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.     

Aspartic proteinases: Fourier transform infrared spectroscopic studies of a model of the active side.

We synthesized and studied by Fourier transform infrared spectroscopy nine monosalts of diamides as models for the active side of aspartic proteinases. One compound, the monosalt of meta-aminobenzoic acid diamide of fumaric acid (m-FUM), shows the same biological activity as pepsin with regard to the splitting of peptide bonds of the Pro-Thi-Glu-Phe-Phe(4-NO2)-Arg-Leu heptapeptide. The monosalt of m-FUM forms with oxindole a complex in which the carboxylic acid group of the monosalt of m-FUM is strongly hydrogen bonded with the O atom of the peptide bond of oxindole. When one water molecule is added to this complex, the strong field of the carboxylate group destabilizes an O-H bond of the water molecule. The distorted water molecule attacks the carbon atom of the peptide group, and the water proton transfers to the peptide N atom. Simultaneously, the C-N bond of the amide group is broken. Hence it is demonstrated that the catalytic mechanism of aspartic acid proteinases is a base catalysis. The results show that for this catalytic mechanism there are sufficient carboxylic and carboxylate groups, as well as a water molecule in the correct arrangement. It was also demonstrated with other monosalts of dicarboxylic acids that well-defined steric conditions of the carboxylic acid and the carboxylate group must be fulfilled to show hydrolytic activity with regard to oxindole molecules.     

Fourier transform infrared spectroscopic study on retinochrome and its primary photoproduct, lumiretinochrome

Structural studies of retinochrome, and its photoproduct, lumiretinochrome, were done by Fourier transform infrared difference spectroscopy. The absorption bands in the carbonyl stretching region which shift in D2O show the changes in the protein part during the photoreaction. Strong absorption bands in the finger-print region show that the all-trans-retinal chromophore in retinochrome isomerizes to the 11-cis-retinal chromophore in lumiretinochrome upon illumination with yellow-green light at 83K.     

Fourier transform infrared spectroscopic analysis of protein secondary structures

Infrared spectroscopy is one of the oldest and well established experimental techniques for the analysis of secondary structure of polypeptides and proteins. It is convenient, non-destructive, requires less sample preparation, and can be used under a wide variety of conditions. This review introduces the recent developments in Fourier transform infrared （FTIR） spectroscopy technique and its applications to protein structural studies. The experimental skills, data analysis, and correlations between the FTIR spectroscopic bands and protein secondary structure components are discussed. The applications of FTIR to the second- ary structure analysis, conformational changes, structural dynamics and stability studies of proteins are also discussed.     

Differential scanning calorimetric and Fourier transform infrared spectroscopic investigations of cerebroside polymorphism

Calorimetric and Fourier transform infrared (FTIR) spectroscopic studies have been made of the polymorphism exhibited by bovine brain cerebroside-water systems, and the effect of cholesterol and dipalmitoylphosphatidylcholine (DPPC) upon this polymorphism was investigated. The conversion of the cerebroside from the thermodynamically stable to the metastable form is found to be accompanied by spectral changes, indicating a decrease in cerebroside headgroup hydration and a rearrangement of the hydrogen-bond network. The incorporation of low concentrations of cholesterol and DPPC into cerebroside bilayers broadens the thermal transitions associated with the cerebroside as a result of the disruption of cerebroside-cerebroside interactions. This disruption is evident in the spectra of cerebroside/cholesterol mixtures.     

Spectroscopic characterization of microorganisms by Fourier transform infrared microspectroscopy

Spectroscopic fingerprints of bacteria were investigated by Fourier transform infrared (FTIR) microspectroscopy for the elucidation of chemical composition and structural information during growth. Good differentiation of six microorganisms was achieved down to the strain level. The inherent compositional and structural differences of cell envelopes and cytoplasm were investigated and utilized to obtain more detailed analysis of the spectroscopic features. Bands or regions of key functional groups were also identified in the original spectra. Microspectroscopic monitoring of bacterial growth demonstrated that FTIR spectroscopy cannot only provide molecular fingerprints of the cell envelope, but also compositional and metabolic information of the cytoplasm under different physiological conditions. This approach could be an effective alternative to traditional nutritional and biochemical methods to monitor and assess the effects of inhibitors and other environmental factors on microbial cell growth.     

