Sites of interaction of a precursor polypeptide on the export chaperone SecB mapped by site-directed spin labeling

Export of protein into the periplasm of Escherichia coli via the general secretory system requires that the transported polypeptides be devoid of stably folded tertiary structure. Capture of the precursor polypeptides before they fold is achieved by the promiscuous binding to the chaperone SecB. SecB delivers its ligand to export sites through its specific binding to SecA, a peripheral component of the membrane translocon. At the translocon the ligand is passed from SecB to SecA and subsequently through the SecYEG channel. We have previously used site-directed spin labeling and electron paramagnetic resonance spectroscopy to establish a docking model between SecB and SecA. Here we report use of the same strategy to map the pathway of a physiologic ligand, the unfolded form of precursor galactose-binding protein, on SecB. Our set of SecB variants each containing a single cysteine, which was used in the previous study, has been expanded to 48 residues, which cover 49% of the surface of SecB. The residues on SecB involved in contacts were identified as those that, upon addition of the unfolded polypeptide ligand, showed changes in spectral line shape consistent with restricted motion of the nitroxide. We conclude that the bound precursor makes contact with a large portion of the surface of the small chaperone. The sites on SecB that interact with the ligand are compared with the previously identified sites that interact with SecA and a model for transfer of the ligand is discussed.     

Analysis of betaB1-crystallin unfolding equilibrium by spin and fluorescence labeling: evidence of a dimeric intermediate

A central step in understanding lens aging is to characterize the thermodynamic stability of its proteins and determine the consequences of changes in the primary sequence on their folding equilibria. For this purpose, destabilized mutations were introduced in betaB1-crystallin targeting the domain interface within the fold of a subunit. Global unfolding was monitored by tryptophan fluorescence while concomitant structural changes at the dimer interface were monitored by fluorescence and spin labels. Both spectral probes report explicit evidence of multi-state unfolding equilibrium. The biphasic nature of the unfolding curves was more pronounced at higher protein concentration. Distinct shifts in the midpoint of the second transition reflect the population of a dimeric intermediate. This intermediate may be a critical determinant for the life-long stability of the beta-crystallins and has important consequences on interactions with alpha-crystallin.     

Saturation transfer EPR measurements of the rotational motion of a strongly immobilized ouabain spin label on renal Na, K-ATPase

The rotational motion of an ouabain spin label with sheep kidney Na,K-ATPase has been measured by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) measurements. Spin-labelled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 ± 0.1 mol of bound ouabain spin label per ATPase β dimer. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (> 99%) of a broad resonance which is characteristic of a strongly immobilized spin label. ST-EPR measurements of the spin labelled ATPase preparations yield effective correlation times for the bound labels of 209 ± 11 μs at 0°C and 44 ± 4 μs at 20°C. These rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements with glutaraldehyde-crosslinked preparations indicated that the observed rotational correlation times predominantly represented the motion of entire Na,K-ATPase-containing membrane fragments, rather than the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The strong immobilization of the ouabain spin label will make it an effective paramagnetic probe of the extracellular surface of the Na,K-ATPase for a variety of NMR and EPR investigations.     

Changes in leaf protein and pigment contents and photosynthetic activities during senescence of detached maize leaves: influence of different ultraviolet radiations

Senescence induced loss in pigments and proteins of detached maize (Zea mays L. cv. Col) leaves was significantly enhanced on the exposure of leaves to different ranges of ultraviolet (UV) radiation. Compared to UV-A (320-400 nm) and UV-B (280-320 nm), the UV-C (200-320 nm) was the most damaging for the pigments and macromolecules. A severe decline in photosystem (PS) 2 mediated photoreduction during senescence of detached leaves exposed to UV irradiation suggested a damage of the system. The PS1 mediated photoreduction of methylviologen with 2,6-dichlorophenol indophenol as electron donor was stimulated by UV-A and UV-B radiations, suggesting a reorganisation of the PS1 complex. These results were fortified by the values of fast and slow kinetics of chlorophyll (Chl) a fluorescence transients. This revised version was published online in June 2006 with corrections to the Cover Date.     

