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1.
Fourier transform infrared spectroscopic studies of Ca(2+)-binding proteins   总被引:2,自引:0,他引:2  
M Jackson  P I Haris  D Chapman 《Biochemistry》1991,30(40):9681-9686
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)  相似文献   

2.
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.  相似文献   

3.
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.  相似文献   

4.
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.  相似文献   

5.
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.  相似文献   

6.
Diffuse reflectance infrared Fourier transform (DRIFT) spectroscopy was used to characterize the product of each step in the preparation of a silica-immobilized N-hydroxysuccinimide (NHS) active ester. The preparation of this NHS active ester linkage was based on a literature procedure for the immobilization of proteins. The DRIFT method was used to guide modification of this literature procedure. The DRIFT method also was used to indicate an impurity entrapped in the 60-A diameter pores of the silica support during the formation of the immobilized active ester. Degradation of the immobilized NHS active ester, stored under either argon or dioxane, can be followed by the DRIFT method. Myoglobin and glycine were allowed to react with the active ester, and the result for this silica support was evaluated by the DRIFT method. Elemental analysis was used to provide information on the loading of the silica-immobilized moieties that were presented for DRIFT analysis.  相似文献   

7.
High-sensitivity differential scanning calorimetry (DSC) and Fourier transform infrared (FTIR) spectroscopy were used to study the interaction of a synthetic alpha-helical hydrophobic transmembrane peptide, Acetyl-Lys2-Gly-Leu24-Lys2-Ala-Amide, and members of a homologous series of n-saturated diacylphosphatidylethanolamines (PEs). In the lower range of peptide mol fractions, the DSC endotherms exhibited by the lipid/peptide mixtures consist of two components. The temperature and cooperativity of the sharper, higher-temperature component are very similar to those of pure PE bilayers and are almost unaffected by variations in the peptide/lipid ratio. However, the fractional contribution of this component to the total enthalpy change decreases with increases in peptide concentration, and this component completely disappears at higher peptide mol fractions. The other component, which is less cooperative and occurs at a lower temperature, predominates at higher peptide concentrations. These two components of the DSC endotherm can be attributed to the chain-melting phase transitions of peptide-nonassociated and peptide-associated PE molecules, respectively. Although the temperature at which the peptide-associated PE molecules melt is progressively decreased by increases in peptide concentration, the magnitude of this shift is independent of the length of the PE hydrocarbon chain. In addition, the width of the phase transition observed at higher peptide concentrations is also relatively insensitive to PE hydrocarbon chain length, except that peptide gel-phase immiscibility occurs in very short- or very long-chain PE bilayers. Moreover, the enthalpy of the chain-melting transition of the peptide-associated PE does not decrease to 0 even at high peptide concentrations, suggesting that this peptide does not abolish the cooperative gel/liquid-crystalline phase transition of the lipids with which it is in contact. The FTIR spectroscopic data indicate that the peptide remains in a predominantly alpha-helical conformation, but that the peptide alpha-helix is subject to small distortions coincident with the changes in hydrophobic thickness that accompany the chain-melting phase transition of the PE bilayer. These data also indicate that the peptide significantly disorders the hydrocarbon chains of adjacent PE molecules in both the gel and liquid-crystalline states relatively independently of lipid hydrocarbon chain length. The relative independence of many aspects of PE-peptide interactions on the hydrophobic thickness of the host bilayer observed in the present study is in marked contrast to the results of our previous study of peptide-phosphatidylcholine (PC) model membranes (Zhang et al. (1992) Biochemistry 31:11579-11588), where strong hydrocarbon chain length-dependent effects were observed.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

