Fourier transform IR spectroscopy of collagen and gelatin solutions: deconvolution of the amide I band for conformational studies

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.     

Raman spectroscopic study of mechanically deformed poly-L-alanine

Raman spectroscopy has been used in investigating the conformational transitions of poly-L -alanine (PLA) induced by mechanical deformation. We see evidence of the alpha-helical, antiparallel beta-sheet, and a disordered conformation in PLA. The disordered conformation has not been discussed in previous infrared and X-ray diffraction investigations and may have local order similar to the left-handed 3₁ poly glycine helix. The amide III mode in the Raman spectrum of PLA is more sensitive than the amide I and II modes to changes in secondary structure of the polypeptide chain. Several lines below 1200 cm^?1 are conformationally sensitive and may generally be useful in the analysis of Raman spectra of proteins. A line at 909 cm^?1 decreases in intensity after deformation of PLA. In general only weak scattering is observed around 900 cm^?1 in the Raman spectra of antiparallel beta-sheet polypeptides. The Raman spectra of the amide N–H deuterated PLA and poly-L -leucine (PLL) in the alpha-helical conformation and poly-L -valine (PLV) in the beta-sheet conformation are presented. Splitting is observed in the amide III mode of PLV and the components of this mode are assigned. The Raman spectrum of an alpha-helical random copolymer of L -leucine and L -glutamic acid is shown to be consistent with the spectra of other alphahelical polypeptides.     

The structural analysis of three‐dimensional fibrous collagen hydrogels by raman microspectroscopy

To investigate molecular effects of 1‐Ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC), EDC/N‐hydroxysuccinimide (NHS), glyceraldehyde cross‐linking as well as polymerization temperature and concentration on the three‐dimensional (3D) collagen hydrogels, we analyzed the structures in situ by Raman microspectroscopy. The increased intensity of the 814 and 936 cm^?1 Raman bands corresponding to the C—C stretch of a protein backbone and a shift in the amide III bands from 1241 cm^?1/1268 cm^?1 in controls to 1247 cm^?1/1283 cm^?1 in glyceraldehyde‐treated gels indicated changes to the alignment of the collagen molecules, fibrils/fibers and/or changes to the secondary structure on glyceraldehyde treatment. The increased intensity of 1450 cm^?1 band and the appearance of a strong peak at 1468 cm^?1 reflected a change in the motion of lysine/arginine CH₂ groups. For the EDC‐treated collagen hydrogels, the increased intensity of 823 cm^?1 peak corresponding to the C—C stretch of the protein backbone indicated that EDC also changed the packing of collagen molecules. The 23% decrease in the ratio of 1238 cm^?1 to 1271 cm^?1 amide III band intensities in the EDC‐modified samples compared with the controls indicated changes to the alignment of the collagen molecules/fibrils and/or the secondary structure. A change in the motion of lysine/arginine CH₂ groups was detected as well. The addition of NHS did not induce additional Raman shifts compared to the effect of EDC alone with the exception of a 1416 cm^?1 band corresponding to a COO^? stretch. Overall, the Raman spectra suggest that glyceraldehyde affects the collagen states within 3D hydrogels to a greater extent compared to EDC and the effects of temperature and concentration are minimal and/or not detectable. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 349–356, 2013.     

A resonance Raman study on the interaction of rifampicin with Escherichia coli RNA polymerase

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.     

Fourier transform-infrared spectroscopic methods for microbial ecology: analysis of bacteria,bacteri-polymer mixtures and biofilms

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.     

Infrared spectra of outer and cytoplasmic membranes of Escherichia coli

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.     

