The effect of changes in gramicidin conformation on bilayer lipid properties.

The effects of two different gramicidin conformations on lipid phase behaviour and dynamics are compared. Samples of chain-perdeuterated dimyristoylphosphatidylcholine containing gramicidin were first prepared with gramicidin in a state having a circular dichroism spectrum generally identified as corresponding to the non-channel conformation. The effects, on bilayer lipid properties, of gramicidin in this conformation were then determined using deuterium nuclear magnetic resonance measurements of acyl chain orientational order and transverse relaxation times as a function of temperature. These samples were then incubated at 65 degrees C to convert the gramicidin to a state with a circular dichroism spectrum of the type generally identified with the channel conformation. The nuclear magnetic resonance measurements were then repeated. In the gel phase, it was found that transverse relaxation time and chain orientational order of the lipid were insensitive to gramicidin conformation. In the liquid crystalline phase, gramicidin in the channel conformation was found to have a slightly larger effect on transverse relaxation and orientational order than gramicidin in the non-channel conformation. The perturbation of the phase behavior by gramicidin was found to be relatively insensitive to gramicidin conformation.     

1H and 31P relaxation rate studies of the interaction of phosphoenolpyruvate and its analogues with avian phosphoenolpyruvate carboxykinase

The interactions of the substrate phosphoenolpyruvate and the substrate analogues (Z)-phosphoenol-alpha-ketobutyrate and (E)-phosphoenol-alpha-ketobutyrate with the enzyme-Mn complex of chicken liver phosphoenolpyruvate carboxykinase have been investigated by 1H and by 31P nuclear relaxation rate studies. Studies of the 1H and the 31P relaxation rates of the ligands in the binary Mn-ligand complexes show that these ligands interact with the metal ion via the phosphate group but not through the carboxylate. An inner sphere coordination complex is formed but the metal-ligand complex is not in the most extended conformation. In the relaxation rate studies of the ligands in the presence of the enzyme, conditions were adjusted so that all of the Mn2+ that was added resided in the ternary enzyme-Mn-ligand complex. The 1H relaxation rates for each of the three ligands were measured at 100 and at 300 MHz. In each case the normalized paramagnetic effects showed that 1/(pT2p) was greater than 1/(pT1p). A frequency dependence of the 1/(pT1p) and 1/(pT2p) values was also measured. The correlation time, tau c, for the Mn-1H interaction was calculated from the frequency dependence of 1/(pT1p) assuming a maximal frequency dependence of tau c and assuming no frequency dependence of tau c and from the T1M/T2M ratios at each frequency. The tau c values for all of the complexes, calculated at 100 MHz, varied from approximately 0.3 to 2.0 ns. These values were used to calculate the Mn-1H distances in each of the ternary complexes. The relaxation rates of 31P were also measured.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence depolarization studies of conformation changes in pyrene-butyryl–fumarase

Measurements of fluorescence depolarization on fumarase labeled with the dye pyrene-butyryl were used to test for previously reported structural changes in this enzymes. These apparent conformation changes were of interest because they seemed to correlate with variation in catalytic activity provoked by changing temperature or pH, or by the presence of a competitive inhibitor. In the present studies, the bound dye pyrene-butyryl and the enzymes were investigated systematically to ensure that simple interpretation of fluorescence depolarization results would be meaningful. This analysis showed that carefully controlled experimental condition were necessary to eliminate a dye component with a short fluorescence lifetime and that it was essential to allow for small variations of lifetime with temperature. Contrary to the previous report, a constant rotational relaxation time of the magnitude expected for a nearly spherical molecule of fumarase was found. No changes were detectable by fluorescence depolarization in the size or shape of pyrene-butyryl–fumarase under the solution conditions tested that caused variation in enzyme activity.     