Ionization state of the coenzyme 5'-phosphate ester in cytosolic aspartate aminotransferase. A Fourier transform infrared spectroscopic study

In order to determine the ionization state of the 5'-phosphate of bound pyridoxal phosphate, a Fourier transform infrared spectroscopic study of cytosolic aspartate aminotransferase has been carried out. Dianionic and monoanionic phosphate monoesters give rise to two bands each in the infrared spectrum [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498]. These bands can be identified in infrared spectra of the free coenzyme in solution. Due to interfering bands arising from the protein, only the band assigned to the symmetric stretching of the dianionic phosphate is observed in holoenzyme solutions. The integrated intensity of this band does not change with pH in the range 5.3-8.6, while for free pyridoxal phosphate, the integrated intensity of the same band changes with pH according to the pK value expected for the 5'-phosphate group in solution. Moreover, the value of the integrated intensity for the bound cofactor is close to the value given by free cofactor at pH 8-9. These results suggest that the 5'-phosphate of the bound cofactor remains mostly dianionic throughout the investigated pH range and disfavor other interpretations in terms of ionization of the phosphate group on the basis of the nuclear magnetic resonance 31P chemical shift-pH titration curve of holoenzyme [Schnackerz, K. D. (1984) in Chemical and Biological Aspects of Vitamin B6 Catalysis (Evangelopoulos, E. A., Ed.) Part A, pp 195-208, Alan R. Liss, New York].(ABSTRACT TRUNCATED AT 250 WORDS)     

Fourier transform infrared spectroscopic studies of Ca(2+)-binding proteins

The secondary structures of calmodulin and parvalbumin are well established from X-ray diffraction and nuclear magnetic resonance spectroscopic studies, which indicate that these proteins are predominantly alpha-helical in character. Recent infrared studies have nevertheless suggested that the helical structures present in these proteins in solution are not the standard alpha-helix but rather some kind of distorted helices [Trewhella, J., et al. (1989) Biochemistry 28, 1294]. The evidence for this was the unusually low amide I frequency for calmodulin and troponin C in 2H2O solution. The studies presented here, however, suggest that the helical structures in these proteins are not significantly distorted, for two reasons. First, distorted helical structures have weaker hydrogen bonds than the standard alpha-helix and would therefore be expected to absorb at a higher rather than a lower frequency. Second, distorted helical structures would absorb at an unusual frequency in H2O solutions which is not the case for the proteins studied here. The band frequency of these proteins is observed to occur at a frequency observed with other proteins known to contain predominantly alpha-helical structures. Quantitative analysis of the FT-IR spectra of calmodulin (67% alpha-helix) and parvalbumin (68% alpha-helix) in H2O in the presence of Ca2+ gives helical contents similar to those reported by X-ray studies. This raises the question as to why these proteins in H2O show a normal frequency for the presence of alpha-helical structures and an abnormal frequency in 2H2O. Addition of deuterated glycerol to the proteins in 2H2O solutions results in a significant shift of absorbance to higher frequency.(ABSTRACT TRUNCATED AT 250 WORDS)     

Attenuated total reflectance Fourier transform infrared analysis of an acyl-enzyme intermediate of alpha-chymotrypsin

Attenuated total reflectance Fourier transform infrared (FTIR) spectroscopy was used to obtain signal enhancement of the spectrum of the trans-cinnamoyl-alpha-chymotrypsin acyl-enzyme intermediate. Dilute solutions (as low as 2.5 mg/ml) of enzyme or stabilized acyl-enzyme intermediate were used to form thin films on a germanium crystal surface. The secondary structure of the enzyme thin film was shown to be consistent with the native secondary structure using deconvoluted FTIR data. A novel subtraction technique was used to eliminate interfering spectra of water vapor and protein in critical regions of analysis for esters. This permitted the difference spectra of the one new ester carbonyl bond to be discerned from the 300 or so amide bonds in the protein. The results suggest that the acyl-enzyme exists in two different conformations. This study demonstrates that ir structural information of enzyme-substrate or enzyme-inhibitor complexes can be obtained with dilute protein solutions.     

Fourier transform infrared spectroscopy as a new tool for characterization of mollicutes

Fourier transform infrared (FT-IR) spectroscopy is a convenient physico-chemical technique to investigate various cell materials. Bacteria of class Mollicutes, identified by conventional methods, as Mycoplasma, Acholeplasma and Ureaplasma genera were characterized using this method. A data set of 74 independent experiments corresponding to fourteen reference strains of Mollicutes was examined by FT-IR spectroscopy to attempt a spectral characterization based on the biomolecular structures. In addition to the separation of Mollicutes within the lipidic region into five main clusters corresponding to the three phylogenetic groups tested, FT-IR spectroscopy allowed a fine discrimination between strains belonging to the same species by using selective spectral windows, particularly in the 1200-900 cm(-1) saccharide range. The results obtained by FT-IR were in good agreement with both taxonomic and phylogenetic classifications of tested strains. Thus, this technique appears to be a useful tool and an accurate mean for a rapid characterization of Mollicutes observed in humans.     