Carbohydrate-induced modulation of cell membrane. VIII. Agglutination with mammalian lectin galectin-1 increases osmofragility and membrane fluidity of trypsinized erythrocytes

Interaction of lectins with cell surface determinants may alter membrane properties. Using trypsinized rabbit erythrocytes as model we tested the capacity of an endogenous lectin in this respect. Galectin-1 is a member of an adhesion/growth-regulatory family known to interact for example with ganglioside GM(1) and also the hydrophobic tail of oncogenic H-Ras. Assays on membrane fluidity and osmofragility detect galectin-1's capacity to increase the parameters. Moreover, it increases susceptibility of erythrocytes to radical damage. These observations indicate the potential of this endogenous lectin to affect membrane properties beyond the immediate interaction with cell surface epitopes.     

Effects of amphotericin B on membrane permeability--kinetics of spin probe reduction

The effect of the polyene antibiotic amphotericin B on the permeability of both unilamellar and multilamellar model membranes is investigated. The method measures the loss of the electron paramagnetic resonance signal of a spin probe, trapped in the aqueous compartment of a lipid dispersion, upon addition of ascorbate ions to the bulk aqueous phase. Amphotericin B causes large increases in the permeability of cholesterol-containing egg phosphatidylcholine membranes, whereas the effects are small in the absence of sterol and do not depend on surface charge. The effect of amphotericin depends upon the antibiotic:sterol mole ratio. The antibiotic appears to be unable to cross the membrane, acting only on the outermost bilayer of a multibilayer dispersion. When a phospholipid in the gel phase is used, amphotericin B causes large increases in permeability, independently of the presence or absence of sterol. It is suggested that the mechanism of action of amphotericin B is different for lipids in the liquid crystalline or gel states.     

Formation of a stable oligomer of beta-2 microglobulin requires only transient encounter with Cu(II)

Beta-2 Microglobulin (beta2m) is a small, globular protein, with high solubility under conditions comparable to human serum. A complication of hemodialysis in renal failure patients is the deposition of unmodified beta2m as amyloid fibers. In vitro, exposure of beta2m to equimolar Cu(2+) under near-physiological conditions can result in self-association leading to amyloid fiber formation. Previously, we have shown that the early steps in this process involve a catalyzed structural rearrangement followed by formation of discrete oligomers. These oligomers, however, have a continued requirement for Cu(2+) while mature fibers are resistant to addition of metal chelate. Here, we report that the transition from Cu(2+) dependent to chelate resistant states occurs in the context of small oligomers, dimeric to hexameric in size. These species require Cu(2+) to form, but once generated, do not need metal cation for stability. Importantly, this transition occurs gradually over several days and the resulting oligomers are isolatable and kinetically stable on timescales exceeding weeks. In addition, formation is enhanced by levels of urea similar to those found in hemodialysis patients. Our results are consistent with our hypothesis that transient encounter of full-length wild-type beta2m with transition metal cation at the dialysis membrane interface is causal to dialysis related amyloidosis.     

EPR study of 1Asp-3Cys ligated 4Fe-4S iron-sulfur cluster in NB-protein (BchN-BchB) of a dark-operative protochlorophyllide reductase complex

Dark-operative protochlorophyllide oxidoreductase, a nitrogenase-like enzyme, contains two [4Fe–4S] clusters, one in the L-protein ((BchL)₂) and the other in the NB-protein ((BchN–BchB)₂). The reduced NB-cluster in the NB-protein, which is ligated by 1Asp/3Cys residues, showed a broad S = 3/2 electron paramagnetic resonance signal that is rather rare in [4Fe–4S] clusters. A 4Cys-ligated NB-cluster in the mutated variant BchB–D36C protein, in which the Asp36 was replaced by a Cys, gave a rhombic normal S = 1/2 signal and lost the catalytic activity. The results suggest that Asp36 contributes to the low redox potential necessary to reduce protochlorophyllide.     