Raman spectroscopic study of tropomyosin denaturation

Raman spectra of the pH denaturation of tropomyosin are presented. In the native state tropomyosin has an alpha-helical content of nearly 90%, but this value drops rapidly as the pH is raised above 9.5. The Raman spectrum of the native state is characterized by a strong amide I line appearing at 1655 cm^?1, very weak scattering in the amide III region around 1250 cm^?1, and a medium-intensity line at 940 cm^?1. When the protein is pH-denatured, a strong amide III line appears at 1254 cm^?1 and the 940 cm^?1 line becomes weak. The intensities of the latter two lines are a sensitive measure of the alpha-helical and disordered chain content. These results are consistent with the helix-to-coil studies of the polypeptides. The Raman spectra of α-casein and prothrombin, proteins thought to have little or no ordered secondary structure, are investigated. The amide III regions of both spectra display strong lines at 1254 cm^?1 and only weak scattering is observed at 940 cm^?1, features characteristic of the denatured tropomyosin spectrum. The amide I mode of α-casein appears at 1668 cm^?1, in agreement with the previously reported spectra of disordered polypeptides, poly-L -glutamic acid and poly-L -lysine at pH 7.0 and mechanically deformed poly-L -alanine.     

The conformation of polycytidylic acid, polyguanylic acid, polyinosinic acid, and their helical complexes in aqueous solution from laser Raman scattering

The Raman spectra of the double helical complexes of poly C–poly G and poly I–poly C at neutral p^H 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.     

The Raman spectra of Bence-Jones proteins. Disulfide stretching frequencies and dependence of Raman intensity of tryptophan residues on their environments

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 (V_L) of BJP (Tod) exhibited a S? S stretching Raman line at 525 cm^?1. Provided that the crystallographic data of the V_L of BJP is applicable to V_L 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.     

Raman spectra of copoly(D,L-alanines)

Raman spectra in the region 1000–150 cm^?1 were measured for copoly(D ,L -alanines) with the D -residue contents, 3, 7, 10, and 20%, and compared with the spectrum of the α-helical poly-L -alanine. The 532- and 378-cm^?1 peaks were assigned to the L -residues with a right-handed α-helix-like local conformation or to the D -residues with a left-handed α-helix-like local conformation. From the intensity of the latter peak the contents of these local conformations were estimated as a function of the D -residue contents for the copolymers. The 264-cm^?1 peak, which has been assigned to the breathing vibration of the α-helical poly-L -alanine, shows a marked decrease in its intensity upon the introduction of the D residues. This result suggests that the overall deformation vibration of the α-helix arises from rather long sequences of the L - and D -alanine residues with the α-helical conformation and that the intensity of this vibration depends on the content of these sequences in the copolymers.     

Raman spectroscopic study of the proteins of egg white

The Raman spectra of ovalbumin, ovomucoid, and conalbumin are reported. Spectral shifts in the conformationally sensitive amide I and amide III lines as a result of thermal denaturation indicate the formation of intermolecular β- sheets. A medium intensity line at 1260 cm^?1 in the spectra of ovomucoid and ribonuclease is demonstrated to contain a substantial contribution from tyrosine residues.     

Raman spectra of chemically modified lysozyme. Oxindole derivatives.

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.     

Raman scattering of collagen, gelatin, and elastin.

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.     

Stoichiometry and Preferential Interaction: Two Components of the Principle for Protein Structure Organization

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.     

Raman spectroscopic study of poly (β-benzyl-L-aspartate) and sequential polypeptides

Poly-β-benzyl-L -aspartate (poly[Asp(OBzl)]) forms either a lefthanded α-helix, β-sheet, ω-helix, or random coil under appropriate conditions. In this paper the Raman spectra of the above poly[Asp(OBzl)] conformations are compared. The Raman active amide I line shifts from 1663 cm^?1 to 1679 cm^?1 upon thermal conversion of poly[Asp(OBzl)] from the α-helical to β-sheet conformation while an intense line appearing at 890 cm^?1 in the spectrum of the α-helix decreases in intensity. The 890 cm^?1 line also displays weak intensity when the polymer is dissolved in chloroform–dichloroacetic acid solution and therefore is converted to the random coil. This line probably arises from a skeletal vibration and is expected to be conformationally sensitive. Similar behavior in the intensity of skeletal vibrations is discussed for other polypeptides undergoing conformational transitions. The Raman spectra of two cross-β-sheet copolypeptides, poly(Ala-Gly) and poly(Ser-Gly), are examined. These sequential polypeptides are model compounds for the crystalline regions of Bombyx mori silk fibroin which forms an extensive β-sheet structure. The amide I, III, and skeletal vibrations appeared in the Raman spectra of these polypeptides at the frequencies and intensities associated with β-sheet homopolypeptides. Since the sequential copolypeptides are intermediate in complexity between the homopolypeptides and the proteins, these results indicate that Raman structure–frequency correlations obtained from homopolypeptide studies can now be applied to protein spectra with greater confidence. The perturbation scheme developed by Krimm and Abe for explaining the frequency splitting of the amide I vibrations in β-sheet polyglycine is applied to poly(L -valine), poly-(Ala-Gly), poly(Ser-Gly), and poly[Asp(OBzl)]. The value of the “unperturbed” frequency, V₀, for poly[Asp(OBzl)] was significantly greater than the corresponding values for the other polypeptides. A structural origin for this difference may be displacement of adjacent hydrogen-bonded chains relative to the standard β-sheet conformation.     

