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PubMed1991

In vivo study of the state of order of the membranes of gram-negative bacteria by Fourier-transform infrared spectroscopy (FT-IR).

Temperature-induced order/disorder transition profiles were obtained from the membranes of intact Gram-negative bacterial cells by FT-IR analysis of the frequency shifts of the acyl chain methylene symmetric stretching band as a monitor. Cells grown at different temperatures yielded distinct transition profiles. At the individual growth temperatures, however, the nearly alike frequency values indicated a very similar 'state of order' of the bacterial membranes. The FT-IR data were complemented by GC analysis of whole cell fatty acid composition. The FT-IR data obtained in vivo gave direct evidence of the adaptation of the 'state of order' and 'fluidity' of bacterial membranes to varying growth temperatures.

Cell Membrane↗
PubMed1991

Fourier transform infrared spectroscopy of aqueous dispersions of phosphatidylserine-cholesterol mixtures.

The effect of cholesterol on vibrational spectra in the non polar and in the polar region of dimyristoyl phosphatidylserine (DMPS) and of phosphatidylserine from bovine spinal cord (PS) has been investigated. The small shifts in the methylene CH stretching frequencies after taking into account the contribution of the cholesterol spectrum were interpreted as a combined effect of cholesterol on the conformation of the chains and of the lesser contributions of the cholesterol methyl groups. Cholesterol also influences the ratio of the trans (1465 cm-1) to the lower wavelength (1457 cm-1) CH2 bending bands. No significant direct effect of cholesterol on the vibration of the polar residues was discerned. The small shift of the carboxylate band observed below the phase transition is probably due to the change in the intermolecular zwitterions when the average distance between the neighboring polar groups increases due to incorporation of cholesterol molecules.

Animals↗
PubMed1983

Polarization of substrate carbonyl groups by yeast aldolase: investigation by Fourier transform infrared spectroscopy.

The infrared spectrum of the complex of D-fructose 1,6-bisphosphate bound to yeast aldolase displays three spectral features between 1700 and 1800 cm-1. One of these (at 1730 cm-1) corresponds to the carbonyl group of enzyme-bound D-fructose 1,6-bisphosphate and/or dihydroxyacetone phosphate. The frequency of this band, which is unaffected by the removal of the intrinsic zinc ion from the enzyme, demonstrates that this carbonyl group is not significantly polarized when the substrate binds to the enzyme. In contrast, the spectral band assigned to the carbonyl group of enzyme-bound D-glyceraldehyde 3-phosphate (at 1706 cm-1) appears at a frequency 24 cm-1 lower than when this substrate is in aqueous solution. This shift indicates considerable polarization of the carbonyl group when D-glyceraldehyde 3-phosphate is bound at the active site. The third spectral feature (at 1748 cm-1), which is observed only in the presence of potassium ion, probably corresponds to an enzymic carboxyl group in a nonpolar environment.

Chemical Phenomena↗
PubMed1993

Orientation of fusion-active synthetic peptides in phospholipid bilayers: determination by Fourier transform infrared spectroscopy.

A group of synthetic peptides having an amino acid sequence related to the N-terminal region of the influenza virus hemagglutinin HA-2 chain can induce phospholipid membrane fusion in a pH-dependent manner. These peptides bind to membranes to form alpha-helices even at pH's where no fusion activity is seen. We determined the orientation of these alpha-helical peptides in lipid multibilayers using attenuated total reflection infrared spectroscopy and found that the peptide alpha-helices took a preferential orientation, the helix axis being about 70 degrees from the normal of the membrane plane, or in other words rather parallel to the membrane plane. The orientation was almost independent of pH and a modification of the N-terminal amino group which reduced the fusion activity of the peptides. The determination was carried out for peptides in lipid multibilayers in dry or hydrated (membranes equilibrated with D2O vapor) conditions. Although a slight decrease in the helix orientation angle from the membrane normal was noticed for a hydrated system, the difference between the results for dry and hydrated conditions was small.

Amino Acid Sequence↗
PubMed1987

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase.