Fourier transform infrared spectroscopy for the characterization of a model peptide-DNA interaction

To better understand the structural basis of protein-DNA interactions, the conformational changes that accompany these interactions need to be described. In order to develop a methodological approach to this problem, Fourier transform infrared spectroscopy (FTIR) with derivative resolution enhancement has been used to identify conformational changes that occur when a 29-residue synthetic peptide binds nonspecifically to heterogeneous cellular DNA in aqueous solution. The peptide sequence was chosen de novo, in order to rationally design a peptide model that would allow the relationship between DNA binding and the stability of protein secondary structure to be studied. Peptide at a concentration of 100-200 microM produces 50% saturation of heterogeneous phage DNA sequences as well as of short synthetic oligonucleotides. FTIR spectra reveal significant changes in peptide and DNA upon binding. Second-derivative spectra resolve the amide I band of native peptide into components located at 1627 (beta-strand), 1658 (alpha-helix), and 1681 (turn or beta-strand) cm-1, with a distinct shoulder at 1647 cm-1 (disordered structure). Assignment of the 1681 cm-1 vibration to a turn conformation is supported by uv CD studies, which indicate significant amounts of turn structure in unbound peptide. Ultraviolet CD also confirms the existence of disordered and beta-strand regions in the free peptide. Upon interacting with DNA the band at 1681 cm-1 (turn) is no longer seen; a new band appears at 1675 cm-1; the 1627 cm-1 band (beta-strand) is considerably reduced in intensity; the position of the alpha-helical (1658 cm-1) component remains unchanged; the shoulder at 1647 cm-1 (disorder) disappears. The new vibration at 1675 cm-1 is characteristic of beta-strand structures. The asymmetric stretch (vAS) of the DNA phosphates shifts from 1223 (unbound) to 1229 cm-1 (bound); the relative intensities of vAS and the PO2- symmetric stretch (vS) are altered upon peptide binding.(ABSTRACT TRUNCATED AT 400 WORDS)     

Attenuated total reflectance Fourier transform mid-infrared spectroscopic quantification of sorbitol and sorbose during a Gluconobacter biotransformation process

Mid-infrared spectroscopy (MIRS) was used to simultaneously detect and predict concentrations of d-sorbitol and l-sorbose during a Gluconobacter suboxydans biotransformation. Quantitative models for both these compounds were developed for the entire time-course of the process and validated externally using samples not included in the original modelling exercise, giving standard errors of prediction of 3.29 and 3.3% for sorbitol and sorbose, respectively, and a correlation coefficient close to 1.     

Secondary structure of streptokinase in aqueous solution: a Fourier transform infrared spectroscopic study.

The secondary structure of streptokinase (Sk) in aqueous solution was quantitatively examined by using Fourier transform infrared (FT-IR) spectroscopy. Resolution enhancement techniques, including Fourier deconvolution and derivative spectroscopy, were combined with band curve-fitting procedures to quantitate the spectral information from the amide I bands. Nine component bands were found under the broad, nearly featureless amide I bands which reflect the presence of various substructures. The relative areas of these component bands indicate an amount of beta-sheet between 30 and 37% and an alpha-helix content of only 12-13% in Sk. Further conformational substructures are assigned to turns (25-26%) and to "random" structures (15-16%). Additionally, the correlation of a pronounced component band near 1640 cm-1 (10-16% fractional area) with the possible presence of 3(10)-helices is discussed.     

Enzymatic acylation of hydroxypropyl cellulose in organic media and determination of ester formation by diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy

Esters were prepared by acylation of hydroxypropyl cellulose with fatty acid catalyzed by immobilized lipase from Candida antarctica in tert-butanol. The nature of the substrates used, the initial water activity of the system, and the molecular weight of the hydroxypropyl cellulose were investigated. Moreover, Fourier transform-infrared (FT-IR) spectroscopy was used for determination of ester content on hydroxypropyl cellulose. Specifically, a linear relationship was established between the peak height assigned to the absorption of the esterified carboxyl groups of the cellulose and the ester content. At optimum reaction conditions, the ester content on the hydroxypropyl cellulose was about 11%.     

Fourier transform infrared spectroscopic studies of lipid-protein interaction in native and reconstituted sarcoplasmic reticulum

