Complex Spectral Changes Research Articles

Emitter as an Intramolecular Probe in a Luciferase Active Center

The distinctive features of firefly luciferase bioluminescence are complex changes in the shape of the spectra and λmax of bioluminescence with varying pH, temperature, and enzyme structure. An analysis of the published data and the authors’ own results leads to the conclusion that the keto–enol tautomerism of the oxyluciferin molecule explains the observed complex spectral changes most reliably. Only one molecule of an electronically excited product is formed in the active center of the enzyme; therefore, the emitter can be considered as an intramolecular probe characterizing the properties of the emitter microenvironment in the active center of the enzyme. The superposition of two or three forms of the emitter, recorded in the bioluminescence spectra, indicates the coexistence of various conformational forms of luciferase, being in dynamic equilibrium in the reaction medium. An analysis of the bioluminescence spectra enables the identification of qualitatively and quantitatively different conformers of the enzyme and their change with varying external conditions and luciferase structure.

Engineering Proton Transfer in Photosynthetic Oxygen Evolution: Chloride, Nitrate, and Trehalose Reorganize a Hydrogen-Bonding Network.

Photosystem II oxidizes water at a Mn4CaO5 cluster. Oxygen evolution is accompanied by proton release through a 35 Å hydrogen-bonding network to the lumen. The mechanism of this proton-transfer reaction is not known, but the reaction is dependent on chloride. Here, vibrational spectroscopy defines the functional properties of the proton-transfer network using chloride, bromide, and nitrate as perturbative agents. As assessed by peptide C═O frequencies, bromide substitution yields a spectral Stark shift because of its increase in ionic radius. Nitrate substitution leads to more complex spectral changes, consistent with an overall increase in hydrogen-bonding interactions with the peptide backbone. The effects are similar to spectral changes previously documented in site-directed mutations in a putative lumenal pathway. Importantly, the effects of nitrate are reversed by the osmolyte, trehalose. Trehalose is known to alter hydrogen-bonding interactions in proteins. Trehalose addition also reverses a shift in an internal hydronium ion signal, consistent with an alteration in its p Ka value and a change in the basicity of bound nitrate. The spectra provide evidence that the proton-transfer pathway contains peptide carbonyl groups, internal water, a hydronium ion, and amino acid side chains. These experiments also show that the proton-transfer pathway functionally adapts to changes in electric field, p Ka, and hydrogen bonding and thereby optimizes proton transfer to the lumen.

A unifying principle underlying the extracellular field potential spectral responses in the human cortex.

Electrophysiological mass potentials show complex spectral changes upon neuronal activation. However, it is unknown to what extent these complex band-limited changes are interrelated or, alternatively, reflect separate neuronal processes. To address this question, intracranial electrocorticograms (ECoG) responses were recorded in patients engaged in visuomotor tasks. We found that in the 10- to 100-Hz frequency range there was a significant reduction in the exponent χ of the 1/f(χ) component of the spectrum associated with neuronal activation. In a minority of electrodes showing particularly high activations the exponent reduction was associated with specific band-limited power modulations: emergence of a high gamma (80-100 Hz) and a decrease in the alpha (9-12 Hz) peaks. Importantly, the peaks' height was correlated with the 1/f(χ) exponent on activation. Control simulation ruled out the possibility that the change in 1/f(χ) exponent was a consequence of the analysis procedure. These results reveal a new global, cross-frequency (10-100 Hz) neuronal process reflected in a significant reduction of the power spectrum slope of the ECoG signal.