Effect of Rocking Movements on Respiration

For centuries, rocking has been used to promote sleep in babies or toddlers. Recent research suggested that relaxation could play a role in facilitating the transition from waking to sleep during rocking. Breathing techniques are often used to promote relaxation. However, studies investigating head motions and body rotations showed that vestibular stimulation might elicit a vestibulo-respiratory response, leading to an increase in respiration frequency. An increase in respiration frequency would not be considered to promote relaxation in the first place. On the other hand, a coordination of respiration to rhythmic vestibular stimulation has been observed. Therefore, this study aimed to investigate the effect of different movement frequencies and amplitudes on respiration frequency. Furthermore, we tested whether subjects adapt their respiration to movement frequencies below their spontaneous respiration frequency at rest, which could be beneficial for relaxation. Twenty-one healthy subjects (24–42 years, 12 males) were investigated using an actuated bed, moving along a lateral translation. Following movement frequencies were applied: +30%, +15%, -15%, and -30% of subjects’ rest respiration frequency during baseline (no movement). Furthermore, two different movement amplitudes were tested (Amplitudes: 15 cm, 7.5 cm; movement frequency: 0.3 Hz). In addition, five subjects (25–28 years, 2 males) were stimulated with their individual rest respiration frequency. Rocking movements along a lateral translation caused a vestibulo-respiratory adaptation leading to an increase in respiration frequency. The increase was independent of the applied movement frequencies or amplitudes but did not occur when stimulating with subjects’ rest respiration frequency. Furthermore, no synchronization of the respiration frequency to the movement frequency was observed. In particular, subjects did not lower their respiration frequency below their resting frequency. Hence, it was not feasible to influence respiration in a manner that might be considered beneficial for relaxation.     

Trehalose effect on low temperature protein dynamics: fluctuation and relaxation phenomena

We performed spectral diffusion experiments in trehalose-enriched glycerol/buffer-glass on horseradish peroxidase where the heme was replaced by metal-free mesoporphyrin IX, and compared them with the respective behavior in a pure glycerol/buffer-glass (Schlichter et al., J. Chem. Phys. 2000, 112:3045-3050). Trehalose has a significant influence: spectral diffusion broadening speeds up compared to the trehalose-free glass. This speeding up is attributed to a shortening of the correlation time of the frequency fluctuations most probably by preventing water molecules from leaving the protein interior. Superimposed to the frequency fluctuation dynamics is a relaxation dynamics that manifests itself as an aging process in the spectral diffusion broadening. Although the trehalose environment speeds up the fluctuations, it does not have any influence on the relaxation. Both relaxation and fluctuations are governed by power laws in time. The respective exponents do not seem to change with the protein environment. From the spectral dynamics, the mean square displacement in conformation space can be determined. It is governed by anomalous diffusion. The associated frequency correlation time is incredibly long, demonstrating that proteins at low temperatures are truly nonergodic systems.     

Solid state NMR and X-ray diffraction studies of alpha-D-galacturonic acid monohydrate

Crystalline alpha-D-galacturonic acid monohydrate has been studied by 13C CPMAS NMR and X-ray crystallography. The molecular dynamics were investigated by evaluating 13C spin-lattice relaxation in the rotating frame (T1rho) and chemical-shift-anisotropy properties of each carbon. Only limited molecular motions can be detected in the low frequency (< 10(4) Hz) range by 13C relaxation time measurements (T1rho) and changes of chemical shift anisotropy properties as a function of temperature. X-ray analysis (at both ambient temperature and 150 K) shows that the acid has the usual chair-shaped, pyranose ring conformation, and that the acid and water molecules are linked, through all their O-H groups, in an extensively hydrogen-bonded lattice.     

Virtual and solution conformations of oligosaccharides

The possibility that observed nuclear Overhauser enhancements and bulk longitudinal relaxation times, parameters measured by 1H NMR and often employed in determining the preferred solution conformation of biologically important molecules, are the result of averaging over many conformational states is quantitatively evaluated. Of particular interest was to ascertain whether certain 1H NMR determined conformations are "virtual" in nature; i.e., the fraction of the population of molecules actually found at any time within the subset of conformational space defined as the "solution conformation" is vanishingly small. A statistical mechanics approach was utilized to calculate an ensemble average relaxation matrix from which (NOE)'s and (T1)'s are calculated. Model glycosidic linkages in four oligosaccharides were studied. The solution conformation at any glycosidic linkage is properly represented by a normalized, Boltzmann distribution of conformers generated from an appropriate potential energy surface. The nature of the resultant population distributions is such that 50% of the molecular population is found within 1% of available microstates, while 99% of the molecular population occupies about 10% of the ensemble microstates, a number roughly equal to that sterically allowed. From this analysis we conclude that in many cases quantitative interpretation of NMR relaxation data, which attempts to define a single set of allowable torsion angle values consistent with the observed data, will lead to solution conformations that are either virtual or reflect torsion angle values possessed by a minority of the molecular population. On the other hand, calculation of ensemble average NMR relaxation data yields values in agreement with experimental results. Observed values of NMR relaxation data are the result of the complex interdependence of the population distribution and NOE (or T1) surfaces in conformational space. In conformational analyses, NMR data can therefore be used to test different population distributions calculated from empirical potential energy functions.     

