FTIR spectroscopic analysis of Saccharomyces cerevisiae cell walls: study of an anomalous strain exhibiting a pink-colored cell phenotype

A new strain, exhibiting an intriguing pink-colored cell phenotype, was obtained after an encoding alpha-glucosidase gene from an archaebacteria Thermococcus hydrothermalis was cloned by functional complementation of a mal11 Saccharomyces cerevisiae mutant TCY70. The possible implications of the alpha-glucosidase on the cell wall were evaluated by infrared spectroscopy and data indicate a 30% decrease in mannoproteins and an increase in beta-glucans. The loss of mannoproteins was confirmed by experiments on cells deprived of peptidomannans. Modifications in the major components of the cell wall did not jeopardize cell viability. Such rapid optical spectroscopic method can be used to screen a wide range of yeast mutants.     

Influence of environment on piroxicam polymorphism: vibrational spectroscopic study

FTIR and FT-Raman spectroscopies were used to evaluate the mechanism of transformation of piroxicam into its different forms (alpha, beta, and monohydrate), depending on the environment. These vibrational techniques allowed us to identify the forms of piroxicam that crystallize from different solvents at different cooling rates and the conformation of the drug in some of its derivatives: piroxicam hydrochloride, piroxicam thallium and sodium salt hemihydrates, and piroxicam sodium salt. The usefulness of Raman spectroscopy in characterizing piroxicam:beta-cyclodextrin (PbetaCD) inclusion compounds was described. The Raman spectrum of 1:2 PbetaCD was discussed in comparison with that of the corresponding piroxicam sodium salt containing inclusion compound (1:2 PNabetaCD) in order to study the influence of the piroxicam derivative used on the structure of the inclusion compound. The Raman results showed that in both of the inclusion compounds the piroxicam mainly assumes the zwitterionic structure typical of a monohydrate; therefore, the kind of derivative used does not affect the conformation of the drug in its inclusion compound. The effect of the method of synthesis utilized (freeze-drying or freeze-thaw cycling) to obtain 1:2.5 PbetaCD was investigated. The inclusion compound obtained by freeze-thaw cycling proved to be more crystalline and to contain a higher amount of the beta form than the freeze-dried inclusion compound. Raman spectroscopy proved to be a useful technique for evaluating the effectiveness of the manufacturing process in relation to the pharmaceutical properties of the drug and to the nondestructive and noninvasive on-line quality control of the industrial products.     

In situ FTIR ATR spectroscopic study of the interaction of immobilized human tumor necrosis factor-alpha with a monoclonal antibody in aqueous environment

By in situ FTIR ATR measurements, the antibody (AB) recognition of human tumor necrosis factor-alpha (TNFalpha) immobilized on the Ge surface of a multiple internal reflection element (MIRE) was investigated. The experiments were performed in aqueous environment in a flow-through cell. After immobilization of TNFalpha on the Ge-MIRE by direct adsorption from aqueous solution, the immobilisate reached stability after about 1 h under flow-through conditions. The remaining sites of the Ge surface were saturated by bovine serum albumin (BSA) in order to prevent unspecific binding of anti-TNFalpha AB which was then added. The obtained FTIR ATR spectra were shown to result exclusively from AB specifically interacting with TNFalpha, since the absence of immunoglobulin binding to BSA adsorbed to the Ge MIRE was verified by a reference experiment. Finally, the stability of all adsorbed protein immobilisates was monitored under flow-through conditions for 10.5 h. The TNFalpha-AB complex showed a decrease of 7.4%, whereas the BSA adsorbate remained stable. IR measurements were performed with polarized light in order to study orientational effects of the immobilized proteins. The dichroic ratios and surface concentrations of all used proteins are available after quantitative analysis of the amide II bands.     

