Spin-labeled polyribonucleotides

Poly (U), poly (C) and poly (A) were spin labeled with N-(2,2,5,5-tetramethyl-3-carbonylpyrroline-1-oxyl)-imidazole. This spin label interacts selectively with 2' OH ribose groups of polynucleotides and does not modify the nucleic acid bases. The extent of spin labeling is not dependent upon the nature of the base and is entirely determined by rigidity of the secondary structure of the polynucleotide. The extent of modification for poly (U), poly (C) and poly (A) was 4.2, 1.7 and 1.5 per cent, respectively, the secondary structure of the polynucleotides being practically unchanged. Some physico-chemical properties of the spin-labeled polynucleotides were investigated by ESR spectroscopy. Rotational correlation times of the spin label and activation energy of its motion were calculated.     

Spin-labeled lipid A

Lipopolysaccharide (LPS), a major component of the outer membranes of gram-negative bacteria, is composed of a polysaccharide chain attached to a lipid A base that contains a disaccharide headgroup with two negative phosphate groups and at least four acyl chains. Lipid A is an essential component of the membranes of a large number of bacteria and is also a substrate for a wide variety of proteins. Here we report the synthesis of a nitroxide spin-labeled lipid A, characterize its localization at the membrane bilayer surface, and demonstrate that it remains a viable substrate for the Escherichia coli lipid flippase MsbA.     

Spin-labeled polyuridylic acid. Formation of an intermediate structure

Temperature-dependent conformational transitions of spin-labeled poly(U) at low temperature in spermidine and cesium chloride buffer have been measured by electron spin resonance spectroscopy. The Arrhenius plot shows the existence of the order-disorder transition at a temperature close to that obtained from absorbance temperature profiles. However, in addition the formation of an intermediate state is observed during the melting of the ordered poly(U) to its random coil.     

Spin-labeled gramicidin a: channel formation and dissociation

Gramicidin A was studied by continuous wave electron spin resonance (CW-ESR) and by double-quantum coherence electron spin resonance (DQC-ESR) in several lipid membranes (using samples that were macroscopically aligned by isopotential spin-dry ultracentrifugation) and vesicles. As a reporter group, the nitroxide spin-label was attached at the C-terminus yielding the spin-labeled product (GAsl). ESR spectra of aligned membranes containing GAsl show strong orientation dependence. In DPPC and DSPC membranes at room temperature the spectral shape is consistent with high ordering, which, in conjunction with the observed high polarity of the environment of the nitroxide, is interpreted in terms of the nitroxide moiety being close to the membrane surface. In contrast, spectra of GAsl in DMPC membranes indicate deeper embedding and tilt of the NO group. The GAsl spectrum in the DPPC membrane at 35 degrees C (the gel to Pbeta phase transition) exhibits sharp changes, and above this temperature becomes similar to that of DMPC. The dipolar spectrum from DQC-ESR clearly indicates the presence of pairs in DMPC membranes. This is not the case for DPPC, rapidly frozen from the gel phase; however, there are hints of aggregation. The interspin distance in the pairs is 30.9 A, in good agreement with estimates for the head-to-head GAsl dimer (the channel-forming conformation), which matches the hydrophobic thickness of the DMPC bilayer. Both DPPC and DSPC, apparently as a result of hydrophobic mismatch between the dimer length and bilayer thickness, do not favor the channel formation in the gel phase. In the Pbeta and Lalpha phases of DPPC (above 35 degrees C) the channel dimer forms, as evidenced by the DQC-ESR dipolar spectrum after rapid freezing. It is associated with a lateral expansion of lipid molecules and a concomitant decrease in bilayer thickness, which reduces the hydrophobic mismatch. A comparison with studies of dimer formation by other physical techniques indicates the desirability of using low concentrations of GA (approximately 0.4-1 mol %) accessible to the ESR methods employed in the study, since this yields non-interacting dimer channels.     

