in this area, greater understanding of the relationship of the structure and function of bioreactor systems is required. In this study, a proteolytic enzyme, papain (EC 3.4.22.2), was covalently coupled onto the surface of a vinyl alcohol/vinyl butyral copolymer (PVB) membrane employing either glutaraldehyde (GA) or 1,1'-carbonyldiimidazole (CDI). Various kinetic and performance properties of the immobilized papain were studied. It was found that these characteristics of the membrane-bound papain depended on the immobilization method. The CDI-immobilized papain bioreactor was used, although the apparent Michaelis constant, Km, of the CDI-immobilized papain was larger than that of the GA-immobilized enzyme. In separate experiments, a six-carbon spacer was also used between the membrane support and the covalently-linked enzyme. It was found that the insertion of the spacer reduced the disturbance of the enzyme system, resulting in a decreased Km, which was now closer to the value for the free enzyme. Electron paramagnetic resonance (EPR) techniques of spin labeling were used for the first time to examine the conformational change and the active site structure of an enzyme covalently immobilized to a membrane. The structural changes of the active site of papain upon immobilization with and without a spacer were in agreement with the functional properties of the enzyme.

Binding Sites↗
PubMed1991

2.9 A resolution structure of an anti-dinitrophenyl-spin-label monoclonal antibody Fab fragment with bound hapten.

The crystal structure of the Fab fragment of the murine monoclonal anti-dinitrophenyl-spin-label antibody AN02 complexed with its hapten has been solved at 2.9 A resolution using a novel molecular replacement method. Prior to translation searches, a large number of the most likely rotation function solutions were subjected to a rigid body refinement against the linear correlation coefficient between intensities of observed and calculated structure factors. First, the overall orientation of the search model and then the orientations and positions of the four Fab domains (VH, VL, CH1 and CL) were refined. This procedure clearly identified the correct orientation of the search model. The refined search model was then subjected to translation searches which unambiguously determined the enantiomer and position in the unit cell of the crystal. The successful search model was refined 2.5 A crystal structure of the Fab fragment of HyHel-5 from which non-matching residues in the variable domains had been removed. HyHel-5 is a murine monoclonal antibody whose heavy and light chains are of the same subclass (gamma 1, kappa, respectively) as AN02. After molecular replacement the structure of the AN02 Fab has been refined using simulated annealing in combination with model building and conjugate gradient refinement to a current crystallographic R-factor of 19.5% for 12,129 unique reflections between 8.0 and 2.9 A. The root-mean-square (r.m.s.) deviation from ideal bond lengths is 0.014 A, and the r.m.s. deviation from ideal bond angles is 3.1 degrees. The electron density reveals the hapten sitting in a pocket formed by the loops of the complementarity determining region. The dinitrophenyl ring of the hapten is sandwiched between the indole rings of Trp96 of the heavy-chain and Trp91 of the light-chain. The positioning of the hapten and general features of the combining site are in good agreement with the results of earlier nuclear magnetic resonance experiments.

Animals↗
PubMed2005

Effects of oxygen saturation on BOLD and arterial spin labelling perfusion fMRI signals studied in a motor activation task.

Effects of oxygen availability on blood oxygenation level dependent (BOLD) and arterial spin labelling (ASL) perfusion functional magnetic resonance imaging (fMRI) signal changes upon motor activation were studied. Mild hypoxic hypoxia was induced by reducing the inspired oxygen content (FIO(2)) to 12%, decreasing blood oxygen saturation (Y) from 0.99 +/- 0.01 to 0.85 +/- 0.03. The fMRI signal characteristics were determined during finger tapping. BOLD activation volume decreased as a function of declining Y in the brain structures involved in execution of the motor task, however, the BOLD signal increase in activated parenchyma was not influenced by Y. ASL fMRI showed that the baseline CBF of 61.8 +/- 3.6 ml/100 g/min was not affected by hypoxic hypoxia. Similar to the BOLD fMRI, the volume of motor cortex areas displaying increase in perfusion by ASL fMRI decreased, but the signal change due to perfusion increase was not influenced in hypoxia. The present fMRI results show distinct patterns of haemodynamic and metabolic responses in the brain to motor task between normoxia and hypoxia. On one hand, neither BOLD nor ASL fMRI signal changes are influenced by hypoxia during motor activation. On the other hand, hypoxia attenuates increase in both BOLD and perfusion fMRI signals upon finger tapping from the levels determined in normoxia. These observations indicate that haemodynamic and metabolic responses may be heterogeneous in brain during execution of motor functions in mild hypoxia.