Fourier transform infrared spectroscopy has been used to monitor lipid-protein interaction and protein secondary structure in native and reconstituted sarcoplasmic reticulum vesicles. Studies of the temperature dependence of the CH₂ symmetric stretching frequency reveal no cooperative phase transitions in purified sarcoplasmic reticulum or in vesicles reconstituted with dioleoylphosphatidylcholine, although a continuous introduction of disorder into the lipid acyl chains is observed as the temperature is raised. In addition, temperature-dependent changes are observed in the Amide I and Amide II vibrations arising from protein peptide bonds. A comparison of lipid order in native sarcoplasmic reticulum and its lipid extract showed that the introduction of protein is accompanied by a slight increase in lipid order. Reconstitution of Ca²⁺-ATPase from sarcoplasmic reticulum with dipalmitoylphosphatidylcholine (lipid/protein ratio 30:1), reveals a perturbed lipid melting event broadened and reduced in midpoint temperature from multilamellar lipid vesicles. The onset of melting (27–28°C) correlates well with the onset of ATPase activity and confirms a suggestion (Hesketh, T.R., Smith G.A., Houslay M.D., McGill, K.A., Birdsall, N.J.M., Metcalfe, J.C. and Warren, G.B. (1976) Biochemistry 15, 4145–4151) that a liquid crystalline environment is a requirement for optimal protein function. Finally, Ca²⁺-ATPase has been reconstituted into binary lipid mixtures of DOPC and acyl-chain perdeuterated DPPC. The effect of protein on the structure and melting behavior of each lipid component was monitored. The protein appears to preferentially interact with the DOPC component.     

Structural investigation of bacteriorhodopsin and some of its photoproducts by polarized Fourier transform infrared spectroscopic methods-difference spectroscopy and photoselection

The direction of selected IR-transition moments of the retinal chromophore of bacteriorhodopsin (BR) and functional active amino acid residues are determined for light- and dark-adapted BR and for the intermediates K and L of the photocycle. Torsions around single bonds of the chromophore are found to be present in all the investigated BR states. The number of twisted single bonds and the magnitude of these torsions decreases in the order K, L, light-adapted BR, dark-adapted BR. In the last, only the C₁₄—C₁₅ single bond is twisted. The orientation of molecular planes and chemical bonds of such protein side chains, which are perturbed during the transition of light-adapted BR to the respective intermediates, are deduced and the results compared with the current three dimensional model of BR. Trp 86 and Trp 185 are found to form a rigid part of the protein, whereas Asp 96 and Asp 115 perform molecular rearrangements upon formation of the L-intermediate.     

Fourier transform infrared and raman spectroscopy for characterization of Listeria monocytogenes strains

The purpose of this study was to characterize the variation in biochemical composition of 89 strains of Listeria monocytogenes with different susceptibilities towards sakacin P, using Fourier transform infrared (FTIR) spectroscopy and Raman spectroscopy. The strains were also analyzed using amplified fragment length polymorphism (AFLP) analysis. Based on their susceptibilities to sakacin P, the 89 strains have previously been divided into two groups. Using the FTIR spectra and AFLP data, the strains were basically differentiated into the same two groups. Analyses of the FTIR and Raman spectra revealed that the strains in the two groups contained differences in the compositions of carbohydrates and fatty acids. The relevance of the variation in the composition of carbohydrates with respect to the variation in the susceptibility towards sakacin P for the L. monocytogenes strains is discussed.     

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.     

Structural properties of acidic phospholipids in complexes with calcitonin: a Fourier transform infrared spectroscopic investigation

The interaction of the polypeptide hormone calcitonin with two acidic phospholipids, dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidic acid (DMPA), was investigated by Fourier-transform infrared spectroscopy. The association of calcitonin with DMPG results in a broadening of the lipid phase transition, accompanied by a marked decrease in the conformational order of the acyl chains at temperatures below the phase transition region. Infrared bands due to carbonyl ester and phosphate group vibrations of DMPG molecules are not significantly affected by the presence of calcitonin. The effect of calcitonin on the conformation of acyl chains in DMPA is much smaller compared with DMPG. The different susceptibility of DMPG and DMPA to perturbation by calcitonin is suggested to be related to different degrees of intermolecular interactions between the headgroups of these two phospholipids.     

Fourier transform infrared spectroscopic imaging and multivariate regression for prediction of proteoglycan content of articular cartilage

Fourier Transform Infrared (FT-IR) spectroscopic imaging has been earlier applied for the spatial estimation of the collagen and the proteoglycan (PG) contents of articular cartilage (AC). However, earlier studies have been limited to the use of univariate analysis techniques. Current analysis methods lack the needed specificity for collagen and PGs. The aim of the present study was to evaluate the suitability of partial least squares regression (PLSR) and principal component regression (PCR) methods for the analysis of the PG content of AC. Multivariate regression models were compared with earlier used univariate methods and tested with a sample material consisting of healthy and enzymatically degraded steer AC. Chondroitinase ABC enzyme was used to increase the variation in PG content levels as compared to intact AC. Digital densitometric measurements of Safranin O-stained sections provided the reference for PG content. The results showed that multivariate regression models predict PG content of AC significantly better than earlier used absorbance spectrum (i.e. the area of carbohydrate region with or without amide I normalization) or second derivative spectrum univariate parameters. Increased molecular specificity favours the use of multivariate regression models, but they require more knowledge of chemometric analysis and extended laboratory resources for gathering reference data for establishing the models. When true molecular specificity is required, the multivariate models should be used.