Visuo-motor synchronization in children of 7-8 years old

The group of 22 children of 7-8 years old and the group of 17 adults participated in the experiment in which they were asked to synchronize their movements (pressing a button) with an isochronous sequence of visual stimuli. The period of the sequence was varied between 500 to 2000 ms with the 300 ms step. Two successive phases of visuo-motor synchronization were studied: the synchronization phase and the initiation phase which corresponds to the process of transition between reacting to a visual stimulus and the stable synchronization. The initiation phase was characterized by the shape and duration of the asynchrony time course (relaxation curve). The statistical properties of asynchrony were analyzed in the framework of the phase correction of the central timer. It is shown that (1) the range of successful visuo-motor synchronization is narrower in children of 7-8 than in adults and it spans from 600-700 ms to approx. 1700 Mc; (2) The initiation phase lasts about the same time in children of 7-8 and adults and typical shapes of individual relaxation curves are similar in both adults and children. (3) Although the statistical properties of asynchrony are comparable in children and adults, the mechanism of phase correction of the central timer operates with a lower value of the correction gain factor in children than in adults. In children, the phase correction process is also characterized by a substantially higher level of the central and motor noise which leads to a higher asynchrony variability and more frequent and longer lasting synchronization losses.     

19F NMR measurements of the rotational mobility of proteins in vivo.

Three glycolytic enzymes, hexokinase, phosphoglycerate kinase, and pyruvate kinase, were fluorine labeled in the yeast Saccharomyces cerevisiae by biosynthetic incorporation of 5-fluorotryptophan. 19F NMR longitudinal relaxation time measurements on the labeled enzymes were used to assess their rotational mobility in the intact cell. Comparison with the results obtained from relaxation time measurements of the purified enzymes in vitro and from theoretical calculations showed that two of the labeled enzymes, phosphoglycerate kinase and hexokinase, were tumbling in a cytoplasm that had a viscosity approximately twice that of water. There were no detectable signals from pyruvate kinase in vivo, although it could be detected in diluted cell extracts, indicating that there was some degree of motional restriction of the enzyme in the intact cell.     

An oscillatory correlation model of auditory streaming

We present a neurocomputational model for auditory streaming, which is a prominent phenomenon of auditory scene analysis. The proposed model represents auditory scene analysis by oscillatory correlation, where a perceptual stream corresponds to a synchronized assembly of neural oscillators and different streams correspond to desynchronized oscillator assemblies. The underlying neural architecture is a two-dimensional network of relaxation oscillators with lateral excitation and global inhibition, where one dimension represents time and another dimension frequency. By employing dynamic connections along the frequency dimension and a random element in global inhibition, the proposed model produces a temporal coherence boundary and a fissure boundary that closely match those from the psychophysical data of auditory streaming. Several issues are discussed, including how to represent physical time and how to relate shifting synchronization to auditory attention.     

Hydrocarbon-nitrosamine synergism as a possible amplifying factor in lung tumorigenesis by tobacco smoke.

Twenty-five coupled relaxation sawtooth oscillators have been investigated for the occurrence of mutual synchronization, using digital computer simulation. It is shown that mutual synchronization occurs only for relatively strong coupling. Synchronization depends heavily upon the precise waveform of the oscillators in the uncoupled state. The results are compared with a number of biological phenomena.     

Aging-related changes of EEG synchronization during a visual working memory task

Differences of EEG synchronization between normal old and young people during a working memory (WM) task were investigated. The synchronization likelihood (SL) is a novel method to assessed synchronization in multivariate time series for non-stationary systems. To evaluate this method to study the mechanisms of WM, we calculated the SL values in brain electrical activity for both resting state and task state. EEG signals were recorded from 14 young adults and 12 old adults during two different states, respectively. SL was used to measure EEG synchronization between 19 electrodes in delta, theta, alpha1, alpha2 and beta frequency bands. Bad task performance and significantly decreased EEG synchronization were found in old group compared to young group in alpha1, alpha2 and beta frequency bands during the WM task. Moreover, significantly decreased EEG synchronization in beta band in the elder was also detected during the resting state. The findings suggested that reduced EEG synchronization may be one of causes for WM capacity decline along with healthy aging.     