Adult↗
PubMed1991

Protein heat denaturation and study of membrane lipid-protein interactions by spin label ESR.

Spinach chloroplast membranes labelled with stearic acid-spin probe-bearing nitroxyl (label) moiety at 5th, 9th, 12th, 13th, 14th or 16th carbon locations with respect to the carboxylic group of stearic acid were studied (in the dark) by electron spin resonance (ESR) spectroscopy. Spectra were recorded at sample temperatures of 5, 30 and 67 degrees C. After heat denaturation of the membrane proteins for 5 min at 67 degrees C, the spectra were re-recorded at 30 and 5 degrees C for comparison. The results unequivocally show that membrane lipid fatty-acyl chains become substantially more rigid after protein heat-denaturation. The data throw light on the degree of lipid-protein interactions at various microlocations along the length of fatty-acyl chains of the membrane lipid matrix.

Chloroplasts↗
PubMed1985

S-100 protein-induced changes in the physical state of synaptosomal particulate fractions as monitored by spin labels.

This report documents changes in the physical state of synaptosomal particulate fractions (SYN) upon binding of S-100 protein, as monitored by spin labels. Studies were conducted on SYN labeled with either 5-doxylstearic acid or 16-doxylstearic acid, which probe the polar region and the hydrophobic core of the lipid bilayer, respectively. S-100 perturbs to some extent both the polar surface and the hydrophobic core of SYN in a time- and temperature-dependent manner. Ca2+ is essential for S-100 to perturb the membranes. K+ almost completely inhibits the S-100 perturbing effect if present in the incubation medium, but fails to reverse the S-100-induced changes if added after S-100 has interacted with SYN. At room temperature and below, the overall S-100 effect registered after about 30 min of association of the protein with SYN is an increase in the fluidity of both the surface and the interior of the membranes. Spectra registered at intervals at room temperature indicate that the S-100 perturbing effect on the membrane surface is practically monophasic, consisting of an increase in fluidity, while that on the membrane interior is multiphasic, consisting of a decrease in fluidity during the first 10 min of association, followed by an increase in fluidity during the subsequent 20 min and a return to starting values during the second 30 min of association. Around 37 degrees C, on the contrary, a decrease in fluidity is registered in both regions. The data suggest that S-100 induces a spatial rearrangement of membrane components (proteins) involved in the specific binding and/or partially penetrates into the lipid bilayer.

Animals↗
PubMed1985

[Use of spin-labelled substrate analogs for a comparative study of microsomal cytochrome P-450 and P-448].

The previously described, iodine-labeled alkylating stable nitroxyl radicals located at different distances between the N-O. group and the iodine atom were used for a comparative study of the structure of microsomal cytochromes P-450 and P-448 active centers. The radicals were shown to change the optical spectra of Fe3+ located in the active site of the enzyme that are similar to those induced by cytochrome P-450 substrates. Some differences in the type of the radicals binding to control, phenobarbital- and 3-methylcholanthrene-induced microsomes were revealed. The alkylating radical substrate analogs covalently bound to microsomal cytochrome P-450 in the vicinity of the active center, resulting in the inhibition of oxidation of type I and II substrates (e. g., aniline and naphthalene). The value of the spectral binding constant (Ks) for naphthalene in the presence of the radical covalently bound to the cytochrome P-450 active center showed a tendency to increase. Using the ESR technique, the interaction between Fe3+ and the radical localized in the active site of cytochrome P-450 was demonstrated. The contribution of Fe3+ to the relaxation of the radicals covalently bound to cytochrome P-450 was evaluated from the values of the spin label ESR spectra saturation curves at 77K. The distances between the N-O. group of these radicals and Fe3+ in the enzyme active center for the three types of microsomes were determined. The data obtained point to structural peculiarities of the active center of cytochrome P-450, depending on the microsomal type.

