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1.
Outer and cytoplasmic membranes of Escherichia coli were prepared by a method based on isopyenic centrifugation on a sucrose gradient. The infrared spectra of solid films of these membranes were studied. The cytoplasmic membrane had an amide I band at 1657 cm?1 and an amide II band at 1548 cm?1. The outer membrane had a broad amide I band at 1631–1657 cm?1 and an amid II band at 1548 cm?1 with a shoulder at 1520–1530 cm?1. Upon deuteration, the amide I band of the cytoplasmic membrane shifted to 1648 cm?1, whereas the band at 1631 cm?1 of the outer membrane remained unchanged. After extraction of lipids with chloroform and methanol, the infrared spectra in the amide I and amide II regions of both membranes remained unchanged. Although the outer membrane specifically contained lipopolysaccharide, this could not account for the difference in the infrared spectra of outer and cytoplasmic membranes. It is concluded that a large portion of proteins in the outer membrane is a β-structured polypeptide, while this conformation is found less, if at all in the cytoplasmic membrane.  相似文献   

2.
The Raman spectra of Bence-Jones proteins (BJP) were measured for their native and denatured states. All of the native BJPs investigated gave amide I at 1670–1675 cm?1 and amide III at 1242–1246 cm?1. Although the amide I was shifted to 1667 cm?1 upon the LiBr, acid, and thermal denaturation, as expected, the amide III frequency was unaltered, indicating that the antiparallel β- and disordered structures of BJP provide amide III at almost the same frequencies. The intensity of the 880-cm?1 line of native BJP was relatively intense compared with that of amino acid mixed solution in which the mole ratios of Trp, Phe, and Tyr were adjusted to reproduce the corresponding ratios of BJP. However, the intensity was evidently reduced upon LiBr, acid, and thermal denaturation, approaching that of the amino acid mixture. Thus, the intensity of the 880-cm?1 line is proposed as a practical probe for the environment of Trp residues. The pH dependence of the intensity of the 880-cm?1 line suggests that one of two buried Trp residues is exposed between pH 4 and 3.2 and the other between pH 3.2 and 1.4. The variable fragment (VL) of BJP (Tod) exhibited a S? S stretching Raman line at 525 cm?1. Provided that the crystallographic data of the VL of BJP is applicable to VL of BJP (Tod), the 525 cm?1 of the S? S stretching frequency should be assigned to a TGG conformation of linkage, but not to the AGT or AGG conformation. This supports Sugeta's model rather than Scheraga's model.  相似文献   

3.
Fourier transform-infrared (FT-IR) spectroscopy has been used to rapidly and nondestructively analyze bacteria, bacteria-polymer mixtures, digester samples and microbial biofilms. Diffuse reflectance FT-IR (DRIFT) analysis for freeze-dried, powdered samples offered a means of obtaining structural information. The bacteria examined were divided into two groups. The first group was characterized by a dominant amide I band and the second group of organisms displayed an additional strong carbonyl stretch at ~ 1740 cm?1. The differences illustrated by the subtraction spectra obtained for microbes of the two groups suggests that FT-IR spectroscopy can be utilized to recognize differences in microbial community structure. Calculation of specific band ratios has enabled to composition of bacteria and extracellular or intracellular storage product polymer mixtures to be determined for bacteria-gum (amide I/carbohydrate C-O-~ 1150 cm?1) and bacteria-poly-β-hydroxybutyrate (amide I/carbonyl ~ 1740 cm?1). The key band ratios correlate with the compositions of the material and provide useful information for the application of FT-IR sepectroscopy to environmental biofilm samples and for distinguishing bacteria grown under differing nutrient conditions. DRIFT spectra have been obtained for biofilms produced by Vibrio natriegens on stainless steel disks. Between 48 and 144 h, an increase in bands at ~ 1740 cm?1 was seen in FT-IR spectra of V. natriegens biofilm. DRIFT spectra of mixed culture effluents of anaerobic digesters show differences induced by shifts in input feedstocks. The use of flow-through attenuated total reflectance has permitted in situ real-time changes in biofilm formation to be monitored and provides a powerful tool for understanding the interactioni within adherent microbial consortia.  相似文献   

4.
Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610?C1,630?cm?1, characteristic of a ??-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ??-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620?cm?1 (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70?C90?% of the native form. Both irradiations at 1,535?cm?1(amide II band) and 1,240?cm?1 (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000?cm?1 afforded only about 60?% recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.  相似文献   