The molecular origin of the inhibition of transducin activation in rhodopsin lacking the 9-methyl group of the retinal chromophore: a UV-Vis and FTIR spectroscopic study

The formation of the active rhodopsin state metarhodopsin II (MII) is believed to be partially governed by specific steric constraints imposed onto the protein by the 9-methyl group of the retinal chromophore. We studied the properties of the synthetic pigment 9-demethyl rhodopsin (9dm-Rho), consisting of the rhodopsin apoprotein regenerated with synthetic retinal lacking the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by the modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equilibrium shift from MII to its immediate precursor, MI. The MII state of 9dm-Rho displays only a partial deprotonation of the retinal Schiff base, leading to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII formation is slowed by 2 orders of magnitude compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respective states in the native pigment, pointing to a more relaxed fit of the retinal chromophore in its binding pocket. The shifted equilibrium between MI and MII is therefore discussed in terms of an increased entropy of the 9dm-Rho MI state due to changed steric interactions.     

Investigating the interaction of saposin C with POPS and POPC phospholipids: a solid-state NMR spectroscopic study

The interaction of Saposin C (Sap C) with negatively charged phospholipids such as phosphatidylserine (PS) is essential for its biological function. In this study, Sap C (initially protonated in a weak acid) was inserted into multilamellar vesicles (MLVs) consisting of either 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-L-serine] (negatively charged, POPS) or 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (neutrally charged, POPC). The MLVs were then investigated using solid-state NMR spectroscopy under neutral pH (7.0) conditions. The (2)H and (31)P solid-state NMR spectroscopic data of Sap C-POPS and Sap C-POPC MLVs (prepared under the same conditions) were compared using the (2)H order parameter profiles of the POPC-d(31) or POPS-d(31) acyl chains as well as the (31)P chemical shift anisotropy width and (31)P T(1) relaxation times of the phospholipids headgroups. All those solid-state NMR spectroscopic approaches indicate that protonated Sap C disturbs the POPS bilayers and not the POPC lipid bilayers. These observations suggest for the first time that protonated Sap C inserts into PS bilayers and forms a stable complex with the lipids even after resuspension under neutral buffer conditions. Additionally, (31)P solid-state NMR spectroscopic studies of mechanically oriented phospholipids on glass plates were conducted and perturbation effect of Sap C on both POPS and POPC bilayers was compared. Unlike POPC bilayers, the data indicates that protonated Sap C (initially protonated in a weak acid) was unable to produce well-oriented POPS bilayers on glass plates at neutral pH. Conversely, unprotonated Sap C (initially dissolved in a neutral buffer) did not interact significantly with POPS phospholipids allowing them to produce well-oriented bilayers at neutral pH.     

Pressure versus temperature unfolding of ribonuclease A: an FTIR spectroscopic characterization of 10 variants at the carboxy-terminal site

FTIR spectroscopy was used to characterize and compare the temperature- and pressure-induced unfolding of ribonuclease A and a set of its variants engineered in a hydrophobic region of the C-terminal part of the molecule postulated as a CFIS. The results show for all the ribonucleases investigated, a cooperative, two-state, reversible unfolding transition using both pressure and temperature. The relative stabilities, among the different sites and different variants at the same site, monitored either through the changes in the position of the maximum of the amide I' band and the tyrosine band, or the maximum of the band assigned to the beta-sheet structure, corroborate the results of a previous study using fourth-derivative UV absorbance spectroscopy. In addition, variants at position 108 are the most critical for ribonuclease structure and stability. The V108G variant seems to present a greater conformational flexibility than the other variants. The pressure- and temperature-denaturated states of all the ribonucleases characterized retained some secondary structure. However, their spectral maxima were centered at different wavenumbers, which suggests that pressure- and temperature-denaturated states do not have the same structural characteristics. Nevertheless, there was close correlation between the pressure and temperature midpoint transition values for the whole series of protein variants, which indicated a common tendency of stability toward pressure and heat.     