The infrared spectrum of oxaloacetate bound in the active site of citrate synthase has been measured in the binary complex and in the ternary complex with the acetyl coenzyme A (CoA) enolate analogue carboxymethyl-CoA. The carbonyl stretching frequency of oxaloacetate in binary and ternary complexes is found at 1697 cm-1, a shift of 21 cm-1 to lower frequency relative to that of the free ligand. The line widths of the carbonyl absorption in enzyme complexes differ from that of the free ligand, decreasing from a value of 20 cm-1 for the free ligand to 10 cm-1 in the binary complex and 7 cm-1 in the ternary complex with carboxymethyl-CoA. The integrated absorbance of the carbonyl absorption in these enzyme complexes is significantly increased over that of the free ligand at the same concentration, increasing approximately 2-fold in the binary complex and approximately 3-fold in the ternary complex. These results indicate strong polarization of the carbonyl bond in the enzyme-substrate complexes and suggest that ground-state destabilization is a major catalytic strategy of citrate synthase.

Acetyl Coenzyme A↗
PubMed1987

Conformational changes in concanavalin A associated with demetallization and alpha-methylmannose binding studied by Fourier transform infrared spectroscopy.

Infrared spectra of concanavalin A have been obtained both in the absence and in the presence of the metal ions, Mn2+ and Ca2+, and the saccharide, alpha-methylmannose. Second derivative calculations have been used to determine the frequencies of the different amide I and II components. In the demetallized protein dissolved in H2O buffer, absorptions in the amide I, II and III regions at 1695 and 1634, 1532 and 1237 cm-1, respectively, are assigned to beta-structure, while absorptions at 1563 and both 1318 and 1343 cm-1 are assigned to turns and bends. After deuterium exchange, the residual amide II maximum in the difference spectrum shifts from 1538 to 1563 cm-1, indicating that exchange is faster in the beta-structure than in the turns. In the presence of Mn2+ and Ca2+, the amide II band component at 1532 cm-1 shifts 4-6 cm-1 to higher wavenumbers, and the amide I band component at 1634 shifts 1 cm-1 in the same direction, both in H2O and 2H2O buffers, suggesting changes in the hydrogen-bonding network of a large portion of the protein, particularly in the beta-sheet regions. The addition of alpha-methylmannose increases the magnitude of exchange from 55% to above 90%. Comparison with existing X-ray crystallographic data has been made, and the usefulness of FT-IR to complement this technique is discussed.

Calcium↗
PubMed1989

Molecular surface characterization of oral streptococci by Fourier transform infrared spectroscopy.

In order to characterize the molecular composition of oral streptococci, infrared transmission spectroscopy on freeze-dried cells dissolved in KBr was used. All infrared spectra show similar absorption bands for the strains studied with the most important absorption bands located at 2930 cm-1 (CH), 1653 cm-1 (AmI), 1541 cm-1 (AmII) and two bands at 1236 cm-1 and 1082 cm-1, which were assigned to phosphate and sugar groups. However, calculation of absorption band ratios normalized with respect to the integrated intensity of the CH stretching region around 2930 cm-1, show significant differences between the strains. Both Streptococcus mitis strains possess high AmI/CH and AmII/CH absorption band ratios compared to the other strains. Streptococcus salivarius HBC12, a mutant strain devoid of all proteinaceous surface appendages, shows significantly lower AmI/CH and AmII/CH band ratios with respect to its parent strain S. salivarius HB. Two positive relationships could be established both between the AmII/CH absorption band ratio and the N/C elemental surface concentration ratio of the strains previously, determined from X-ray photoelectron spectroscopy (XPS) and also between AmI/CH and the fraction of carbon atoms at the surface involved in amide bonds, determined by XPS as well. From this comparison, it is concluded that transmission infrared spectroscopy can be employed as a technique to study the molecular surface composition of freeze-dried microorganisms.

Amides↗
PubMed1989

Fourier-transform infrared spectroscopy studies of lipid/protein interaction in pulmonary surfactant.

The thermotropic behavior of intact bovine lung surfactant and its hydrophobic extract has been monitored via the temperature dependence of the 2850 cm-1 phospholipid acyl chain CH2 symmetric stretching frequencies in the IR spectrum. A broad, reversible, melting event was noted from about 15 to 40 degrees C in both the lipid extract and the native surfactant. Slight protein-induced disordering of the lipid acyl chains was evident. The melting event was confirmed by differential scanning calorimetry. The major surfactant protein, a 30-36-kDa class of glycoprotein (SP-A), has been isolated from bovine lung lavage and purified by affinity chromatography. SP-A was reconstituted into a binary lipid mixture of acyl chain perdeuterated dipalmitoylphosphatidylcholine/dipalmitoylphosphatidylglycerol (DPPC-d62/DPPG, 85:15 w/w), a ratio which approximates that in surfactant. Use of DPPC-d62 permitted the FT-IR determination of the effect of protein on the thermotropic behavior of individual phospholipids in the binary mixture. High levels of SP-A induced an ordering of the phospholipids, as shown by an increase in the transition temperature of DPPC-d62 compared to the lipid model. In contrast, a mixture of the other surfactant proteins induced a progressive disordering of the phospholipids and disruption of the cooperativity of the melting event. Transition widths of about 3 degrees, 9 degrees, and 27 degrees were noted for protein:lipid ratios of 0, 1:1, and 2:1 (w/w), respectively. Possible roles for the various proteins in surfactant function are discussed in light of these data.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1992