Label-Free Characterization of Cancer-Activated Fibroblasts Using Infrared Spectroscopic Imaging

Glandular tumors arising in epithelial cells comprise the majority of solid human cancers. Glands are supported by stroma, which is activated in the proximity of a tumor. Activated stroma is often characterized by the molecular expression of α-smooth muscle actin (α-SMA) within fibroblasts. However, the precise spatial and temporal evolution of chemical changes in fibroblasts upon epithelial tumor signaling is poorly understood. Here we report a label-free method to characterize fibroblast changes by using Fourier transform infrared spectroscopic imaging and comparing spectra with α-SMA expression in primary normal human fibroblasts. We recorded the fibroblast activation process by spectroscopic imaging using increasingly tissue-like conditions: 1), stimulation with the growth factor TGFβ1; 2), coculture with MCF-7 human breast cancerous epithelial cells in Transwell coculture; and 3), coculture with MCF-7 in three-dimensional cell culture. Finally, we compared the spectral signatures of stromal transformation with normal and malignant human breast tissue biopsies. The results indicate that this approach reveals temporally complex spectral changes and thus provides a richer assessment than simple molecular imaging based on α-SMA expression. Some changes are conserved across culture conditions and in human tissue, providing a label-free method to monitor stromal transformations.

Spectral changes during dipping in low-mass X-ray binaries due to highly-ionized absorbers

X-ray observations have revealed that many microquasars and low-mass X-ray binaries (LMXBs) exhibit narrow absorption features identified with resonant absorption from Fe XXV and Fe XXVI and other abundant ions. In many well studied systems there is evidence for blue-shifts, indicating outflowing plasmas. We succesfully model the changes in both the X-ray continuum and the Fe absorption features during dips from all the bright dipping LMXBs observed by XMM-Newton (EXO 0748-676, XB 1254-690, X 1624-490, MXB 1659-298, 4U 1746-371 and XB 1916-053) as resulting primarily from an increase in column density and a decrease in the ionization state of a highly-ionized absorber in a similar way as was done for 4U 1323-62. This implies that the complex spectral changes in the X-ray continua observed from the dip sources as a class can be most simply explained primarily by changes in the highly ionized absorbers present in these systems. There is no need to invoke unusual abundances or partial covering of extended emission regions. Outside of the dips, the absorption line properties do not vary strongly with orbital phase. This implies that the ionized plasma has a cylindrical geometry with a maximum column density close to the plane of the accretion disk. Since dipping sources are simply normal LMXBs viewed from close to the orbital plane this implies that ionized plasmas are a common feature of LMXBs.

Differences in the spectra of anomalous cosmic ray helium nuclei in two solar magnetic polarity cycles

We compare the spectra of anomalous He nuclei measured in the heliosphere by the IMP, Voyager, and Pioneer spacecraft at heliospheric distances ranging from 1 to over 80 AU in the time period from 1977 to 2003. Striking differences in the energy spectra of these nuclei are found between positive and negative solar magnetic polarity cycles. These differences are such that the spectra observed in negative polarity cycles appear to be greatly deficient in particles with energies of less than ∼15 MeV but have an excess intensity above >25 MeV, relative to the spectra observed in positive cycles. These spectral differences are complex and may be related to both solar modulation effects inside of the heliospheric termination shock as well as differences occurring at or near the termination shock itself. At the modulation minimum in 1998 when V2 was at a distance of 56 AU and V1 at 71 AU, a high intensity of low‐energy anomalous He particles was observed with a peak in the spectrum at ∼5 MeV/nuc. In late 2001 after the solar magnetic polarity had changed from positive to negative and the 11‐year solar modulation had increased, the previous high intensities of the anomalous He nuclei seen in 1998 were greatly reduced at both V1 and V2, and the energy of the peak intensity had moved up to ∼20–25 MeV/nuc. However, in both polarity periods the highest‐energy part of this spectrum above ∼30 MeV/nuc is similar with a slope of −2.8 ± 0.2. The details of these complex spectral changes are described in this paper.

Photochemical reactions of (eta 5-cyclopentadienyl)bis(t-butylacrylate) rhodium with silanes: dynamics of isomer interconversion via Rh(eta 2-silane) species.