Consequences of counterion mutation in sensory rhodopsin II of Natronobacterium pharaonis for photoreaction and receptor activation: an FTIR study

In many retinal proteins the proton transfer from the Schiff base to the counterion represents a functionally important step of the photoreaction. In the signaling state of sensory rhodopsin II from Natronobacterium pharaonis this transfer has already occurred, but in the counterion mutant Asp75Asn it is blocked during all steps of the photocycle. Therefore, the study of the molecular changes during the photoreaction of this mutant should provide a deeper understanding of the activation mechanism, and for this, we have applied time-resolved step-scan FTIR spectroscopy. The photoreaction is drastically altered; only red-shifted intermediates are formed with a chromophore strongly twisted around the 14-15 single bond. In addition, the photocycle is shortened by 2 orders of magnitude. Nevertheless, a transition involving only protein changes similar to that of the wild type is observed, which has been correlated with the formation of the signaling state. However, whereas in the wild type this transition occurs in the millisecond range, it is shortened to 200 micros in the mutant. The results are discussed with respect to the altered electrostatic interactions, role of proton transfer, the published 3D structure, and physiological activity.     

Altered hydrogen bonding of Arg82 during the proton pump cycle of bacteriorhodopsin: a low-temperature polarized FTIR spectroscopic study

Light-driven proton transport in bacteriorhodopsin (BR) is achieved by dynamic rearrangement of the hydrogen-bonding network inside the membrane protein. Arg82 is located between the Schiff base region and proton release group, and has a major influence on the pK(a) values of these groups. It is believed that Arg82 changes its hydrogen-bonding acceptors during the pump cycle of BR, stages of which are correlated with proton movement along the transport pathway. In this study, we compare low-temperature polarized FTIR spectra of [eta(1,2)-(15)N]arginine-labeled BR in the 2750-2000 cm(-1) region with those of unlabeled BR for the K, L, M, and N intermediates. In the K-minus-BR difference spectra, (15)N-shifted modes were found at 2292 (-)/2266 (+) cm(-1) and at 2579 (-)/2567 (+) cm(-1). The former corresponds to strong hydrogen bonding, while the latter corresponds to very weak hydrogen bonding. Both N-D stretches probably originate from Arg82, the former oriented toward water 406 and the latter toward the extracellular side, and both hydrogen bonds are somewhat strengthened upon retinal photoisomerization. This perturbation of arginine hydrogen bonding is entirely relaxed in the L intermediate where no (15)N-isotope shifts are observed in the difference spectrum. In the M intermediate, the frequency is not significantly altered from that in BR. However, the polarized FTIR spectra strongly suggest that the dipolar orientation of the strongly hydrogen bonded N-D group of Arg82 is changed from perpendicular to parallel to the membrane plane. Such a change is presumably related to the motion of the Arg82 side chain from the Schiff base region to the extracellular proton release group. Additional bands corresponding to weak hydrogen bonding were observed in both the M-minus-BR and N-minus-BR spectra. Changes in hydrogen-bonding structures involving Arg82 are discussed on the basis of these FTIR observations.     

Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii beta-lactamase: a time-resolved FTIR spectroscopic study

Time-resolved infrared difference spectroscopy has been used to show that the carbonyl group of the acylenzyme reaction intermediate in the Citrobacter freundii beta-lactamase-catalyzed hydrolysis of methicillin can assume at least four conformations. A single-turnover experiment shows that all four conformations decline during deacylation with essentially the same rate constant. The conformers are thus in exchange on the reaction time scale, assuming that deacylation takes place only from the conformation which is most strongly hydrogen bonded or from a more minor species not visible in these experiments. All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethyl ester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to free solution. The polarity of the carbonyl group environment in the conformers varies from 'ether-like' to strongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme. From the absorption intensities, it is estimated that the conformers are populated approximately proportional to the hydrogen bonding strength at the carbonyl oxygen. A change in the difference spectrum at 1628 cm-1 consistent with a perturbation (relaxation) of protein beta-sheet occurs slightly faster than deacylation. Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation in the acyl group is a plausible explanation of the change seen at 1628 cm-1. A turnover reaction supports the above conclusions and shows that the conformational relaxation occurs as the substrate is exhausted and the acylenzymes decline. The observation of multiple conformers is discussed in relation to the poor specificity of methicillin as a substrate of this beta-lactamase and in terms of X-ray crystallographic structures of acylenzymes where multiple forms are not apparently observed (or modeled). Infrared spectroscopy has shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactions in physiological turnover conditions as well as for determination of ordering, hydrogen bonding, and protein perturbation.     