EPR studies of phospholipid bilayers after lipoperoxidation. 1. Inner molecular order and fluidity gradient

The molecular order of fatty acyl chains in oriented lipid bilayers on solid support (SPB), made of either natural or synthetic phospholipids oxidized by Fenton reagent and probed with spin labeled lecithin (5-DSPC) was studied by means of EPR spectrometry. Phospholipids (ASPC, EYPC, mitochondrial extract) were oxidized as either aqueous buffer/methanol dispersions or reverse-phase evaporation vesicles (REV) suspensions. Oxidation was preliminarily revealed both by assaying MDA and by detecting conjugated dienes. Oxidized phospholipid species was quantified by preparative TLC. The degree of order in oriented lipid bilayers of samples containing oxidized phospholipids was estimated by the loss of EPR spectral anisotropy, and an empirical index of the related bilayer disorder was calculated from the second derivative spectra. Bilayers made of each non-oxidized phospholipid species from either ethanol solutions or REV suspensions showed the highest anisotropy, while the increasing presence of oxidized lipids in the samples resulted in progressive loss of EPR spectral anisotropy. In contrast, vesicles containing 40% of the oxidized species maintained an unaltered fluidity gradient, while REV formation was hindered by oxidized phospholipid percentages higher than 45% for ASPC and EYPC, and 35% for Mitochondrial lipids (MtL). It is concluded that the early stages of lipoperoxidation bring about disordering of the phospholipid bilayer interior rather than fluidity alterations, and that prolonged oxidation may result in loss of structural and chemical properties of the bilayer until the structure no longer holds.     

"Distant spin trapping": a method for expanding the availability of spin trapping measurements

The technique of spin trapping is used to study a wide range of free radicals in various systems, including those generated in vitro and in vivo. But unfortunately, EPR spectrometers are not always immediately accessible at the site of experimentation, and therefore it is important to find a method that can preserve a radical adduct over longer periods of time. We describe here an alternative method in which the samples can be frozen and transported for EPR measurements at another site. Various spin adducts of DEPMPO were frozen and measured at 0 degrees C at various intervals after freezing to determine their stability in the frozen state. The radical adducts were generated by established methods and stored at two different temperatures; -196 degrees C (liquid nitrogen) and -80 degrees C (dry ice). The experiments were carried out in an aqueous solution with and without a model of reducing environment (2 mM ascorbate). The results indicate that it is feasible to store and transport spin adducts for subsequent analysis. We conclude that this approach, which we term "distant spin trapping", makes it feasible to transport samples to another site for EPR measurements. This should significantly expand the ability to use spin trapping in biology and medicine.     

The main thylakoid membrane lipid monogalactosyldiacylglycerol (MGDG) promotes the de-epoxidation of violaxanthin associated with the light-harvesting complex of photosystem II (LHCII)

In higher plants, the major part of the xanthophyll cycle pigment violaxanthin (Vx) is non-covalently bound to the main light-harvesting complex of PSII (LHCII). Under saturating light conditions Vx has to be released from its binding site into the surrounding lipid phase, where it is converted to zeaxanthin (Zx) by the enzyme Vx de-epoxidase (VDE). In the present study we investigated the influence of thylakoid lipids on the de-epoxidation of Vx, which was still associated with the LHCII. We isolated LHCII with different concentrations of native, endogenous lipids and Vx by sucrose gradient centrifugation or successive cation precipitation. Analysis of the different LHCII preparations showed that the concentration of LHCII-associated Vx was correlated with the concentration of the main thylakoid lipid monogalactosyldiacylglycerol (MGDG) associated with the complexes. Decreases in the MGDG content of the LHCII led to a diminished Vx concentration, indicating that a part of the total Vx pool was located in an MGDG phase surrounding the LHCII, whereas another part was bound to the LHCII apoproteins. We further studied the convertibility of LHCII-associated Vx in in-vitro enzyme assays by addition of isolated VDE. We observed an efficient and almost complete Vx conversion in the LHCII fractions containing high amounts of endogenous MGDG. LHCII preparations with low concentrations of MGDG exhibited a strongly reduced Vx de-epoxidation, which could be increased by addition of exogenous, pure MGDG. The de-epoxidation of LHCII-associated Vx was saturated at a much lower concentration of native, endogenous MGDG compared with the concentration of isolated, exogenous MGDG, which is needed for optimal VDE activity in in-vitro assays employing pure isolated Vx.     