The compound CpRh(C(2)H(3)CO(2)(t)Bu)(2) 1 has been synthesised as a mixture of two pairs of interconverting isomers which differ in the relative orientations of the alkene substituents. The four isomers have been fully characterised by NMR spectroscopy. When complex 1 is photolysed in the presence of a silane, HSiR(2)R'R(2)R'= Et(3), Me(3), HEt(2), (OMe)(3) and Me(2)Cl] the corresponding Si-H oxidative addition products CpRh(SiR(2)R')(H)(C(2)H(3)CO(2)(t)Bu) and CpRh(H)(2)(SiR(2)R')(2) are formed. The Rh(III) complexes CpRh(SiR(2)R')(H)(C(2)H(3)CO(2)(t)Bu) exist in two isomeric forms of comparable energy which interconvert in an intramolecular process that does not involve a reversible [1,3] hydride or [1,3] silyl migration. The hydride (1)H NMR resonances for these species consequently broaden before coalescing into a single peak. For R(2)R'= Et(3), the activation parameters for interchange from the major to minor isomer were Delta H++= 60.2 +/- 2 kJ mol(-1) and Delta S++= 8 +/- 9 J mol(-1) K(-1), while for R(2)R'= Me(3) and Et(2)H, Delta H++= 61.5 +/- 1 kJ mol(-1), Delta S++= 6 +/- 5 J mol(-1) K(-1), and Delta H++= 61.8 +/- 3 kJ mol(-1), Delta S++= 12 +/- 9 J mol(-1) K(-1) respectively for conversion from the major isomer to the minor. For these complexes an eta(2)-Rh-H-Si transition state or intermediate is consistent with the evidence. When R(2)R'=(OMe)(3) and Me(2)Cl the change in appearance of the hydride resonances is more complex, with the activation parameters for interchange from the major to minor isomer for the former species being Delta H++= 78.3 +/- 2 kJ mol(-1) and Delta S++= 30 +/- 7 J mol(-1) K(-1) while for Me(2)Cl the barrier proved too high to measure before decomposition occurred. The complex spectral changes could be simulated when a discrete eta(2)-Rh-H-Si intermediate was involved in the isomer interconversion process and hence silane rotation in all these systems is proposed to involve two isomers of CpRh(eta(2)-HSiR(2)R')(C(2)H(3)CO(2)(t)Bu).

Temporal variability of Mrk 421 from XMM–Newton observations

We present the results of a detailed spectral and temporal analysis of the currently available XMM-Newton obser- vations of the bright BL Lac object Mrk 421 using mainly the EPIC-PN data. The source was found in various intensity states differing by up to a factor of five in count rates. In general, the source is more variable and shows a harder spectrum during higher intensities than when it is in lower states. The spectrum is very complex and cannot be fitted adequately by a broken power law or a continuously curved model. We find that the flux variations on time scales of >few thousand seconds are associ- ated with significant and sometimes very complex spectral changes. The spectral variability rate is not the same in all cases and is correlated with the source flux state: the spectral variations per unit time increase with the source flux. The Cross-Correlation analysis shows that the soft and hard band light curves are often well correlated near zero lag, in other cases the hard band variations lead the soft band variations by typically ∼5 min, in two cases we find the soft band leading the hard band variations. The delays appear to be correlated to the flares' duration: the shorter the flare, the smaller the delay.

Calcium-Dependent Stabilization of the Central Sequence Between Met 76 and Ser 81 in Vertebrate Calmodulin