5.
The Raman spectra of collagen, gelatin, and elastin are presented. The Raman lines in the latter two spectra are assigned by deuterating the amide N-H groups in gelatin and by studying the superposition spectra of the constituent amino acids. Two lines appear at 1271 and 1248 cm?1 in the spectra of collagen and gelatin that can be assigned to the amide III mode. Possibly, the appearance of two amide III lines is related to the biphasic nature of the tropocollagen molecule, i.e., proline-rich (nonpolar) and proline-poor (polar) regions distributed along the chain. The melting, or collagen-to-gelatin transition, in water-soluble calf skin collagen is studied and the 1248-cm?1 amide III line is assigned to the 31 helical regions of the tropocollagen molecule. Elastin is thought to be mostly random and the Raman spectrum confirms this assertion. Strong amide I and III lines appear at 1668 and 1254 cm?1, respectively, and only weak scattering is observed at 938 cm?1. These features have been shown to be characteristic of the disordered conformation in proteins.  相似文献   

6.
Examination of the secondary structure of proteins by deconvolved FTIR spectra   总被引:70,自引:0,他引:70  
D M Byler  H Susi 《Biopolymers》1986,25(3):469-487
Fourier transform ir (FTIR) spectra of 21 globular proteins have been obtained at 2 cm?1 resolution from 1600 to 1700 cm?1 in deuterium oxide solution. Fourier self-deconvolution was applied to all spectra, revealing that the amide I band of each protein except casein consists of six to nine components. The components are observed at 11 well-defined frequencies, although all proteins do not exhibit components at every characteristic frequency. The root mean square (RMS) deviation of 124 individual values from the 11 average characteristic frequencies is 1.9 cm?1. The observed components are assigned to helical segments, extended beta-segments, unordered segments, and turns. Segments with similar structures do not necessarily exhibit band components with identical frequencies. For instance, the lower frequency beta-structure band can vary within a range of approximately 15 cm?1. The relative areas of the individual components of the deconvolved spectra were determined by a Gauss–Newton, iterative curve-fitting procedure that assumed Gaussian band envelopes for the deconvolved components. The measured areas were used to estimate the percentage of helix and beta-structure for each of 21 globular proteins. The results are in good general agreement with values derived from x-ray data by Levitt and Greer. The RMS deviation between 22 values (alpha- and beta-content of 11 beta-rich proteins measured by both techniques) is 2.5 percentage points; the maximum absolute deviation is 4 percentage points.  相似文献   

7.
The technique of resonance Raman spectroscopy has been used to investigate the interaction of the antibiotic rifampicin with Escherichia coli RNA polymerase. Spectra were analyzed by generating the first derivative of each recorded spectrum using the Savitsky-Golay algorithm. The only band that shifted significantly in the resonance Raman spectrum of rifampicin upon the formation of the drug-core polymerase complex was the amide III band. It underwent an 8 cm?1 shift from 1306 cm?1 in aqueous solution to 1314 cm?1. A comparable shift was observed for the rifampicin-holoenzyme complex. Thus, the interaction of the sigma subunit with the core polymerase does not significantly alter the manner in which rifampicin interacts with RNA polymerase. The nature of this shift has been analyzed further by recording the resonance Raman spectrum of rifampicin in a variety of solvents with different hydrogen-bonding ability. In non-hydrogen-bonding solvents (benzene and carbon disulfide) the amide III band was observed at approximately 1220 cm?1; in dimethyl sulfoxide, a weak hydrogen-bond acceptor, 1274 cm?1; in water, a strong hydrogen-bonding solvent, 1306 cm?1; and finally, in triethylamine, a stronger hydrogen-bonding solvent than water, it was observed at 1314 cm?1. Thus, as the hydrogen-bonding ability of the solvent increased, the amide III band shifted to higher frequency. Based on these results, the rifampicin binding site in RNA polymerase provides a stronger hydrogen-bonding environment for the amidic proton of rifampicin than is encountered when rifampicin is free in aqueous solution.  相似文献   