Potential of 13C and 15N labeling for studying protein-protein interactions using Fourier transform infrared spectroscopy.

In this study, we examine the interaction between two bacterial proteins, namely HPr and IIAmtl of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system, using FTIR spectroscopy. In an interaction involving a 1:1 molar ratio of these two proteins, when they are unlabeled, the overlap of absorbance of the amide I band arising from the peptide group vibrations of the two proteins is such that it is not possible to determine the contribution which each protein makes to the absorbance. Uniform 15N labeling has little effect on the frequency of the amide I band although there is a significant shift of the amide II band. However, we show that uniform (90%) 13C labeling produces a large shift of bands associated with the carbonyl moiety, especially the amide I band. This opens up windows in different regions of the infrared spectrum. Thus, when the same mixture of the two bacterial proteins is made where one of the proteins is uniformly 13C-labeled (in our case HPr), the amide I maxima of this protein shifts by approximately 45 cm-1 toward lower frequency and reveals the previously overlapped amide I band of the unlabeled IIAmtl. This application of 13C labeling shows the potential of studying protein-protein interactions using FTIR spectroscopy. With thoughtful selection of systems and labeling strategies, numerous studies with proteins should be possible. These could include, among others, enzyme-substrate and protein-ligand interactions.

Bacterial Proteins↗
PubMed1990

Quantitative determination of hydrocarbon chain conformational order in bilayers of saturated phosphatidylcholines of various chain lengths by Fourier transform infrared spectroscopy.

The infrared spectra of aqueous dispersions of a homologous series of symmetric-chain, disaturated phosphatidylcholines, with fatty acyl chain lengths ranging from 12 to 19 carbons, have been measured at comparable reduced temperatures in their liquid-crystalline phases. The infrared spectra of these compounds contain bands that are dependent on the conformation of the fatty acyl chains. In particular, in the 1400-1300-cm-1 spectral region, there are bands due to CH2 wagging which are specific for the different types of gauche conformers. Thus, gauche-trans-gauché sequences (or kinks) give a band at 1367 cm-1, end-gauche conformers a band at 1341 cm-1, and double-gauche conformers a band at 1355 cm-1. The intensities of these bands were determined and normalized to the intensity of the conformation-insensitive band due to symmetric methyl bending at 1378 cm-1. The intensities of the different "gauche" bands yield a "per chain" intensity, which is directly related to the concentration of the different types of conformational defects. We find that, within experimental error, the concentration of end-gauche and double-gauche conformers is relatively low and practically invariant with chain length when a series of homologous phosphatidylcholines are compared at the same reduced temperature. In contrast, the concentration of gauche-trans-gauché sequences (kink defects) is much higher and increases as the chain length increases. For dipalmitoylphosphatidylcholine we find that there are about 1.2 kink, 0.5-0.6 end-gauche, and 0.4 double-gauche conformers per hydrocarbon chain.(ABSTRACT TRUNCATED AT 250 WORDS)

Fourier Analysis↗
PubMed1988

A technique for monitoring mammalian cell growth and inhibition in situ via Fourier transform infrared spectroscopy.

A single culture of Chinese hamster ovary cells was grown on germanium attenuated total reflectance (ATR) crystals and continuously monitored in situ via ATR/Fourier transform infrared (FT-IR) spectroscopy for approximately 60 h. The cells were seeded into a specially designed flow cell which controlled physiological conditions, flow rate, and addition of growth medium or metabolic inhibitors. Infrared spectra were taken at 20-min intervals until a confluent monolayer was formed. Several strong bands are evident in the spectra which can be generally ascribed to molecular features of cellular components. Cell growth kinetics were measured as a function of infrared band intensity over time and exhibited the normal lag phase, logarithmic growth, and stationary phase on reaching confluence. Spectra of growing cells, normalized to the area under the spectral region 1800-1000 cm-1, were subtracted from reference spectra of confluent cells at 60 h. Difference spectra showed that the largest differences were observed between confluent cells and cells in early growth stages. Differences may reflect cell morphological changes, biochemical activity, and degree of ATR crystal exposure to the bulk medium. ATR/FT-IR spectroscopy of living Chinese hamster ovary cells was also used in a toxicological study to monitor the effects of hydroxyurea, an inhibitor of DNA synthesis. Delayed growth was observed in the cell growth curve of the hydroxyurea-treated cells during the course of treatment as compared to the control culture.