Spin-label electron paramagnetic resonance (EPR) provides optimal resolution of dynamic and conformational heterogeneity on the nanosecond time-scale and was used to assess the structure of the sequence between Met 76 and Ser 81 in vertebrate calmodulin (CaM). Previous fluorescence resonance energy transfer and anisotropy measurements indicate that the opposing domains of CaM are structurally coupled and the interconnecting central sequence adopts conformationally distinct structures in the apo-form and following calcium activation. In contrast, NMR data suggest that the opposing domains of CaM undergo independent rotational dynamics and that the sequence between Met 76 and Ser 81 in the central sequence functions as a flexible linker that connects two structurally independent domains. However, these latter measurements also resolve weak internuclear interactions that suggest the formation of transient helical structures that are stable on the nanosecond time-scale within the sequence between Met 76 and Asp 80 in apo-CaM (H. Kuboniwa, N. Tjandra, S. Grzekiek, H. Ren, C. B. Klee, and A. Bax, 1995, Nat. Struct. Biol. 2:768–776). This reported conformational heterogeneity was resolved using site-directed mutagenesis and spin-label EPR, which detects two component spectra for 1-oxyl-2,2,5,5-tetramethylpyrroline-3-methyl)-methanethiosulfonate spin labels (MTSSL) bound to CaM mutants T79C and S81C that include a motionally restricted component. In comparison to MTSSL bound within stable helical regions, the fractional contribution of the immobilized component at these positions is enhanced upon the addition of small amounts of the helicogenic solvent trifluoroethanol (TFE). These results suggest that the immobilized component reflects the formation of stable secondary structures. Similar spectral changes are observed upon calcium activation, suggesting a calcium-dependent stabilization of the secondary structure. No corresponding changes are observed in either the solvent accessibility to molecular oxygen or the maximal hyperfine splitting. In contrast, more complex spectral changes in the line-shape and maximal hyperfine splitting are observed for spin labels bound to sites that undergo tertiary contact interactions. These results suggest that spin labels at solvent-exposed positions within the central sequence are primarily sensitive to backbone fluctuations and that either TFE or calcium binding stabilizes the secondary structure of the sequence between Met 76 and Ser 81 and modulates the structural coupling between the opposing domains of CaM.

Contribution of extracellular Glu residues to the structure and function of bacteriorhodopsin. Presence of specific cation-binding sites.

Single and multiple mutants of extracellular Glu side chains of bacteriorhodopsin were analyzed by acid and calcium titration, differential scanning calorimetry, and thermal difference spectrophotometry. Acid titration spectra show that the second group protonating with Asp(85) is revealed in E204Q in the absence of Cl(-) but is not observed in the triple mutant E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q. The results point to Glu(9) as the second group protonating cooperatively with Asp(85). Comparison of the apparent pK(a) of Asp(85) protonation in water and in the deionized forms and results of calcium titration suggest that cation-binding sites are of low affinity in the multiple Glu mutants. Like for deionized wild type bacteriorhodopsin, differential scanning calorimetry reveals a lack of the pretransition in the multiple mutants, whereas in E9Q it appears at lower temperature and with lower cooperativity. Additionally, at neutral pH the band at 630 nm arising from cation release upon temperature increase is absent for the multiple mutants. Based on these results, we propose the presence of two cation-binding sites in the extracellular region of bacteriorhodopsin having as ligands Glu(9), Glu(194), Glu(204), and water molecules.

Real-time additive synthesis with envelope parameter stremas: an alternative to sampling

A sampling synthesizer uses a set of recordings for sound synthesis. A shortcoming of the sampling technique is the inability to produce complex spectral changes associated with the performer’s actions during a note. Additive synthesis, on the other hand, represents each sound as a collection of sine wave components, or partials. A wide variety of modifications are possible with additive synthesis, including frequency shifting, time dilation, cross synthesis, and sound morphing. Our real-time additive synthesis uses a set of recordings, like a traditional sampling synthesizer. Unlike a traditional sampling synthesizer, our additive synthesis models timbres as collections of bandwidth-enhanced partials (sine waves with noise) with time-varying parameters. The timbres are encoded in envelope parameter streams that can be manipulated in real time. These envelope parameter streams, which provide amplitude, frequency, phase, and bandwidth information for each partial, are the counterpart of sample streams in sampling synthesis. The talk will include demonstrations using the continuum fingerboard.

Reactions of glutamate semialdehyde aminotransferase (glutamate-1-semialdehyde 2,1 aminomutase) with vinyl and acetylenic substrate analogues analysed by rapid scanning spectrophotometry.

