Rhodopsin-lumirhodopsin phototransition of bovine rhodopsin investigated by Fourier transform infrared difference spectroscopy

The rhodopsin-lumirhodopsin transition has been investigated by Fourier transform infrared difference spectroscopy using isotope-labeled retinals. In the transition, two protonated carboxyl groups are involved. Another carbonyl band, located at 1725 cm-1 in rhodopsin, is shifted to 1731.5 cm-1 in lumirhodopsin. This line is tentatively assigned to a carbonyl stretching vibration of a peptide bond adjacent to the nitrogen of a proline residue. The C=N stretching vibration of rhodopsin could unequivocally be assigned to a band at 1659 cm-1. In contrast to rhodopsin and bathorhodopsin, the C=N stretching vibration of lumirhodopsin is at a low position, i.e., at 1635 cm-1, and exhibits only a downshift of 4 cm-1 upon deuteriation of the nitrogen. The C15-H rocking vibration of rhodopsin is assigned to the unusual high position of 1456 cm-1 and shifts into the normal region upon formation of lumirhodopsin. From these results, it is concluded that, whereas the environment of the Schiff base in rhodopsin, bathorhodopsin, and isorhodopsin is approximately the same, large changes occur with the formation of lumirhodopsin. From the assignment of the C10-C11 stretching vibration in bathorhodopsin and lumirhodopsin, a 10-s-cis geometry of lumirhodopsin can be excluded.     

Carrageenophyte identification by second-derivative Fourier transform infrared spectroscopy

The second-derivative mode of the Fourier transform I.R. spectra of dried algal material has been applied to distinguish the carrageenans-producingStenogramme interrupta from the isomorphous speciesRhodymenia howeana. Spectra of the tetrasporophyteS. interrupta showed bands assigned to a -carrageenan type polysaccharide, while the gametophytic and cystocarpic plants showed the characteristic absorptions of -and -carrageenans. Results were confirmed by hot water extraction of samples of the three nuclear phases ofS. interrupta and characterization of the extracts by chemical analysis.Author for correspondence     

Heat-induced secondary structure and conformation change of bovine serum albumin investigated by Fourier transform infrared spectroscopy

Fourier transform infrared (FT-IR) spectra were measured for an aqueous solution (pD = 5.40) of defatted monomer bovine serum albumin (BSA) over a temperature range of 25-90 degrees C to investigate temperature-induced secondary structure and conformation changes. The curve fitting method combined with the Fourier self-deconvolution technique allowed us to explore details of the secondary structure and conformation changes in defatted BSA. Particularly striking in the FT-IR spectra was an observation of the formation of an irreversible intermolecular beta-sheet of BSA on heating above 70 degrees C. A band at 1630 cm(-1) in the spectra was assigned to short-segment chains connecting alpha-helical segments. The transition temperature for the short-segment chains connecting alpha-helical segments is lower by 17-18 degrees C, when compared to those of the alpha-helix, turn, and intermolecular beta-sheet structures of BSA, suggesting that the alpha-helix and turn structures of BSA are cooperatively denatured on heating. Moreover, the results give an important feature in heat-induced denaturation of BSA that the conformation changes occur twice around both 57 and 75 degrees C. The appearance of two peaks is interpreted by the collapse of the N-terminal BSA domain due to the crevice in the vicinity between domains I and II at low-temperature transition and by the change in cooperative unit composed of the other two BSA domains at high-temperature transition.     

Fourier transform infrared (FTIR) spectroscopy

Fourier transform infrared (FTIR) spectroscopy probes the vibrational properties of amino acids and cofactors, which are sensitive to minute structural changes. The lack of specificity of this technique, on the one hand, permits us to probe directly the vibrational properties of almost all the cofactors, amino acid side chains, and of water molecules. On the other hand, we can use reaction-induced FTIR difference spectroscopy to select vibrations corresponding to single chemical groups involved in a specific reaction. Various strategies are used to identify the IR signatures of each residue of interest in the resulting reaction-induced FTIR difference spectra. (Specific) Isotope labeling, site-directed mutagenesis, hydrogen/deuterium exchange are often used to identify the chemical groups. Studies on model compounds and the increasing use of theoretical chemistry for normal modes calculations allow us to interpret the IR frequencies in terms of specific structural characteristics of the chemical group or molecule of interest. This review presents basics of FTIR spectroscopy technique and provides specific important structural and functional information obtained from the analysis of the data from the photosystems, using this method.     