Animals↗
PubMed1993

Fourier transform infrared spectroscopy and electrochemistry of the primary electron donor in Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers: vibrational modes of the pigments in situ and evidence for protein and water modes affected by P+ formation.

Protein electrochemistry in an ultra-thin-layer electrochemical cell suitable for UV/vis and IR spectroscopy has been used to characterize the vibrational modes of the primary electron donors of Rhodobacter sphaeroides and Rhodopseudomonas viridis reaction centers in their neutral and cation radical states (P and P+, respectively). The P-->P+ redox transitions could be well separated from redox reactions of other cofactors according to their redox midpoint potential. The IR difference bands of the primary electron donor bacteriochlorophylls all titrate in unison and exhibit the correct midpoint potential. Comparison of the difference spectra with those of isolated bacteriochlorophylls a and b in organic solvents of different polarity and proton activity [Mäntele, W., Wollenweber, A. M., Nabedryk, E., & Breton, J. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 8468-8472] leads to similar conclusions on the binding and interaction of the pigments within the protein matrix as previously obtained from light-induced P+Q-/PQ difference spectra. Equilibration of the reaction centers in D2O leads to few but distinct shifts of bands and changes of band intensities at 1662, 1634, and 1526 cm-1 (Rhodobacter sphaeroides) and 1694, 1664, 1648, 1630, and 1532 cm-1 (Rhodopseudomonas viridis) as well as to smaller deviations at other wavenumbers. The H-->D-sensitive band at 1662 cm-1 is interpreted in terms of a histidine NH2+ bending mode. A second H/D-sensitive difference band around 1648 cm-1 in the Rhodopseudomonas viridis reaction center may be associated with the peptide C = O of one of the amino acids surrounding P [eventually of the histidine(s) ligating the Mg] which is affected by P+ formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Cations↗
PubMed1991

Studies of peptides forming 3(10)- and alpha-helices and beta-bend ribbon structures in organic solution and in model biomembranes by Fourier transform infrared spectroscopy.

In order to examine the potential correlation between infrared absorption spectra and 3(10)- and alpha-helices and beta-bend ribbon structures, the secondary structures of synthetic peptides known to contain pure 3(10)-helices, mixed 3(10)/alpha-helices, and pure beta-bend ribbon structures, based upon X-ray diffraction and NMR studies, have been investigated by using FTIR spectroscopy incorporating resolution-enhancement techniques. Studies of the peptides known to contain a stable 3(10)-helix in CDCl3 show the main amide I band of fully stable 3(10)-helices occurs at 1666-1662 cm-1. Resolution-enhancement methods revealed small contributions at 1681-1678 and 1646-1644 cm-1, while the amide II band occurs at 1533-1531 cm-1. Peptides known to contain both alpha- and 3(10)-helices in their structure exhibit bands characteristic of both types of conformation. Peptides known to fold into the beta-bend ribbon structure show an amide I band maximum at 1648-1645 cm-1 with the amide II band at 1538-1536 cm-1. Incorporation of these peptides into model membrane structures, e.g., DMPC vesicles, in aqueous buffer sometimes produces changes in the peptide secondary structure. Those peptides which possess a 3(10)-helical structure in CDCl3 solution change the secondary structure in DMPC vesicles to predominantly alpha-helical, plus a contribution from short, unstable 3(10)-helix and/or beta-turns. Those peptides which contain a combination of alpha- and 3(10)-helical structures in CDCl3 solution tend to retain some 3(10)-helical structure within the lipid environment, although the overall H-bonding pattern is altered. Those peptides which form a beta-bend ribbon structure appear to be largely unaffected in the membrane environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