Membrane interactions of a new class of anticancer agents derived from arylchloroethylurea: a FTIR spectroscopic study.

We have investigated the interaction between a new class of antineoplastic agents derived from arylchloroethylurea (CEU) and different lipids such as dimyristoylphosphatidylcholine (DMPC) in the absence and presence of 30 mol% of cholesterol, dimyristoylphosphatidylglycerol (DMPG) and a mixture made of 1-palmitoyl-2-oleylphosphatidylcholine (POPC) and DMPC by Fourier transform infrared (FTIR) spectroscopy. The results indicate that the drugs incorporate in the bilayer and cause a decrease of the phase transition temperature and an increase of the conformational disorder of the lipid acyl chains. These effects are dependent on the nature (degree of branching, length of the alkyl chain and presence of a sulfur atom), as well as on the position of the R substituent and are related to the cytotoxicity of the drugs. More specifically, the more cytotoxic drugs, such as 4-sec-butyl CEU, are those having a bulky branched substituent and those for which the disordering effect on the lipid bilayer is the greatest. On the other hand, the disordering effect is small for the long chain CEUs, such as 4-n-hexadecyl CEU, which have been shown to have weak cytotoxic activity.     

PI transfer protein: the specific recognition of phospholipids and its functions

Phosphatidylinositol transfer proteins (PITPs) can bind specifically and transfer a single phosphatidylinositol (PI) molecule between phospholipid membranes in an ATP-independent manner in vitro. PITPs exist in all the eukaryotic systems from yeast to human. PITP plays an essential role in intracellular vesicle flow and inositol lipid signaling. The crystal structure of yeast PITP Sec14p reveals a large hydrophobic pocket to accommodate the acyl chains of phospholipid molecules. At the opening of the pocket, a hydrogen bond network may render Sec14p the binding specificity to PI molecules. The structure suggests that the PI-binding ability may play an important role in the in vivo function of PITPs.     

Unraveling the substrate-metal binding site of ferrochelatase: an X-ray absorption spectroscopic study

Ferrochelatase (EC 4.99.1.1), the terminal enzyme of the heme biosynthetic pathway, catalyzes the insertion of ferrous iron into the protoporphyrin IX ring. Ferrochelatases can be arbitrarily divided into two broad categories: those with and those without a [2Fe-2S] center. In this work we have used X-ray absorption spectroscopy to investigate the metal ion binding sites of murine and Saccharomyces cerevisiae (yeast) ferrochelatases, which are representatives of the former and latter categories, respectively. Co(2+) and Zn(2+) complexes of both enzymes were studied, but the Fe(2+) complex was only studied for yeast ferrochelatase because the [2Fe-2S] center of the murine enzyme interferes with the analysis. Co(2+) and Zn(2+) binding to site-directed mutants of the murine enzyme were also studied, in which the highly conserved and potentially metal-coordinating residues H207 and Y220 were substituted by residues that should not coordinate metal (i.e., H207N, H207A, and Y220F). Our experiments indicate four-coordinate zinc with Zn(N/O)(3)(S/Cl)(1) coordination for the yeast and Zn(N/O)(2)(S/Cl)(2) coordination for the wild-type murine enzyme. In contrast to zinc, a six-coordinate site for Co(2+) coordinated with oxygen or nitrogen was present in both the yeast and murine (wild-type and mutated) enzymes, with evidence of two histidine ligands in both. Like Co(2+), Fe(2+) bound to yeast ferrochelatase was coordinated by approximately six oxygen or nitrogen ligands, again with evidence of two histidine ligands. For the murine enzyme, mutation of both H207 and Y220 significantly changed the spectra, indicating a likely role for these residues in metal ion substrate binding. This is in marked disagreement with the conclusions from X-ray crystallographic studies of the human enzyme, and possible reasons for this are discussed.     