Aniline Compounds↗
PubMed2001

Cerebral perfusion during anesthesia with fentanyl, isoflurane, or pentobarbital in normal rats studied by arterial spin-labeled MRI.

The influence of anesthetic agents on cerebral blood flow (CBF) was tested in normal rats. CBF is quantified with arterial spin-labeled MRI in rats anesthetized with either an opiate (fentanyl), a potent inhalation anesthetic agent (isoflurane), or a barbiturate (pentobarbital) using doses commonly employed in experimental paradigms. CBF values were found to be about 2.5-3 times lower in most regions analyzed during anesthesia with either fentanyl (with N(2)O/O(2)) or pentobarbital vs. isoflurane (with N(2)O/O(2)), in agreement with findings utilizing invasive measurement techniques. CBF was heterogeneous in rats anesthetized with isoflurane (with N(2)O/O(2)), but relatively homogeneous in rats anesthetized with either fentanyl (with N(2)O/O(2)) or pentobarbital, also in agreement with studies using other techniques. Magn Reson Med 46:202-206, 2001.

Anesthesia, General↗
PubMed2004

Formation of weak polyelectrolyte multilayers studied by spin labeling.

Multilayers of alternately adsorbing poly(allylamine) (PAH) and poly(acrylic acid) (PAA) of opposite charges on silica have been studied by the spin labeling technique, as a function of pH. The two polyelectrolytes have been labeled independently by a nitroxide free radical. Its electron paramagnetic resonance spectrum is mainly sensitive to the local Brownian motion and shows lines typical of two different environments, namely, loops protruding in solution with a fast motion and trains adsorbed on the solid with a hindered motion. These two parts have been evaluated for each of the polymer layers separately, and the thickness of the coatings has been described more precisely by characterizing the four contributions existing, for example, for a bilayer. Complexation is demonstrated by the loss of loops and tails belonging to the first polyelectrolyte. The overall picture emerging from the data is explained in terms of compensation of charges and entropy of confinement.

Journal Article↗
PubMed1998

Effects of spin labeled derivatives of podophyllotoxin on cell cycle and macromolecular synthesis in human lymphoid leukemia Molt 4B cells.

AIM: To examine the effect of the spin labeled derivatives of podophyllotoxin, N-podophyllic acid-N"-[4-(2,2,6,6-tetramethyl-1-piperidinyloxy)] thiosemicarbazide (GP4) and 4-[4"-(2",2",6",6"-tetramethyl-1"-piperidinyloxy) amino]-4'-demethylepipodophyllotoxin (GP7) on the cell cycle and macromolecular synthesis of human lymphoid leukemia Molt 4B cells in vitro. METHODS: MTT assay, 3H incorporation, and flow cytometer were used. RESULTS: GP4, GP7, and etoposide 0.02-100 mmol.L-1 cultured for 48 h inhibited the proliferation of human lymphoid leukemia Molt 4B cells. IC50 values of GP4, GP7, and etoposide were 0.11, 4.7, and 1.6 mmol.L-1, respectively. DNA and protein syntheses were obviously suppressed by GP4, GP7, and etoposide 10 mmol.L-1 for 48 h. After Molt 4B cells were treated with GP4, GP7, and etoposide 10 mmol.L-1 for 6 and 12 h, the mitotic index was increased by GP4 and reduced by GP7 and etoposide. According to flow cytometric BrdU/DNA analysis, GP4 slightly retarded S phase and mainly arrested cell cycle progression in G2/M phase, whereas GP7 similar to etoposide induced cells accumulated at S phase and retarded the cells in G2 phase. CONCLUSION: GP4 and GP7 inhibit the proliferation of Molt 4B cells, but the mechanisms are different.

Antineoplastic Agents, Phytogenic↗