Visuomotor synchronization in adults and seven- to eight-year-old children

A group of 22 seven- to eight-year-old children and a group of 17 adults participated in the experiment in which they synchronized their movements (pressing a button) with an isochronous sequence of visual stimuli. The period of the sequence was varied between 500 to 2000 ms at a step of 300 ms. Two consecutive phases of visuomotor synchronization were studied: the initiation phase, which corresponds to the process of transition between responding to a visual stimulus to stable synchronization with them, and the synchronization phase. The initiation phase was characterized by the shape and duration of the asynchrony time course (relaxation curve). The statistical properties of asynchrony were analyzed in terms of the phase correction of the central timer. It was shown that (1) the range of successful visuomotor synchronization was narrower in seven- to eight-year-old children than in adults (from 600–700 ms to ∼1700 ms); (2) the initiation phase duration was about the same in seven- to eight-year-old children and adults, and typical shapes of individual relaxation curves were similar in both adults and children; (3) although the statistical properties of asynchrony were comparable in children and adults, the mechanism of phase correction of the central timer operated at a lower correction gain factor in children than in adults. In children, the phase correction process was also characterized by a substantially higher level of the central and motor noise, which leads to higher asynchrony variability and more frequent and longer synchronization losses.     

Interaction of the complex between cloacin and its immunity protein and of cloacin with the outer and cytoplasmic membranes of sensitive cells.

Longitudinal and transverse proton relaxation rates for water in the hydration spheres of Gd(III) bound to the non-immune rabbit IgG fragments Fc (C-terminal half of heavy-chain dimer), pFc' (C-terminal quarter of heavy-chain dimer) and Fab (N-terminal half of heavy and light chain) have been measured at a number of frequencies and temperatures using pulsed nuclear magnetic resonance spectrometry. For the fragments Fc and pFc', a full computer analysis showed that the results could be fitted by parameters of similar magnitude to those found previously for IgG. In contrast to the results for the other complexes the Fab -Gd(III) complex showed no slow exchange contribution to the relaxation rates. Under these circumstances it was found possible to obtain an accurate value for the hydration number (q) from measurements of the longitudinal and transverse relaxation rates at a chosen frequency such that the product of the nuclear Larmor frequency (omega1) and the correlation time for the dipolar relaxation processes (tauc) was approximately unity. Water-proton relaxation rates were also determined for the complex of Gd(III) with the Fv fragment of the mouse myeloma protein MOPC 315. A computer analysis of the results revealed a slow exchange contribution to the rates and this gave errors in the variable parameters similar to those observed previously for IgG, Fc and pFc'. The conclusions drawn from the different systems are discussed in terms of the present state of application of the proton relaxation enhancement technique in biology.     

Ligand-induced changes in the structure and dynamics of a human class Mu glutathione S-transferase

Glutathione transferases are detoxification enzymes that catalyze the addition of glutathione (GSH) to a wide variety of hydrophobic compounds. Although this group of enzymes has been extensively characterized by crystallographic studies, little is known about their dynamic properties. This study investigates the role of protein dynamics in the mechanism of a human class mu enzyme (GSTM2-2) by characterizing the motional properties of the unliganded enzyme, the enzyme-substrate (GSH) complex, an enzyme-product complex [S-(2,4-dinitrobenzyl)glutathione, GSDNB], and an enzyme-inhibitor complex (S-1-hexylglutathione, GSHEX). The kinetic on- and off-rates for these ligands are 10-20-fold lower than the diffusion limit, suggesting dynamic conformational heterogeneity of the active site. The off-rate of GSDNB is similar to the turnover number for its enzymatic formation, suggesting that product release is rate-limiting when 1-chloro-2,4-dinitrobenzene is the substrate. The dynamic properties of GSTM2-2 were investigated over a wide range of time scales using (15)N nuclear spin relaxation, residual dipolar couplings, and amide hydrogen-deuterium exchange rates. These data show that the majority of the protein backbone is rigid on the nanosecond to picosecond time scale for all forms of the enzyme. The presence of motion on the millisecond to microsecond time scale was detected for a small number of residues within the active site. These motions are likely to play a role in facilitating substrate binding and product release. The residual dipolar couplings also show that the conformation of the active site region is more open in solution than in the crystalline environment, further enhancing ligand accessibility to the active site. Amide hydrogen-deuterium exchange rates indicate a reduction in the dynamic properties of several residues near the active site due to the binding of ligand. GSH binding reduces the exchange rate of a number of residues in proximity to its binding site, while GSHEX causes a reduction in amide-exchange rates throughout the entire active site region. The location of the dinitrobenzene (DNB) ring in the GSDNB-GSTM2-2 complex was modeled using chemical shift changes that occur when GSDNB binds to the enzyme. The DNB ring makes a number of contacts with hydrophobic residues in the active site, including Met108. Replacement of Met108 with Ala increases the turnover number of the enzyme by a factor of 1.7.     