8.
E G Bendit 《Biopolymers》1966,4(5):539-559
A number of basic features of the infrared spectrum of keratin have been confirmed and some new features have been found. In the 3-μ region, the amide A frequency of helical material in α-keratin at 3286 cm.?1 is close to the expected value, but that of the crystalline phase in α-keratin, near 3270 cm.?1, is lower than had previously been reported. The noncrystalline phase absorbs in the vicinity of 3300 cm.?1 or above, and this causes the low-intensity component of the amide A band in both α- and β-keratin to occur at higher frequencies than those of the high-intensity component. In the 6-μ region, the amide II frequency of noncrystalline material is below 1525 cm.?1. Keratin denatured in lithium bromide, after washing out the reagent, appears to have a considerable helix content, possibly as much as that of the original protein. Hydration causes significant spectral changes. In the 6-mu; region, the frequency of the amide I band of crystalline material is lowered, while that of the amide II band is increased, both by a few wavenumbers; the amide II frequency of noncrystaline material is also increased by a few wavenumbers. In the 3-μ region, no significant change is observed in the amide A frequency of crystalline material, while the frequency of the noncrystaline material is reduced. These spectral changes are interpreted in terms of a weak association of water with main-chain carbonyl groups in the crystalline phase, while in the noncrystaline phase it is thought likely that water molecules form hydrogen-bond bridges between polypetide chains. The absorption coefficient of the amide A band and the integrated absorption intensities of the amide A, I, and II bands do not vary appreciably in the three forms of keratin investigated.  相似文献   

9.
The Raman spectra of the double helical complexes of poly C–poly G and poly I–poly C at neutral pH are presented and compared with the spectra of the constituent homopolymers. When a completely double-helical structure is formed in solution a strong sharp band at 810–814 cm?1 appears which has previously been shown to be due to the A-type conformation of the sugar–phosphate backbone chain. By taking the ratio of the intensity of the 810–814 cm?1 band to the intensity of the 1090–1100 cm?1 phosphate vibration, one can obtain an estimate of the fraction of the backbone chain in the A-type conformation for both double-stranded helices and self-stacked single chains. This type of information can apparently only be obtained by Raman spectroscopy. In addition, other significant changes in Raman intensities and frequencies have been observed and tabulated: (1) the Raman intensity of certain of the ring vibrations of guanine and hypoxanthine bases decrease as these bases become increasingly stacked (Raman hypochromism), (2) the Raman band at 1464 cm?1 in poly I is asigned to the amide II band of the cis-amide group of the hypoxanthine base. It shifts in frequency upon base pairing to 1484 cm?1, thus permitting the determination of the fraction of I–C pairs formed.  相似文献   

10.
Laser Raman spectroscopy study indicates that in concentrated fresh acidic solution (30 mg/ml), glucagon remains predominantly α-helix and not random-coil. The splitting of the amide III band into three components in the crystal at 1262, 1275, and 1295 cm?1 is due to the α-conformation as expected. The presence of a small fraction of β-conformation is demonstrated by the appearance of the weak band at 1230 cm?1 in the fresh solution. This study also established the frequencies of amide III′ bands for the α- and β-conformations of glucagon: 957 and 988 cm?1 for α and β forms, respectively. The conformations of acidic and basic glucagon solutions are apparently different.  相似文献   

11.
K J Payne  A Veis 《Biopolymers》1988,27(11):1749-1760
The ir spectra of lathyritic rat skin collagen and calf skin gelatin solutions at a variety of temperatures were obtained using Fourier transform ir spectroscopy and a 9-reflection, 2-pass ZnSe prism sample cell. The spectra were then deconvolved (based on Kauppinnen's method) and the behavior of the amide I band at ~ 1650 cm?1 observed in detail. Throughout the temperature range studied (4–50°C), three component absorption peaks within the amide I band (at 1633, 1643, and 1660 cm?1) are common to the spectra irrespective of the degree of triple helix content of the sample. Changes in the relative intensities of these component peaks are, however, conformationally dependent. During denaturation of the triple helix, the dominant 1660-cm?1 component in the native collagen spectrum diminishes and the 1633-cm?1 peak becomes relatively intensified. The inherently strong basicity of the carbonyl group of the proline residues together with the frequent occurrence of this imino acid in the X position of the Gly-X-Y triplet of collagen largely accounts for the ?30-cm?1 shift of the amide I band during denaturation. Temperature and conformationally dependent changes in the fine structure of the amide I band from dilute solutions of collagen can be monitored in a reproducible and quantitative fashion.  相似文献   

12.
New techniques in laser Raman spectroscopy are used to obtain spectra of aqueous solutions of lysozylme for frequency shifts as small as 5 cm?1. In addition, Raman measurements are made on two crystalline forms of hen egg white lysozyme. The spectra obtained from the solution and from the crystal are found to be similar for frequencies above 100 cm?1. However, a low-frequency band at 25 cm?1 observed in crystalline lysozyme is not found in the solution, indicating that this band cannot be attributed to an internal molecular vibration.  相似文献   