Substrate binding and enzyme function investigated by infrared spectroscopy

Protein conformational changes triggered by molecule binding are increasingly investigated by infrared spectroscopy often using caged compounds. Several examples of molecule-protein recognition studies are given, which focus on nucleotide binding to proteins. The investigation of enzyme mechanisms is illustrated in detail using the Ca(2+)-ATPase of the sarcoplasmic reticulum membrane as an example. It is shown that infrared spectroscopy provides valuable information on general aspects of enzyme function as well as on molecular details of molecule-protein interactions and the mechanism of catalysis.     

Fourier transform infrared spectroscopic characterization of a photolabile precursor of glutamate

Recently, it has been demonstrated that Fourier transform infrared spectroscopy (FTIR) detects conformational changes in the glutamate receptor ligand-binding domain that are associated with agonist binding. Combined with flash photolysis, this observation offers the prospect of following conformational changes at individual protein and agonist moieties in parallel and with high temporal resolution. Here, we demonstrate that gamma(alpha-carboxy-2-nitrobenzyl) glutamate (caged glutamate) does not interact with the protein, and that following photolysis with UV light the FTIR difference spectrum indicated changes in the protein tertiary and secondary interactions. These changes were similar to those observed for the protein upon addition of free glutamate. Thus, caged glutamate and its photolysis by-products are inert in this system, whereas the released glutamate exhibits full activity. Difference spectra of caged glutamate and of reaction analogs permitted identification of and correction for FTIR signals arising from the photolytic reaction and confirmed that its products are indeed glutamate and 2-nitrosophenyl glyoxalic acid.     

Analysis of human tear fluid by Fourier transform infrared spectroscopy

The purpose of this research is to find some useful spectroscopic factors in human tear fluid contents to monitor diurnal changes of the physicochemical ocular conditions noninvasively. All tear fluid samples were collected with glass microcapillary tubes from both eyes of three donors and analyzed by Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR). We measured the peak intensities at 2852, 1735, 1546, and 1242 cm(-1), and the peak intensity ratios among those peaks in the second derivative spectra. We found significant diurnal and individual variations in those peak intensities for tear fluid obtained from right and left eyes. Among these variations, we observed significant changes in tear samples between right and left eyes. In this case the peak intensity ratio between 1242 (phosphate ester) and 2852 cm(-1) (fatty acid methylene) of right eye tear fluid was increased in the afternoon (1600 to 1900 h), while that of left eye tear fluid did not change significantly. In the ratio between 1242 (phosphate ester) and 1546 cm(-1) (amide II), the difference was not observed between both eyes. We conclude that the difference in diurnal variations of biochemical constituents between right and left eye tear fluids could be monitored noninvasively and nondestructively by FTIR technique and this method could be useful in the future for tear diagnoses.     

Rapid identification of coagulase-negative staphylococci by Fourier transform infrared spectroscopy

Coagulase-negative staphylococci (CNS), frequently associated with both community-acquired and nosocomial bloodstream infections, must be distinguished from Staphylococcus aureus for clinical purposes. Conventional methods are too laborious and time-consuming and often lack sensitivity to CNS. Fourier transform infrared (FTIR) spectroscopy combined with the use of a universal growth medium (Que-Bact Universal Medium No. 2) and chemometrics was evaluated for its potential as a rapid and simple clinical tool for making this distinction. FTIR spectra of 11 methicillin-sensitive and 11 methicillin-resistant CNS isolates as well as 25 methicillin-sensitive, 47 methicillin-resistant, 34 borderline oxacillin-resistant and 35 glycopeptide intermediate S. aureus isolates were obtained from dried films of stationary-phase cells grown on the universal medium. Principal component analysis (PCA), self-organizing maps, and the K-nearest neighbor algorithm were employed to cluster the different phenotypes based on similarity of their FTIR spectra. PCA of the first-derivative normalized spectral data from a single narrow region (2888-2868 cm(-1)) yielded complete differentiation of CNS from both methicillin-sensitive and methicillin-resistant S. aureus. The rate of correct classification was somewhat reduced, from 100% to 90%, after inclusion of borderline oxacillin-resistant and glycopeptide intermediate S. aureus strains in the data set. Differentiation based on the data in broader spectral regions was much less reliable. The results of this study indicate that with proper spectral region selection, FTIR spectroscopy and cluster analysis may provide a simple and accurate means of CNS species identification.     

Quantitative analysis of cellulose nitrates by Fourier transform infrared spectroscopy

Quantitative express analysis of nitrogen content in cellulose nitrates by Fourier transform infrared spectroscopy has been developed. The slope of the dependence of the ratio of the band intensity (and area) to sample weight in a tablet, on the nitrogen content in a sample was used to find the reduced extinction coefficients for quantitative analysis of nitrogen content in cellulose nitrate samples by IR spectroscopy. The results were compared with the nitrogen content values in the same samples determined by the ferrosulfate method.     