Interaction between the Frontal and Left Parietotemporal Areas in Verbal Thinking

The synchronization of the rhythmical components of evoked potentials (EP) was studied during verbal-task solving. A novel method of the calculation of Wavelet curve correlation was used to reveal synchronization between the evoked rhythmical components in short time intervals. This method was applied to earlier EP records, which were conducted during the search for verbal associations and revealed the successive activation of the frontal and left parietal cortical areas. Two stages of task solving were identified. Independently of the task type, the first stage was characterized by a diffuse synchronization in a broad frequency band below 22 Hz immediately after the stimulus presentation. This stage results in a realization of the verbal stimulus. The second stage was manifested in a localized synchronization between the frontal and left temporal (Wernicke's) areas in the narrow frequency band about 17 Hz only during search for associations. This specific and local synchronization took place earlier than the diffuse activation of the left temporal cortex. This stage appears to reflect the information transmission from the frontal cortex to the left parietotemporal area.     

Anomalies of nanosecond ultrasonic relaxation in the lipid bilayer transition

The frequency dependence of ultrasonic velocity as well as absorption in a suspension of sonicated dipalmitoylphosphatidylcholine vesicles was measured by a differential ultrasonic resonator. The frequency was scanned between 1.3 and 13 MHz and the temperature was varied from 25 to 47°C. A pronounced relaxation was observed in the time range of 10 ns. The data were analyzed assuming a single relaxation which appeared to be a good approximation. The relaxation time as well as relaxation strength increased anomalously in the vicinity of the gel-to-liquid crystal transition of 41.5°C. This result represents the first definite evidence of the critical slowing down in the lipid bilayer and is discussed in terms of the Landau theory of phase transition. The possible biological significance of the mechanical relaxation is also presented.     

Analysis of restriction enzyme-induced DNA double-strand breaks in Chinese hamster ovary cells by pulsed-field gel electrophoresis: implications for chromosome damage.

Restriction enzymes can be electroporated into mammalian cells, and the induced DNA double-strand breaks can lead to aberrations in metaphase chromosomes. Chinese hamster ovary cells were electroporated with PstI, which generates 3' cohesive-end breaks, PvuII, which generates blunt-end breaks, or XbaI, which generates 5' cohesive-end breaks. Although all three restriction enzymes induced similar numbers of aberrant metaphase cells, PvuII was dramatically more effective at inducing both exchange-type and deletion-type chromosome aberrations. Our cytogenetic studies also indicated that enzymes are active within cells for only a short time. We used pulsed-field gel electrophoresis to investigate (i) how long it takes for enzymes to cleave DNA after electroporation into cells, (ii) how long enzymes are active in the cells, and (iii) how the DNA double-strand breaks induced are related to the aberrations observed in metaphase chromosomes. At the same concentrations used in the cytogenetic studies, all enzymes were active within 10 min of electroporation. PstI and PvuII showed a distinct peak in break formation at 20 min, whereas XbaI showed a gradual increase in break frequency over time. Another increase in the number of breaks observed with all three enzymes at 2 and 3 h after electroporation was probably due to nonspecific DNA degradation in a subpopulation of enzyme-damaged cells that lysed after enzyme exposure. Break frequency and chromosome aberration frequency were inversely related: The blunt-end cutter PvuII gave rise to the most aberrations but the fewest breaks, suggesting that it is the type of break rather than the break frequency that is important for chromosome aberration formation.     

Salt induced deprotonation of initially salt-free transfer RNA.

¹H NMR double resonance studies of valinomycin in (CD₃)₂SO were conducted at 90 MHz (FT-mode) and 250 MHz (correlation mode) to determine the mechanism of intramolecular nuclear Overhauser effects (NOE). These studies set specific constraints on any model for the conformation of valinomycin in (CD₃)₂SO and illustrate that NOE experiments of this type hold considerable promise for conformational studies of peptides, proteins and other biomolecules. The NOE's are positive at the lower frequency and negative at the higher frequency. Consideration of the theoretical dependence of the NOE on the proton-proton internuclear correlation time and on the resonance frequency indicates that these results are explained by a predominantly dipolar relaxation mechanism.