13.
Infrared spectra of polypeptides were measured in the region of 1800–400 cm?1. For the α-helical form, disordered form, and antiparallel-chain β-form, amide V band- arising from N-H out-of-plane bending models were observed at 610–620, around 650, and 700–705 cm?1, respectively, and amide V′ bands arising from N-D out-of-plane bending modes were observed at 455–465, around 510, and a 515–530 cm?1, respectively. These correlations are useful for conformation diagnoses, particularly for copolyamino-acids or proteins which are not oriented. The nature of low-frequency amide bands are discussed with reference to potential energy distributions calculated for the α-helical form and β form.  相似文献   

14.
Raman spectra are presented for sarcoplasmic reticulum membranes. Interpretation of the 1000–1130 cm?1 region of the spectrum indicates that the sarcoplasmic reticulum membrane may be more fluid than erythrocyte membranes that have been examined by the same technique. The fluidity of the membrane also manifests itself in the amide I portion of the membrane spectrum with a strong 1658 cm?1 band characteristic of CC stretching in hydrocarbon side chains exhibiting cis conformation. This band is unaltered in intensity and position in H2O and in 2H2O thus obscuring amide I protein conformation. Of particular interest is the appearance of strong, resonantly enhanced bands at 1160 and 1527 cm?1 attributable to membrane-associated carotenoids.  相似文献   

15.
Abstract

The effect of pressure on the conformational structure of amyloid β (1–40) peptide (Aβ(1–40)), exacerbated with or without temperature, was determined by Fourier transform infrared (FT-IR) microspectroscopy. The result indicates the shift of the maximum peak of amide I band of intact solid Aβ(1–40) from 1655 cm?1 (α-helix) to 1647–1643 cm?1 (random coil) with the increase of the mechanical pressure. A new peak at 1634 cm?1 assigned to β-antipar- allel sheet structure was also evident. Furthermore, the peak at 1540 cm?1 also shifted to 1527 (1529) cm?1 in amide II band. The former was assigned to the combination of α-helix and random coil structures, and the latter was due to β-sheet structure. Changes in the composition of each component in the deconvoluted and curve-fitted amide I band of the compressed Aβ(1–40) samples were obtained from 33% to 22% for α-helix/random coil structures and from 47% to 57% for β-sheet structure with the increase of pressure, respectively. This demonstrates that pressure might induce the conformational transition from α-helix to random coil and to β-sheet structure. The structural transformation of the compressed Aβ(1–40) samples was synergistically influenced by the combined effects of pressure and temperature. The thermal-induced formation of β-sheet structure was significantly dependent on the pressures applied. The smaller the pressure applied the faster the β-sheet structure transformed. The thermal-dependent transition temperatures of solid Aβ(1–40) prepared by different pressures were near 55–60 °C.  相似文献   

16.
Intensities and other spectral parameters of infrared amide I and II bands of α-helical polypeptides in solutions have been determined for poly(γ-benzylglutamate), poly(γ-ethylglutamate), and polymethionine in chloroform, polylysine, poly(glutamic acid), and fibrillar protein tropomyosin from rabbit muscles in heavy water. The majority of spectral parameters are characteristic. The half-width of the amide I band was found to vary in the range of 15–40 cm?1 for different polypeptides in the different solutions. The correlation between this parameter of the amide I band and the stability of the α-helix was estimated. A new weak band near 1537 cm?1 of unknown origin was observed for the hydrogen form of polypeptides in the α-helical state.  相似文献   

17.
The α-helical from of poly(L -glutamic acid) [α-poly(Glu)] gives rise to the same amide I and III lines as α-poly(γ-benzyl-L -glutamate) at 1652 and 1296 cm?1, respectively. The latter is a superposition of the amide III line near 1290 cm?1 and a line deu to vibrational made of CH2 groups of the side chain near 1300 cm?1. A line at 924 cm?1 is tentatively identified as characteristics of α-poly(Glu). Both the β1- and β2- forms of poly(Glu) give rise to characteristic of β-amide. III frequencies that are similar because of their similar backbone structures. Differences in the conformations of their side chains and in the environments of the backbone are reflected in the region 800–1200 cm?1 and in the amide I. A line at 1042 cm?1 and a pair at 1021 and 1059 cm?1 are tentatively assigned as characteristic of β1-poly(Glu) and β2-poly(Glu), respectively. The α-β2 transition in poly(L -Glu78L -Val22) is shown by the appearance of all the β2-characteristic lines in the thermally transformed sample. The same features observed in poly(L -Glu95L -Val5) also indicate that the α-β2 transition of poly(Glu) is facilitated by the presence of L -valine and that the content of L -valine is not critical for this purpose. Investigation of the Raman spectra of the calcium, strontium, barium and sodium slats of poly(Glu) shows that these salts, under the conditions of preparation used, all the have random-coil conformations.  相似文献   