Far-infrared spectra of polyalanines with alpha-helical and beta-form structures

Far-infrared spectra of poly-L -alanines having the α-helical conformation and the β-form structure were measured. The spectra of glycine–L -alanine copolymer, silk fibroin, and copoly-D ,L -alanines with different D :L compositions were also measured. In addition to the bands so far reported, four bands at 190, 107, 120, and 90 cm^?1were found for the α-helix conformation and the two bands at 442 and 247 cm^?1 were found for the β form. The 442 cm^?1 band consists of the parallel 432 cm^?1 and perpendicular 445 cm^?1 bands. The 247 cm^?1 band is well defined and has strong dichroism parallel to the direction of stretching. These two bands appear also for silk fibroin and glycine–L -alanine copolymer. All the far-infrared bands of copoly-D ,L -alanines can be interpreted as α-helix bands, the three peaks at 580, 478, and 420 cm^?1 being ascribed to the D -residue incorporated into the right-handed α-helix or to the L -residue in the left-handed α-helix.     

Relation of certain infrared bands to conformational changes of cellulose and cellulose oligosaccharides

The i.r. spectra of D-glucose and cellulose oligosaccharides up to cellopentaose have been compared with those of cellulose at various temperatures between that of liquid nitrogen and ～250°. Significant changes in frequency and intensity of the bands at ～3400 cm^?1 were observed. The a_1372cm^?1/a_2900cm^?1 ratio for each carbohydrate studied decreased gradually as the temperature was increased above ambient. The change of the band intensifies at 1429 and 893 cm^?1 with temperature was also investigated. The observed spectral changes are assumed to be associated with changes of hydrogen bonding.     

Low-frequency infrared bands and chain conformations of polypeptides

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.     

Effect of Mid-infrared Free-Electron Laser Irradiation on Refolding of Amyloid-Like Fibrils of Lysozyme into Native Form

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.     

Vibrational Raman optical activity of alanyl peptide oligomers: A new perspective on aqueous solution conformation

The vibrational Raman optical activity (ROA) spectra of di- and tri-L -alanine in the range 650–1750 cm^?1 have been measured in H₂O and D₂O solution at high, neutral, and low pH and pD. Corresponding ROA spectra for tetra- and penta-L -alanine have also been obtained, but over a more restricted set of pH and pD conditions. There are similarities with the ROA spectrum of L -alanine below ～ 1200 cm^?1, but the spectra are very different above this wavenumber due to the influence of the vibrational coordinates of the peptide group. The similar overall appearance of the di-, tri-, and tetrapeptide ROA under selected conditions of pH and pD, and of all four peptide ROA spectra in DCl and HCl solutions, in the backbone skeletal stretch region ～ 1050–1200 cm^?1 and the extended amide III region ～ 1250–1350 cm^?1, suggests that the backbone conformation is approximately the same in all four structures. One difference, however, is a shift of a large positive ROA band in H₂O at ～ 1341 cm^?1 in the dipeptide, assigned to C_α–H and in-plane N–H deformations, down to ～ 1331 cm ^?1 in the tripeptide and to ～ 1315 cm^?1 in the tetrapeptide and pentapeptide (the last in HCl due to insufficient solubility in H₂O), which indicates increasing delocalization of the corresponding normal mode with increasing chain length. Our results do not support the suggestion that stabilizing interactions of the zwitterionic end groups in tri-L -alanine at neutral pH leads to a different solution structure to that at high pH. © 1994 John Wiley & Sons, Inc.