Structure of frataxin iron cores: an X-ray absorption spectroscopic study

X-ray absorption spectroscopy at the iron K-edge indicates that the iron cores of human and yeast frataxin polymers assembled in vitro are identical to each other and are similar but not identical to ferritin cores. Both frataxin polymers contain ferrihydrite, a biomineral composed of ferric oxide/hydroxide octahedra. The ferrihydrite in frataxin is less ordered than iron cores of horse spleen ferritin, having fewer face-sharing Fe-Fe interactions but similar double corner-sharing interactions. The extended X-ray absorption fine structure (EXAFS) analysis agrees with previous electron microscopy data showing that frataxin cores are composed of very small ferrihydrite crystallites.     

Modification of phospholipids in erythrocyte membranes by phospholipase D. A fluorescence and ESR spectroscopic study

The conversion of more than 65% of the phospholipids in human erythrocyte membranes to phosphatidyl-methanol and phosphatidic acid by incubation with phospholipase D and methanol increased the dissociation constant of the fluorescence probe ANS compared to untreated membranes, but did not affect the number of binding sites and the limiting fluorescence enhancement at maximal binding (Imax). On the contrary, the cationic fluorescence probe dansylcadaverin showed additional binding sites without a change in Kd and an increase of Imax upon incubation with phospholipase D treated erythrocyte membranes compared to incubations of membranes with the original phospholipid pattern. The characteristic temperature-dependence of the quenching of the membrane protein fluorescence by a membrane-bound nitroxide-labeled stearic acid was not influenced by the modification of the phospholipids. A slight reduction of the order parameter, S, determined by ESR-spectroscopy with the same nitroxide spin-labeled fatty acid incorporated into modified membranes compared to controls was found at 40 degrees C, but not at 25 degrees C. The results were interpreted as an indication of membrane domains that retained their physical properties and lipid composition during the incubation with phospholipase D.     

Navigating abstract virtual environment: an eeg study

Perceptions of different environments are different for different people. An abstract designed environment, with a degree of freedom from any visual reference in the physical world requests a completely different perception than a fully or semi-designed environment that has some correlation with the physical world. Maximal evidence on the manner in which the human brain is involved/operates in dealing with such novel perception comes from neuropsychology. Harnessing the tools and techniques involved in the domain of neuropsychology, the paper presents nee evidence on the role of pre-central gyrus in the perception of abstract spatial environments. In order to do so, the research team developed three different categories of designed environment with different characteristics: (1) Abstract environment, (2) Semi-designed environment, (3) Fully designed environment, as experimental sample environments. Perception of Fully-designed and semi-designed environments is almost the same, [maybe] since the brain can find a correlation between designed environments and already experienced physical world. In addition to this, the response to questionnaires accompanied with a list of buzzwords that have been provided after the experiments, also describe the characteristics of the chosen sample environments. Additionally, these results confirm the suitability of continuous electroencephalography (EEG) for studying Perception from the perspective of architectural environments.     

The vanadium environment in blood cells of Ascidia ceratodes is divergent at all organismal levels: an XAS and EPR spectroscopic study

K-edge X-ray absorption and EPR spectroscopies were used to test the variation in blood cell vanadium between and within specimens of the tunicate Ascidia ceratodes from Bodega Bay, California. Intracellular vanadium was speciated by fitting the XAS spectra of whole blood cells with linear combinations of the XAS spectra of models. Blood cell samples representing one specimen each, respectively, revealed 92.5 and 38.7% of endogenous vanadium as [V(H(2)O)(6)](3+), indicating dissimilar distributions. Conversely, vanadium distributions within blood cell samples respectively representing one and six specimens proved very similar. The derived array of V(III) complexes was consistent with multiple intracellular regions that differ both in pH and c(sulfate), both within and between specimens. No systematic effect on vanadium distribution was apparent on mixing blood cells. EPR and XAS results indicated at least three forms of endogenous vanadyl ion, two of which may be dimeric. An inverse linear correlation was found between soluble and complexed forms of vanadyl ion, implying co-regulation. The EPR A value of endogenous vanadyl ion [A(0)=(1.062+/-0.008)x10(-2) cm(-1)] was marginally different from that representing Monterey Bay A. ceratodes [A(0)=(1.092+/-0.006) x10(-2) cm(-1)]. Comparisons indicate that Bodega Bay A. ceratodes maintain V(III) in a more acidic intracellular environment on average than do those from Monterey Bay, showing variation across populations. Blood cell vanadium thus noticeably diverges at all organismal levels among A. ceratodes.     