Spin-labeled erythrocyte membranes: direct identification of nitroxide-conjugated proteins

The covalent incorporation of a spin-labeled analog of N-ethyl maleimide into erythrocyte membrane proteins has been monitored by electron paramagnetic resonance spectroscopy and the individually labeled proteins detected by immunoblotting techniques, using an anti-nitroxide antibody, following electrophoretic separation of the membrane components. Spin-label was primarily found in the high molecular weight bands (I and II) with no incorporation in proteins with molecular weights less than 35,000. Increasing the reaction time between the spin-label and ghosts altered both the observed labeling pattern and the epr spectra with an increase in the proportion of strongly-immobilized species.     

Solvent Interactions and Protein Dynamics in Spin-labeled T4 Lysozyme

Abstract

Aspects of T4 lysozyme dynamics and solvent interaction are investigated using atomically detailed Molecular Dynamics (MD) simulations. Two spin-labeled mutants of T4 lysozyme are analyzed (T4L-N40C and T4L-K48C), which have been found from electronic paramagnetic resonance (EPR) experiments to exhibit different mobilities at the site of spin probe attachment (N- and C-terminus of helix B, respectively). Similarities and differences in solvent distribution and diffusion around the spin label, as well as around exposed and buried residues within the protein, are discussed. The purpose is to capture possible strong interactions between the spin label (ring) and solvent molecules, which may affect EPR lineshapes. The effect of backbone motions on the water density profiles is also investigated. The focus is on the domain closure associated with the T4 lysozyme hinge-bending motion, which is analyzed by Essential Dynamics (ED). The N-terminus of helix B is found to be a “hinge” residue, which explains the high degree of flexibility and motional freedom at this site.     

Anhydrobiosis: Inside yeast cells

Under natural conditions yeast cells as well as other microorganisms are regularly subjected to the influence of severe drought, which leads to their serious dehydration. The dry seasons are then changed by rains and there is a restoration of normal water potential inside the cells. To survive such seasonal changes a lot of vegetative microbial cells, which belong to various genera and species, may be able to enter into a state of anhydrobiosis, in which their metabolism is temporarily and reversibly suspended or delayed. This evolutionarily developed adaptation to extreme conditions of the environment is widely used for practical goals – for conservation of microorganisms in collections, for maintenance and long storage of different important strain-producers and for other various biotechnological purposes. This current review presents the most important data obtained mainly in the studies of the structural and functional changes in yeast cells during dehydration. It describes the changes of the main organelles of eukaryotic cells and their role in cell survival in a dry state. The review provides information regarding the role of water in the structure and functions of biological macromolecules and membranes. Some important intracellular protective reactions of eukaryotic organisms, which were revealed in these studies and may have more general importance, are also discussed. The results of the studies of yeast anhydrobiosis summarized in the review show the possibilities of improving the conservation and long-term storage of various microorganisms and of increasing the quality of industrially produced dry microbial preparations.     

Biosurfactant-rhamnolipid effects on yeast cells

AIM: The aim of this work was to study the effect of the novel surfactant PS from Pseudomonas sp. S-17 on Saccharomyces cerevisiae 83-20 yeast cells and to compare it with the effect of the well known surfactant Triton X-100. METHODS AND RESULTS: The effect of surfactants was investigated on the cells during growth, and on the separated cells. The cell-permeabilizing effect of surfactants was studied by following the release of protein and some enzyme activities. The biosurfactant did not affect the culture growth kinetics, and altered the polypeptide profiles of cells and membrane proteins in the same way as Triton X-100. CONCLUSION: Results of this study demonstrate that biosurfactant PS and Triton X-100 have a similar type of action, mainly surface located, and that they do not affect the intracellular structures of yeast cells. SIGNIFICANCE AND IMPACT OF THE STUDY: A novel surfactant PS was isolated from Pseudomonas sp. S-17. A mild effect of PS on yeast cells was demonstrated. The results indicate the ecological safety of the biosurfactant and its potential use in the development of environmentally-benign and efficient cleaning technologies.     