18.
Raman spectra have been recorded for native and selenium substituted adrenodoxin in dilute solution. Adrenodoxin shows three bands at 397, 350 and 297 cm?1, all polarized, which can be associated with the iron-sulfur core. Selenium substitution leaves the 350 cm?1 band essentially unshifted, but the other two bands disappear and are replaced by new bands at 355 and 263 cm?1. The 350 cm?1 band is assigned to stretching of iron-sulfur (cysteine) bonds, while the 397 and 297 cm?1 bands are associated with vibrations of the labile sulfur atoms. The iron-selenium charge transfer bands were observed at 438 and 480 nm for the oxidized form and at 580 nm for the reduced form. The reduced selena-adrenodoxin displayed absorption maxima at 4, 450 and 5, 550 cm?1, which can be assigned to the d-d transitions of high-spin ferrous ion. From this data and the reported g-values of electron paramagnetic resonance signals, the spin-orbit coupling constants were calculated to be 170 and 210 cm?1 for the respective d-d transitions.  相似文献   

19.
The Raman spectra of oxidation products of lysozyme have been investigated. The protein was oxidized by N-bromosuccinimide and dimethyl sulfoxide/HCl. Depending on the experimental conditions one to six tryptophan residues are oxidized to oxindole. The most prominent difference between the spectra of lysozyme and its oxindole derivatives is the strong band at 1017 cm?1 which displaces the tryptophan peak at 1010 cm?1. Other tryptophan bands are also weakened corresponding to the number of the tryptophan side chains destroyed. Shifts are observed in the amide I and in the amide III regions sensitive to conformational changes. These shifts indicate conformational differences in the higher oxidized species and in the native enzyme, although the amide III maxima overlap with a strong oxindole band. Similar effects are observed in the range of the C-C stretching vibrations of the peptide backbone. If more than one tryptophan side chain is oxidized changes have also been found in the S-S stretching range. The evaluation of this effect is difficult because of the strong oxindole vibration appearing in this region. In species oxidized by great excess of N-bromosuccinimide the tyrosine vibrations can no longer be detected, indicating the modification of this amino acid too.  相似文献   

20.
The Raman spectra of β-lactoglobulin in the crystalline, freeze-dried, and solution states are compared. The spectra of the freeze-dried and crystalline proteins were practically identical. The conformationally sensitive amide III line appearing at 1242 cm?1 increased in intensity 30% upon dissolution of the protein in water which is interpreted as a conformational change in the disordered chains of the protein. This result appears to be a phenomenon for globular proteins containing a large disordered chain fraction. The alkaline denaturation of β-lactoglobulin was studied. When the pH was increased from 6.0 to 11.0, the amide III line shifted from 1242 to 1246 cm?1, broadened, and decreased in intensity. This is consistent with the conversion of β-sheet regions in β-lactoglobulin to the disordered conformation, as has been proposed by other investigators. At pH 13.5 the amide III shifts to 1257 cm?1 characteristic of a completely disordered protein, indicating that any remaining “core” of β-sheet has been randomized. Several changes in the intensities of the tyrosine and tryptophan vibrations accompany the denaturation. As the pH is increased from 6.0 (native state) to 11.0 (denatured state) the intensity ratio of two tyrosine ring vibrations, I855 cm?1/I830 cm?1, decreases from 1.0:0.9 to 1.0:1.3. The same ratio for a copolymer consisting of 95% glutamic acid and 5% tyrosine at pH 7.0, where the polymer forms a random coil exposing the tyrosine to the aqueous environment, is 1.0:0.62. This ratio more closely resembles that corresponding to β-lactoglobulin at pH 6.0 (native state) than pH 11.0 (denatured state) suggesting that the average tyrosine in the denatured state may be in a more hydrophobic environment than in the native state. A time-dependent polymerization of the denatured protein reported by other investigators and observed by us may account for the change in the tyrosine environment. A tryptophan vibration appearing at 833 cm?1 in the spectrum of the native state becomes weak as the pH is increased to 11.0. The intensity of this line may also reflect the local environment of the tryptophan residue.  相似文献   

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