FTIR spectroscopic evidence for the involvement of an acidic residue in quinone binding in cytochrome bd from Escherichia coli

In this work, FTIR difference spectroscopy is used to search for possible binding partners and protonable groups involved in the binding of the quinol to cytochrome bd from Escherichia coli. In addition, the electrochemically induced FTIR difference spectra are compared for preparations of the enzyme isolated from cells grown at different oxygen levels in which the quinone content of the membrane is altered. On this basis, difference signals can be tentatively attributed to the vibrational modes of the different quinones types that are associated with the enzyme depending on growth conditions. Furthermore, vibrational modes due to the redox-dependent reorganization of the protein vary depending on the quinone associated with the isolated enzyme. Of particular interest are the observations that a mode at 1738 cm(-1) is decreased and a mode at 1595 cm(-1) is increased as observed in direct comparison to the data obtained from samples grown anaerobically. These signals indicate a change in the protonation state of an aspartic or glutamic acid. Since these changes are observed when the ubiquinone ratio in the preparation increases, the data provide evidence for the modulation of the binding site by the interacting quinone and the involvement of an acidic group in the binding site. The tentative assignments of the vibrational modes are supported by electrochemically induced FTIR difference spectra of cytochrome bd in the presence of the specific quinone binding site inhibitors heptylhydroxyquinoline-N-oxide (HQNO) or 2-methyl-3-undecylquinolone-4. Whereas HQNO leads to strong shifts in the FTIR redox difference spectrum, 2-methyl-3-undecylquinolone-4 induces a specific shift of a mode at 1635 cm(-1), which likely originates from the displacement of the C=O group of the bound quinone.     

Characterization of gramicidin A in an inverted micellar environment. A combined high-performance liquid chromatographic and spectroscopic study.

We have investigated the conformational adaptability of gramicidin A incorporated into reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water, a so far unexplored "host" membrane-mimetic model system for this peptide. A high-performance liquid chromatographic strategy previously developed for the study of gramicidin in phospholipid vesicles and normal micelles [Ba?ó et al. (1989) FEBS Lett. 250, 67; Ba?ó et al. (1991) Biochemistry 30, 886] has been successfully extended to this system. The method has permitted the separation of peptide conformational species, namely, double-stranded dimers and monomers, and an accurate quantitation of their proportion in the inverted micellar environment. It has been demonstrated that, once inserted in the micelle, the double-stranded dimers undergo a dissociation process toward a thermodynamically stable monomeric configuration, whose monomerization rate constant (k1) is dependent in a bell-shaped manner on the water:surfactant mole ratio, w0. A tight correlation between k1 and the double-stranded dimer backbone conformation has been found from the comparison of chromatographic and circular dichroism data. In addition, fluorescence experiments indicate that the peptide tryptophans are in a rather nonpolar environment, with a restricted accessibility to water-soluble quenchers such as acrylamide.     

Selenium alters the lipid content and protein profile of rat heart: an FTIR microspectroscopic study

Cardiovascular disease is one of the most important causes of morbidity and mortality in Western countries. In addition, it is well documented that selenium (Se) deficiency has been linked to cardiovascular diseases. This study was undertaken to present the effect of sodium selenite on left and right myocardia, and small veins of normal control rat heart at molecular level by using Fourier transform infrared (FTIR) microspectroscopy. The results mainly reveal that, Se treatment causes an increase in lipid content both in the saturated and unsaturated lipids, and an alteration in protein profile with a decrease in alpha-helix and an increase in beta-sheet structure of the rat heart which might be reflecting a slight subtoxic effect of selenium supplementation on normal rat heart at the dose used in this study.