The reactions occurring when glutamate-1-semialdehyde amino-transferase (glutamate-1-semialdehyde 2,1 aminomutase, EC 5.4.3.8) was treated with two potential mechanism-based inactivators, namely 4-aminohex-5-enoate and 4-aminohex-5-ynoate, have been investigated by monitoring rapid transient changes in the absorption spectrum of the enzyme's prosthetic group, pyridoxal 5'-phosphate. In both cases a short-lived chromophore absorbing maximally at about 500 nm was formed in a few milliseconds. In the case of the vinyl analogue (4-aminohex-5-enoate) this chromophore, considered to be a quinonoid intermediate, converted rapidly into the pyridoxamine phosphate form of the co-enzyme in a single turnover which was accompanied by negligible inactivation. However, slow inactivation of the enzyme by this compound was observed when the enzyme was made to undergo multiple turnovers by including the efficient aldehyde substrate, succinic semialdehyde. The acetylenic compound, aminohexynoate, produced more complex spectral changes with the consecutive formation of compounds absorbing maximally at 496 nm, 450 nm, 564 nm and 330 nm. The enzyme was 90% inactivated by aminohexynoate within 10 s and thereafter lost no further activity unless aldehyde substrate was added. Mechanisms and kinetic constants consistent with the observations are proposed for each compound. The observation that the acetylenic compound is a much more potent inactivator than its vinyl analogue is attributed to the occurrence of a conjugated allene as intermediate.

Detection of simple and complex changes of spectral shape

In most of the previous studies (see Green, 1987) concerning the detection of a change in spectral shape, or "profile analysis," the listener's task was to detect an increment to a single component of an otherwise equal-amplitude, multicomponent background. An important theoretical issue is whether listeners' sensitivity to more complex spectral changes can be predicted from these results. In the present investigation, the sensitivity of a single group of listeners to a wide variety of simple and complex spectral changes was determined. After collecting the data, it was noted that almost all the thresholds could be predicted by a simple calculation scheme that assumed detection of a change in spectral shape occurs when the addition of the signal to the flat, multicomponent background produces a sufficient difference in level between only two regions of the spectrum. Unfortunately, this scheme, while successful for our limited set of data, fails to account for other "profile" data, namely, those obtained when the number of components is altered.

Frequency effects in profile analysis and detecting complex spectral changes

Seven experiments on the detectability of intensity changes in complex multitonal acoustic spectra are reported. Two general questions organize the experimental efforts. The first question is how the detectability of a change in a flat (equal energy) spectrum depends on the frequency region where a single intensive change is made. The answer is that frequency region plays a relatively minor role. Frequency changes in the midregion of the spectrum are the easiest to hear, but thresholds increase by only about 5 dB over the range from 200 to 5000 Hz. For all frequencies, the psychometric function is of the form d' = k(delta p), where k is a constant and delta p is the change in pressure. The second question is how can we predict the detectability of complex changes over the entire frequency range from the detectability of change at each separate region. Thresholds for detecting a change from a flat spectrum to a spectrum whose amplitude varies in sinusoidal ("rippled") fashion over logarithmic frequency are measured at different frequencies of ripple. The thresholds are found to be independent of ripple frequency and are 7 dB higher than predicted on the basis of an optimum combination rule.

Interactions of divalent and trivalent cations with antibiotic U-20,661, a DNA binding agent

The absorption maximum of antibiotic U-20,661 undergoes a hathochromic shift from 440 mμ in acidic solution to 530 mμ in strong basic solution. Divalent cation species in ten- to hundredfold molar excess over the antibiotic have been found to induce identical shifts to 530 mμ in neutral solutions. An isosbestic point was apparent at 475 mμ. Monovalent cations did not cause such an effect. The trivalent cations Fe 3+ or Al 3+ induced complex spectralchanges which differed significantly from each other as well as from the changes observed with divalent cations. The extent of the binding capacity of the antibiotic to DNA remained unaffected by the presence of di- or trivalent cations.