TOAC spin labels in the backbone of alamethicin: EPR studies in lipid membranes

Alamethicin is a 19-amino-acid residue hydrophobic peptide that produces voltage-dependent ion channels in membranes. Analogues of the Glu(OMe)(7,18,19) variant of alamethicin F50/5 that are rigidly spin-labeled in the peptide backbone have been synthesized by replacing residue 1, 8, or 16 with 2,2,6,6-tetramethyl-piperidine-1-oxyl-4-amino-4-carboxyl (TOAC), a helicogenic nitroxyl amino acid. Conventional electron paramagnetic resonance spectra are used to determine the insertion and orientation of the TOAC(n) alamethicins in fluid lipid bilayer membranes of dimyristoyl phosphatidylcholine. Isotropic (14)N-hyperfine couplings indicate that TOAC(8) and TOAC(16) are situated in the hydrophobic core of the membrane, whereas the TOAC(1) label resides closer to the membrane surface. Anisotropic hyperfine splittings show that alamethicin is highly ordered in the fluid membranes. Experiments with aligned membranes demonstrate that the principal diffusion axis lies close to the membrane normal, corresponding to a transmembrane orientation. Combination of data from the three spin-labeled positions yields both the dynamic order parameter of the peptide backbone and the intramolecular orientations of the TOAC groups. The latter are compared with x-ray diffraction results from alamethicin crystals. Saturation transfer electron paramagnetic resonance, which is sensitive to microsecond rotational motion, reveals that overall rotation of alamethicin is fast in fluid membranes, with effective correlation times <30 ns. Thus, alamethicin does not form large stable aggregates in fluid membranes, and ionic conductance must arise from transient or voltage-induced associations.     

Nucleotide-Induced Flexibility Change in Neck Linkers of Dimeric Kinesin as Detected by Distance Measurements Using Spin-Labeling EPR

Using dipolar continuous-wave and pulsed electron paramagnetic resonance methods, we have determined the distribution of the distances between two spin labels placed on the middle of each of the neck linkers of dimeric kinesin. In the absence of microtubules, the distance was centered at 3.3 nm, but displayed a broad distribution with a width of 2.7 nm. This broad distribution implies that the linkers are random coils and extend well beyond the 2.5-nm distance expected of crystal structures. In the presence of microtubules, two linker populations were found: one similar to that observed in the absence of microtubules (a broad distribution centered at 3.3 nm), and the second population with a narrower distribution centered at 1.3-2.5 nm. In the absence of nucleotide but in the presence of microtubules, ∼ 40% of the linkers were at a distance centered at 1.9 nm with a 1.2-nm width; the remaining fraction was at 3.3 nm, as before. This suggests that neck linkers exhibit dynamics covering a wide distance range between 1.0 and 5.0 nm. In the presence of ATP analogs adenosine 5′-(β,γ-imido)triphosphate and adenosine 5′-(γ-thio)triphosphate, 40-50% of the spins showed a very narrow distribution centered at 1.6 nm, with a width of 0.4-0.5 nm. The remaining population displayed the broad 3.3-nm distribution. Under these conditions, a large fraction of linkers are docked firmly onto a motor core or microtubule, while the remainder is disordered.We propose that large nucleotide-dependent flexibility changes in the linkers contribute to the directional bias of the kinesin molecule stepping 8 nm along the microtubule.     