8.
Fourier transform infrared spectroscopy has been applied to the study of the secondary structure of the Ca2+-ATPase of sarcoplasmic reticulum. An attempt is made to quantitatively assess the various secondary structures present. Values of 45% alpha-helix, 32% beta-sheet and 23% turns were obtained. A comparison is made of these results and those obtained using other techniques such as CD and Raman spectroscopy. The various assumptions inherent in the present procedure are discussed. The effect of various ligands, e.g. Ca2+, vanadate, ATP and phosphate, upon the structure were investigated. Upon binding these ligands no marked spectral changes were observed.  相似文献   

9.
The orientation of the protein secondary structures in porin is investigated by Fourier transform infrared (FTIR) linear dichroism of oriented multilayers of porin reconstituted in lipid vesicles. The FTIR absorbance spectrum shows the amide I band at 1,631 cm-1 and several shoulders around 1,675 cm-1 and at 1,696 cm-1 indicative of antiparallel beta-sheets. The amide II is centered around 1,530 cm-1. The main dichroic signals peak at 1,738, 1,698, 1,660, 1,634, and 1,531 cm-1. The small magnitude of the 1,634 cm-1 and 1,531 cm-1 positive dichroism bands demonstrates that the transition moments of the amide I and amide II vibrations are on the average tilted at 47 degrees +/- 3 degrees from the membrane normal. This indicates that the plane of the beta-sheets is approximately perpendicular to the bilayer. From these IR dichroism results and previously reported diffuse x-ray data which revealed that a substantial number of beta-strands are nearly perpendicular to the membrane, a model for the packing of beta-strands in porin is proposed which satisfies both IR and x-ray requirements. In this model, the porin monomer consists of at least two beta-sheet domains, both with their plane perpendicular to the membrane. One sheet has its strands direction lying nearly parallel to the membrane normal while the other sheet has its strands inclined at a small angle away from the membrane plane.  相似文献   

10.
Kubo S  Kadla JF 《Biomacromolecules》2005,6(5):2815-2821
Hydrogen bonding plays an important role in the thermal and mechanical properties of biopolymers. To investigate hydrogen bond formation in lignin, an abundant natural polymer found in plants, Fourier transform infrared (FTIR) analysis of various lignin model compounds was performed. Four monomeric model compounds and one dimeric model compound were studied under various conditions. FTIR analysis revealed aliphatic hydroxyl groups form stronger hydrogen bonds than phenolic hydroxyl groups. Further, the dimeric biphenyl-type structure formed significantly stronger intermolecular hydrogen bonds as compared to the other monomeric model compounds. Results from the model compound studies were used to explain the observed complex hydrogen-bonding system present in both softwood and hardwood technical lignins. Together with chemical analysis, we discuss the difference in hydrogen bonding between hardwood and softwood lignin and the observed differences in the glass transition temperature.  相似文献   

11.
Aggregation of the hydrophobic peptide derivative Boc-Ala-Ile-Ile-Gly-OMe (1) was examined in methanol solution and in solvated film states. Formation of the peptide by self-assembly was evidenced using fluorescence [Mg salt of 8-anilino-naphthalenesulfonic acid (ANS) as an external probe] and circular dichroism (CD) spectroscopic techniques. In solution, peptide 1 formed as a stable aggregate at a concentration around 3 x 10(-4)m. The peptide gelled into a thin film for which we carried out CD and Fourier transform infrared (FTIR) measurements. Our spectroscopic study on peptide films at differing methanol concentrations indicates that the helical content of the peptide decreases with decreasing methanol concentration in solvated films. However, by reducing the methanol concentration we were able to observe a conformational transition from a predominantly helical turn to a beta-sheet structure via a random coil conformation. Our study focused on the aggregation of the alpha-helical turn-forming peptide derivative, which shows conformational transition on changing solvent concentration in the film form.  相似文献   

12.
Fourier transform infrared and laser Raman spectroscopies were used to study the effects of dodecylpyridinium bromide on the conformation of haemoglobin, myoglobin, bovine serum albumin, ribonuclease, ovalbumin, lysozyme, trypsin and beta-lactoglobulin in aqueous solution. Addition of the cationic detergent caused a decrease in alpha-helix conformation in highly helical proteins. At low detergent concentrations stabilization of beta-sheet conformation was observed.  相似文献   