Dielectric properties of yeast cells

Summary Dielectric measurements were made on suspensions of intact yeast cells over a frequency range of 10 kHz to 100 MHz. The suspensions showed typical dielectric dispersions, which are considered to be caused by the presence of cytoplasmic membranes with sufficiently low conductivity. Since the conductivity of the cell wall was found to be of nearly the same value as that of the suspending medium, composed of KCl solutions in a range from 10 to 80mm, the cell wall may be ignored in establishing an electrical model of the cells suspended in such media. An analysis of the dielectric data was carried out by use of Pauly and Schwan's theory. The membrane capacitance was estimated to be 1.1±0.1 F/cm², which is compared with values reported so far for most biological membranes. The conductivity of the cell interior was almost unchanged with varying KCl concentrations and showed low values owing to the presence of less conducting particles, presumably intracellular organelles. The relatively low dielectric constant of about 50 obtained for the cell interior, in comparison with values of aqueous solutions, may be attributed also to the presence of intracellular organelles and proteins.     

Sugar-induced apoptosis in yeast cells

Sugars induce death of Saccharomyces cerevisiae within a few hours in the absence of additional nutrients to support growth; by contrast, cells incubated in water or in the presence of other nutrients without sugar remain viable for weeks. Here we show that this sugar-induced cell death (SICD) is characterized by rapid production of reactive oxygen species (ROS), RNA and DNA degradation, membrane damage, nucleus fragmentation and cell shrinkage. Addition of ascorbic acid to sugar-incubated cells prevents SICD, indicating that SICD is initiated by ROS. The lack of a protection mechanism against SICD suggests that sugars use to be the limiting nutrients for yeast and are probably depleted before all other nutrients. Being the limiting nutrient, sugars became the growth-stimulating agent, signaling the presence of sufficient nutrients for growth, but in the absence of the complementing nutrients they induce apoptotic death.     

Vanadium uptake by yeast cells

During incubation with vanadyl, Saccharomyces cerevisiae yeast cells were able to accumulate millimolar concentrations of this divalent cation within an intracellular compartment. The intracellular vanadyl ions were bound to low molecular weight substances. This was indicated by the isotropic nature of the electron paramagnetic resonance (EPR) spectra of the respective samples. Accumulation of intracellular vanadyl was dependent on presence of glucose during incubation. It could be inhibited by various di- and trivalent metal cations. Of these cations lanthanum displayed the strongest inhibitory action. If yeast cells were exposed to more than 50 microM vanadyl sulfate at a pH higher than 4.0, a potassium loss into the medium was detected. The magnitude of this potassium loss suggests a damage of the plasma membrane caused by vanadyl. Upon addition of vanadate to yeast cells surface-bound vanadyl was detectable after several minutes by EPR. This could be the consequence of extracellular reduction of vanadate to vanadyl. The reduction was followed by a slow accumulation of intracellular vanadium, which could be inhibited by lanthanum or phosphate. Therefore, permeation of vanadyl into the cells can be assumed as one mechanism of vanadium accumulation by yeast during incubation with vanadate.     

Manganese accumulation in yeast cells

Summary Manganese accumulation was studied by room-temperature electron spin resonance (ESR) spectroscopy inSaccharomyces cerevisiae grown in the presence of increasing amounts of MnSO₄. Mn²⁺ retention was nearly linear in intact cells for fractions related to both low-molecular-mass and macromolecular complexes (free and bound Mn²⁺, respectively). A deviation from linearity was observed in cell extracts between the control value and 0.1 mM Mn²⁺, indicating more efficient accumulation at low Mn²⁺ concentrations. The difference in slopes between the two straight lines describing Mn²⁺ retention at concentrations lower and higher than 0.1 mM, respectively, was quite large for the free Mn²⁺ fraction. Furthermore it was unaffected by subsequent dialyses of the extracts, showing stable retention in the form of low-molecular-mass complexes. In contrast, the slope of the line describing retention of bound Mn²⁺ at concentrations higher than 0.1 mM became less steep after subsequent dialyses of the cell extracts. This result indicates that the macromolecule-bound Mn²⁺ was essentially associated with particulate structures. In contrast to Cu²⁺, Mn²⁺ had no effect on the major enzyme activities involved in oxygen metabolism except for a slight increase of cyanide-resistant Mn-superoxide dismutase activity, due to dialyzable Mn²⁺ complexes.