Dark production of reactive oxygen species in photosystem II membrane particles at elevated temperature: EPR spin-trapping study

In our study, EPR spin-trapping technique was employed to study dark production of two reactive oxygen species, hydroxyl radicals (OH) and singlet oxygen (¹O₂), in spinach photosystem II (PSII) membrane particles exposed to elevated temperature (47 °C). Production of OH, evaluated as EMPO-OH adduct EPR signal, was suppressed by the enzymatic removal of hydrogen peroxide and by the addition of iron chelator desferal, whereas externally added hydrogen peroxide enhanced OH production. These observations reveal that OH is presumably produced by metal-mediated reduction of hydrogen peroxide in a Fenton-type reaction. Increase in pH above physiological values significantly stimulated the formation of OH, whereas the presence of chloride and calcium ions had the opposite effect. Based on our results it is proposed that the formation of OH is linked to the thermal disassembly of water-splitting manganese complex on PSII donor side. Singlet oxygen production, followed as the formation of nitroxyl radical TEMPO, was not affected by OH scavengers. This finding indicates that the production of these two species was independent and that the production of ¹O₂ is not closely linked to PSII donor side.     

EPR kinetic studies of the S−1 state in spinach thylakoids

The Y_Z decay kinetics in a formal S₋₁ state, regarded as a reduced state of the oxygen evolving complex, was determined using time-resolved EPR spectroscopy. This S₋₁ state was generated by biochemical treatment of thylakoid membranes with hydrazine. The steady-state oxygen evolution of the sample was used to optimize the biochemical procedure for performing EPR experiments. A high yield of the S₋₁ state was generated as judged by the two-flash delay in the first maximum of oxygen evolution in Joliot flash-type experiments. We have shown that the Y_Z re-reduction rate by the S₋₁ state is much slower than that of any other S-state transition in hydrazine-treated samples. This slow reduction rate in the S₋₁ to S₀ transition, which is in the order of the S₃ to S₀ transition rate, suggests that this transition is accompanied by some structural rearrangements. Possible explanations of this unique, slow reduction rate in the S₋₁ to S₀ transition are considered, in light of earlier observations by others on hydrazine/hydroxylamine reduced PS II samples.     

Diurnal courses of net photosynthesis and photosystem II quantum efficiency of submerged Lagarosiphon major under natural light conditions

An optode device for net-photosynthesis measurements, based on oxygen-depending quenching of fluorescence from O₂-specific sensors, and PAM fluorometry have been used to study diurnal courses of net-photosynthesis and the F_v/F_m ratio of the submerged plant Lagarosiphon major. Plants were pre-cultivated and studied in large mesocosm flow-through outdoor tanks under 50% and 80% shade cloth, respectively. Growth under the different shade cloths resulted in similar light compensation points (∼20 μmol photons m⁻² s⁻¹), but strongly different light saturation levels, with about 150 μmol m⁻² s⁻¹ for plants grown under 80% shade cloth and about 350 μmol m⁻² s⁻¹ for plants grown under 50% shade cloth. Plants under both growth conditions showed a transient reduction of the maximum F_v/F_m value in the afternoon (down to 70% of the morning control values under 80% shade cloth and down to 85% under 50% shade cloth), which was not accompanied by a reduction of the net photosynthetic rate. This indicated that the fluorescence parameter F_v/F_m must not be a reliable indicator of the rate of photosynthesis under all conditions. The new photo-optical device became evidenced as a valuable tool not only for laboratory experiments, but also for field studies of gas exchange of submerged plants.     