13.
We report the results of a microspectroscopy study on the Fourier transform infrared (FT-IR) absorption spectra of Caenorhabditis elegans, collected from the different parts of a single intact specimen--pharynx, intestine and tail regions. The principal absorption bands were assigned to the molecular species present in C. elegans, with an excellent reproducibility for the pharynx spectrum. These results enabled us to explore if FT-IR microspectroscopy could offer a new tool for nematode identification. As an example, the discrimination among four well characterised nematode taxa is reported. The FT-IR results completely match those obtained by Blaxter and colleagues through molecular biology [Nature 392 (1998) 71].  相似文献   

14.
S B Dev  L Walters 《Biopolymers》1990,29(1):289-299
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)  相似文献   

15.
Glycophorin has been isolated in pure form from human erythrocyte membranes and reconstituted into lipid vesicles composed of binary mixtures of bovine brain phosphatidylserine (PS) and acyl-chain perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62). The effect of protein on lipid melting behavior and order has been monitored with differential scanning calorimetry and Fourier transform infrared spectroscopy (FT-IR). The phase diagram for PS/DPPC-d62 is consistent with that previously reported for PS/DPPC (Stewart et al. (1979) Biochim. Biophys. Acta 556, 1-16) and indicates that acyl chain perdeuteration does not greatly alter the lipid mixing characteristics. The use of deuterated lipid allows the examination of lipid order by FT-IR of each lipid component in the binary mixtures as well as in the ternary (lipid/lipid/protein) systems. Addition of glycophorin to a 30:70 PS/DPPC-d62 binary lipid mixture results in a preferential glycophorin/PS interaction leading to bulk lipid enriched in DPPC-d62. This is revealed in two ways: first, through cooperative calorimetric transitions increased in temperature from the binary lipid system and second, through FT-IR melting curves of the DPPC-d62 component which shows transitions increased in both onset and completion temperatures in the presence of protein. In addition, non-cooperative melting events are observed at temperatures below the onset of phase separation. The FT-IR data are used to assign these non-cooperative events to the melting of the PS component. For the 50:50 lipid mixture with protein, two transitions are observed in the DSC experiments. The IR results indicate that both lipid components are involved with the lower temperature event.  相似文献   

16.
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.  相似文献   

17.
Several aspects of the application of Fourier transform infrared spectroscopy (FTIR) in high-pressure studies on proteins are reviewed. Basic methodological considerations regarding spectral band assignments, quantitative analysis, and choice of pressure calibrants are also placed within the scope of this paper. This work attempts to evaluate recent developments in the field of high-pressure FTIR of proteins and its prospects for future. Particular attention is paid to the phenomenon of protein aggregation.  相似文献   

18.
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.  相似文献   

19.
Modern protein Fourier transform infrared (FT-IR) spectroscopy has proven to be a versatile and sensitive technique, applicable to many aspects of protein characterization. The major practical drawback for the FT-IR spectroscopy of proteins is the large absorbance band of water, which overlaps the amide I resonances. D2O is often substituted for H2O in infrared experiments. Removal of water from protein samples can be complicated and tedious and potentially lead to denaturation, aggregation, or sample loss. Solvent removal by dialysis is difficult for suspensions and sols. A new method called the D2O dilution technique (Ddt) is described which simplifies the sample preparation step and improves the solvent subtraction. The effect of the D2O concentration on the IR spectrum of aqueous solutions of several model proteins was studied. Dilution of aqueous samples with D2O yields good quality spectra. The Ddt has been evaluated for quantitative analysis using standard proteins and its applicability to solutions and suspensions of a genetically engineered malaria antigen is demonstrated. Use of resolution-enhancement techniques with spectra in mixed solvents has also been investigated.  相似文献   

20.
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.  相似文献   

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