Probing conformational changes in the nicotinic acetylcholine receptor by Fourier transform infrared difference spectroscopy.

We have developed a Fourier transform infrared (FTIR) difference method for probing conformational changes that occur upon the binding of ligands to the nicotinic acetylcholine receptor (nAChR). Our approach is to deposit reconstituted nAChR membranes in a thin film on the surface of a germanium internal reflection element, acquire FTIR spectra in the presence of bulk aqueous solution using attenuated total reflection, and then trigger conformational changes by sequentially flowing a buffer either with or without an agonist past the film surface. Using the fluorescent probe, ethidium bromide, it is demonstrated that the method of nAChR film deposition does not affect the ability of the receptor to undergo the resting-to-desensitized state transition. The difference of FTIR spectra of nAChR films recorded in the presence and absence of agonists reveal highly reproducible infrared bands that are not observed in the difference of spectra recorded with only buffer flowing past the film surface. Some of the bands are assigned to changes in protein secondary structure and to changes in the structure of individual amino acid residues. Bands arising from the vibrations of the agonist bound to the receptor are also observed. The results demonstrate that FTIR difference spectroscopy can detect structural changes in the nAChR that occur upon the binding of ligands. The technique will be an effective method for investigating nAChR structure and function as well as receptor-drug interactions.     

Sensitivity of Deinococcus radiodurans to gamma-irradiation: a novel approach by Fourier transform infrared spectroscopy

Deinococcus radiodurans is a red-pigmented coccus known to be particularly resistant to both chemical and radiative agents. Fourier transform infrared (FT-IR) spectroscopy was used as a convenient and easy-to-run method to monitor damage induced in this bacterium by ionizing radiations. First, stationary-phase cultures were submitted to increasing doses of gamma-irradiation ((137)Cs source). Beyond a threshold of 11 kGy, striking changes occurred in spectra of irradiated samples compared with unirradiated ones, especially in the 1750-900 cm(-1) region, which is spectroscopically assigned to amide I and II components, nucleotide bases, the phosphodiester backbone, and the sugar ring. Second, bacterial cultures were postirradiation reincubated. After a reincubation time of 15 h, the oxidative stress was in part overwhelmed, and the growth of D. radiodurans again occurred, although some biocellular components remained altered. Consequently, FT-IR analysis is an accurate means to rapidly visualize biomolecular changes undergone by cells both after gamma-irradiation and during the repair mechanism.     

Characterization of an RNA bulge structure by Fourier transform infrared spectroscopy

There may be several advantages associated with an antisense oligonucleotide that induces a bulged structure into its RNA target molecule. Many structures of RNA bulges are elucidated from single-stranded RNA models. However, a two-component system is the minimum requirement for a realistic antisense model. We have used Fourier transform infrared spectroscopy to investigate a single-stranded RNA oligonucleotide with known NMR solution structure, constructed to model a five nucleotide bulge, and its two-component oligonucleotide counterpart. The infrared spectra show A-helical base-paired stems and non-base-paired loops in both systems. The nucleosides are mainly in an anti-conformation. Both N-type and S-type of sugar puckers can be inferred from the infrared region sensitive to sugar conformations. The S-type of sugar pucker is likely to be associated with the nucleotides in the bulge. The FTIR results display an overall structural similarity between the two model systems.     

Conformational changes of bacteriorhodopsin detected by Fourier transform infrared difference spectroscopy

We report the first Fourier transform infrared difference spectra of purple membrane. Evidence is presented that alterations in the vibrations of both the retinylidene chromophore and the protein groups of bacteriorhodopsin associated with photocycling can be detected. This method provides a new tool for probing the conformational changes occurring in bacteriorhodopsin during the proton pump cycle.     