High-light induced singlet oxygen formation in cytochrome b6f complex from Bryopsis corticulans as detected by EPR spectroscopy

Electron paramagnetic resonance (EPR) spectroscopy was used to detect the light-induced formation of singlet oxygen (¹O₂*) in the intact and the Rieske-depleted cytochrome b₆f complexes (Cyt b₆f) from Bryopsis corticulans, as well as in the isolated Rieske Fe–S protein. It is shown that, under white-light illumination and aerobic conditions, chlorophyll a (Chl a) bound in the intact Cyt b₆f can be bleached by light-induced ¹O₂*, and that the ¹O₂* production can be promoted by D₂O or scavenged by extraneous antioxidants such as l-histidine, ascorbate, β-carotene and glutathione. Under similar experimental conditions, ¹O₂* was also detected in the Rieske-depleted Cyt b₆f complex, but not in the isolated Rieske Fe–S protein. The results prove that Chl a cofactor, rather than Rieske Fe–S protein, is the specific site of ¹O₂* formation, a conclusion which draws further support from the generation of ¹O₂* with selective excitation of Chl a using monocolor red light.     

Conversion of the g = 4.1 EPR signal to the multiline conformation during the S2 to S3 transition of the oxygen evolving complex of Photosystem II

The oxygen evolving complex of Photosystem II undergoes four light-induced oxidation transitions, S₀-S₁,…,S₃-(S₄)S₀ during its catalytic cycle. The oxidizing equivalents are stored at a (Mn)₄Ca cluster, the site of water oxidation. EPR spectroscopy has yielded valuable information on the S states. S₂ shows a notable heterogeneity with two spectral forms; a g = 2 (S = 1/2) multiline, and a g = 4.1 (S = 5/2) signal. These oscillate in parallel during the period-four cycle. Cyanobacteria show only the multiline signal, but upon advancement to S₃ they exhibit the same characteristic g = 10 (S = 3) absorption with plant preparations, implying that this latter signal results from the multiline configuration. The fate of the g = 4.1 conformation during advancement to S₃ is accordingly unknown. We searched for light-induced transient changes in the EPR spectra at temperatures below and above the half-inhibition temperature for the S₂ to S₃ transition (ca 230 K). We observed that, above about 220 K the g = 4.1 signal converts to a multiline form prior to advancement to S₃. We cannot exclude that the conversion results from visible-light excitation of the Mn cluster itself. The fact however, that the conversion coincides with the onset of the S₂ to S₃ transition, suggests that it is triggered by the charge-separation process, possibly the oxidation of tyr Z and the accompanying proton relocations. It therefore appears that a configuration of (Mn)₄Ca with a low-spin ground state advances to S₃.     

The multiple roles of light-harvesting chlorophyll a/b-protein complexes define structure and optimize function of Arabidopsis chloroplasts: A study using two chlorophyll b-less mutants

The multiple roles of light-harvesting chlorophyll a/b-protein complexes in the structure and function of Arabidopsis chloroplasts were investigated using two chlorophyll b-less mutants grown under metal halide lamps with a significant far-red component. In ch1-3, all six light-harvesting proteins of photosystem (PS) II were greatly decreased; in ch1-3lhcb5, Lhcb5 was completely absent while the other five proteins were further decreased. The thylakoids of ch1-3 were less negatively-charged than the wild type, and those of ch1-3lhcb5 were even less so. Despite the expected weaker electrostatic repulsion, however, thylakoids in leaves of the mutants were not well stacked, an effect we attribute to lower van der Waals attraction, lower electrostatic attraction between opposite charges, and the absence or instability of PSII supercomplexes and peripheral light-harvesting trimers. The quantum yield of oxygen evolution in leaves decreased from 0.109 (wild type) to 0.087 (ch1-3) and 0.081 (ch1-3lhcb5) O₂ (photon absorbed)^− 1; we attribute this decrease to an excessive spillover from PSII to PSI, a limited PSII antenna, and increased light-independent thermal dissipation in PSII in the mutants. Destabilization of the donor side of PSII, indicated by slower electron donation to the redox-active tyrosine Y_Z in ch1-3, probably enhanced PSII susceptibility to photoinactivation, increased the non-functional PSII complexes in vivo, and further inactivated PSII complexes in vitro. The evolution of chlorophyll b-containing chloroplasts seems to fine-tune oxygenic photosynthesis.