High-Resolution Fourier Transform Infrared Spectroscopy of Nitrosyl Fluoride

The high-resolution FTIR spectrum of FNO has been recorded and the rovibrational structure of the essentially unperturbed nu3, 2nu3, and nu1 + nu3 cold bands and the 2nu3 <-- nu3, nu2 + nu3 <-- nu3, and nu1 + nu2 + nu3 <-- nu3 hot bands has been analyzed. The perturbed bands nu1 and nu1 + nu2 have been assigned and the data analyzed by fitting the transitions with the dark states nu2 + 2nu3 and 2nu2 + 2nu3. Parameters for both light and dark states together with Coriolis coupling parameters have been determined. Copyright 1998 Academic Press. Copyright 1998Academic Press

Journal Article↗
PubMed1999

High-Resolution Fourier Transform Infrared Spectroscopy of Vinyl Alcohol: Rotational Analysis of the nu(13) CH(2) Wagging Fundamental at 817 cm(-1).

The first high-resolution infrared spectra of the unstable molecule vinyl alcohol are reported. The spectra have been obtained using a new precursor, 2-chloroethanol, which when pyrolyzed at 1050 degrees C gives strong infrared spectra of vinyl alcohol free of interfering hydrocarbon absorption bands. In this work, we have analyzed the strong nu(13) fundamental at 817 cm(-1) and substantially improved the ground state rotational constants by a simultaneous fitting of previous microwave data and a large number of infrared combination differences. The 13(1) upper state was found to be perturbed by the 15(2) "dark" state at 775.7 cm(-1), and a complete analysis of the perturbed rotational structure has been achieved using an interacting two-state model. Further small perturbations at high K(a) and J have been identified as interactions with the nu(10) and nu(12) fundamentals at 948 and 960 cm(-1), respectively. Copyright 1999 Academic Press.

Journal Article↗
PubMed1996

Determination of alpha-amylase activity using Fourier transform infrared spectroscopy.

A new method for the determination of alpha-amylase activity in aqueous solutions and human serum with FTIR-spectroscopy is proposed. The chemical reaction catalyzed by the enzyme under study can be followed directly when applying FTIR-spectroscopic detection also in the case, where no colored or electrochemical active species are generated or consumed during the course of the reaction of alpha-amylase with simple starch. Therefore the determination of the alpha-amylase activity could successfully be performed by recording two FTIR-spectra, one immediately after mixing the sample and a substrate (starch-) solution and the other after a 20 min reaction time. From these two FTIR-spectra a difference spectrum was calculated hereby eliminating an unspecific absorption of the matrix. The intensities of the resulting difference spectra corresponded to the extent of the reaction which took place during the investigated time interval and hence could be related to the activity of the enzyme in the sample. The developed method is linear from 80 to 1400 U/l (r.s.d.=5% for 700 U/l) in aqueous solutions and was also successfully applied to the determination of alpha-amylase activity in human serum where a linear working range from 100 to 800 U/l (r.s.d.=11% for 150 U/l) was achieved.

Journal Article↗
PubMed1993

Structure of the apolipoprotein A-IV/lipid discoidal complexes: an attenuated total reflection polarized Fourier transform infrared spectroscopy study.

Discoidal lipid particles were prepared from a reaction mixture containing apo A-IV and dimyristoylphosphatidylcholine (DMPC) or dipalmitoylphosphatidylcholine (DPPC) in the molar ratio of 185:1 (lipid/protein). The complexes were isolated by gel filtration and characterized in terms of composition and size. Infrared attenuated total reflection spectroscopy was used to estimate the secondary structure of apolipoprotein A-IV and the orientation of its amphipathic alpha-helices with respect to the lipid hydrocarbon chains. In addition, infrared spectra were analyzed in terms of the conformation and organization of different regions of the lipid molecules in the particles. This approach has been applied successfully to reconstituted HDL particles prepared from a reaction mixture containing DPPC and apo A-I in the molar ratio of 150:1 (Wald, J.H., Goormaghtigh, E., De Meutter, J., Ruysschaert, J.M. and Jonas, A. (1990) J. Biol. Chem. 265, 20044-20050). Apo A-IV helicity increased for the protein bound to DMPC or DPPC but the increase was more pronounced for the apo A-IV/DMPC particles. In both complexes, the alpha helical amphipathic segments of the protein were parallel to the lipid acyl chains and no significant modification of the overall organization of the lipid molecules in the lipid bilayer was observed. The presence of apo A-IV seems only to affect the conformation of the lipid hydrocarbon chains in close contact with the protein in the discoidal particles.

1,2-Dipalmitoylphosphatidylcholine↗