Structural analysis of DNA-chlorophyll complexes by Fourier transform infrared difference spectroscopy

Porphyrins and metalloporphyrins are strong DNA binders. Some of these compounds have been used for radiation sensitization therapy of cancer and are targeted to interact with cellular DNA. This study was designed to examine the interaction of calf thymus DNA with chlorophyll a (CHL) in aqueous solution at physiological pH with CHL/DNA(phosphate) ratios (r) of 1/160, 1/80, 1/40, 1/20, 1/10, and 1/5. Fourier transform infrared (FTIR) difference spectroscopy was used to characterize the nature of DNA-pigment interactions and to establish correlations between spectral changes and the CHL binding mode, binding constant, sequence selectivity, DNA secondary structure, and structural variations of DNA-CHL complexes in aqueous solution. Spectroscopic results showed that CHL is an external DNA binder with no affinity for DNA intercalation. At low pigment concentration (r = 1/160, 1/80, and 1/40), there are two major binding sites for CHL on DNA duplex: 1) Mg-PO2 and 2) Mg-N7 (guanine) with an overall binding constant of K = 1.13 x 10(4) M-1. The pigment distributions are 60% with the backbone PO2 group and 20% with the G-C base pairs. The chlorophyll interaction is associated with a major reduction of B-DNA structure in favor of A-DNA. At high chlorophyll content (r = 1/10), helix opening occurs, with major spectral alterations of the G-C and A-T bases. At high chlorophyll concentration (1/5), pigment aggregation is observed, which does not favor CHL-DNA complexation.     

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.     

Low-temperature Fourier transform infrared spectroscopy of photoactive yellow protein

The photocycle intermediates of photoactive yellow protein (PYP) were characterized by low-temperature Fourier transform infrared spectroscopy. The difference FTIR spectra of PYP(B), PYP(H), PYP(L), and PYP(M) minus PYP were measured under the irradiation condition determined by UV-visible spectroscopy. Although the chromophore bands of PYP(B) were weak, intense sharp bands complementary to the 1163-cm(-1) band of PYP, which show the chromophore is deprotonated, were observed at 1168-1169 cm(-1) for PYP(H) and PYP(L), indicating that the proton at Glu46 is not transferred before formation of PYP(M). Free trans-p-coumaric acid had a 1294-cm(-1) band, which was shifted to 1288 cm(-1) in the cis form. All the difference FTIR spectra obtained had the pair of bands corresponding to them, indicating that all the intermediates have the chromophore in the cis configuration. The characteristic vibrational modes at 1020-960 cm(-1) distinguished the intermediates. Because these modes were shifted by deuterium-labeling at the ethylene bond of the chromophore while labeling at the phenol part had no effect, they were attributed to the ethylene bond region. Hence, structural differences among the intermediates are present in this region. Bands at about 1730 cm(-1), which show that Glu46 is protonated, were observed for all intermediates except for PYP(M). Because the frequency of this mode was constant in PYP(B), PYP(H), and PYP(L), the environment of Glu46 is conserved in these intermediates. The photocycle of PYP would therefore proceed by changing the structure of the twisted ethylene bond of the chromophore.     

Protein structure by Fourier transform infrared spectroscopy: second derivative spectra

Second derivative Fourier transform infrared spectra of the proteins ribonuclease A, hemoglobin, and beta-lactoglobulin A (native and denatured) have been obtained in deuterium oxide solution from 1350 to 1800 cm-1. The relationship of the original spectra to their second derivatives is briefly discussed. In the second derivative spectra, clearly resolved peaks are observed which can be associated with the alpha-helix, beta-strands, and turns. No protein spectra with such resolution have heretofore been reported. Tentative assignments are proposed, and the observed peaks are related to the secondary structure of the proteins studied. The data appear to present the first direct spectroscopic evidence of turns in a native protein.     

Structural and functional asymmetry of the nucleotide-binding domains of P-glycoprotein investigated by attenuated total reflection Fourier transform infrared spectroscopy.

The dynamic changes occurring during the catalytic cycle of MDR3 P-glycoprotein (Pgp) and the role of each nucleotide-binding domain (NBD) in the transport process were investigated using attenuated total reflection Fourier transform infrared spectroscopy. For this purpose, wild-type Pgp and two mutations of homologous residues in each NBD were studied. On the one hand, we demonstrate here that, during its catalytic cycle, Pgp does not undergo secondary structure changes, but only modifications in its stability and accessibility to the external environment. On the other hand, amide H/D exchange kinetics demonstrate that homologous mutations in the two NBDs affect, in a different way, the dynamic properties of Pgp and also the dynamic changes occurring during ATP hydrolysis. These observations led to the conclusion that the NBDs have an asymmetric structure and different functions in the catalytic cycle of Pgp. Our data suggest that the release of drug from the membrane into the extracellular environment is due to decreased stability and/or increased accessibility to the external medium of the membrane-embedded drug-binding site(s). NBD1 would play an important role in this first restructuring of the membrane-embedded domains. NBD2 would be directly implicated in the subsequent restructuring of the membrane-embedded binding sites by which they recover their initial stability and accessibility to the membrane. It is proposed that this restructuring step would allow the binding and transport of another molecule of substrate.