Laser Raman investigations of intact single muscle fibers. Protein conformations

Raman spectra, in the frequency region of the protein vibrations, of intact single muscle fibers of the giant barnacle are presented. Strong bands at 1521 and 1156 cm-1 in the spectra are attributed to resonance-enhanced Raman bands of membrane-bound beta-carotene. Many bands of the myofibrillar proteins are also observed, and at least three spectral features confirm that these proteins adopt a predominantly alpha-helical structure: (1) the amide I band at 1648 cm-1, (2) the weak scattering in the amide III region, and (3) a strong skeletal C-C stretching band at 939 cm-1. Deuterated fibers have also been examined in order to find the exact shape of the amide III band. The presence in the fibers of paramyosin, which is only found in catch muscles, is also apparent from the spectra.     

Effects of organic solutes on the Raman spectra of barnacle muscle fibers

The Raman spectra observed from barnacle muscle fibers are quite complex because the cytoplasm of these cells contains several proteins and solutes. An extraction procedure was used to separate organic solutes from the contractile proteins. Glycine, trimethylamine oxide, taurine, and alanine were found to contribute to the Raman spectra of barnacle muscle fibers, while spectra of lobster fibers reveal the presence of betaine in addition. We have observed that the increase in osmolarity of the intracellular fluid caused by the augmentation of the salinity of sea water (density, 1.023-1.030) in which the barnacles were kept, induces a reduction of intensity of the amide I band. To distinguish among the different parameters which are modified by the sea water salinity, observations were made on glycerinated barnacle muscle fibers. The reduction of intensity of the amide I band in the Raman spectra of glycerinated muscle fibers was also observed with the addition of taurine (0.08 M) in the external relaxing solution. Therefore, under these experimental conditions, the Raman scattering intensity in the amide I region assigned to the alpha-helix conformation (1645-1650 cm-1) is increased when the concentration of organic electrolytes is reduced. However, as no significant decrease of the scattering intensity in the 1660-1670 cm-1 region where the amide I bands of either beta-sheet or disordered conformations normally appear was observed, the increase of intensity of the amide I band centered at 1645 cm-1 is assigned to a change of orientation of alpha-helical segments of the myosin molecules. Our results suggest that organic solutes influence the position of the S-2 segments relative to the thick filaments.     

Raman spectroscopic investigations of sarcoplasmic reticulum membranes.

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 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.     

Raman, infrared, and circular dichroism spectroscopic studies on metallothionein: a predominantly "turn"-containing protein

Raman and IR spectra of rabbit liver metallothionein 1 (MT-1) containing 7 mol of either cadmium or zinc ions reveal high-lying amide III bands between 1290 and 1330 cm-1, indicative of beta-turns. A comparison of the splitting pattern in the amide III region below 1290 cm-1 and in the amide I band between 1600 and 1700 cm-1, with the normal-mode calculations of Lagant et al. [Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984a) Eur. J. Biochem. 139, 137-148; Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984b) Eur. J. Biochem. 139, 149-154; Lagant, P., Vergoten, G., Fleury, G., & Loucheux-Lefebvre, M. (1984c) J. Raman Spectrosc. 15, 421-423] and Krimm and Bandekar [Krimm, S., & Bandekar, J. (1980) Biopolymers 19, 1-29], suggests that metal-bound (holo) MT-1 consists largely of beta-turns of type II. In contrast, the metal-free (apo) protein displays a predominantly unordered conformation. The Raman spectra of the holoproteins below 1000 cm-1 are characterized by several unusual skeletal stretching and bending modes. The spectral pattern between 760 and 800 cm-1 in conjunction with the splitting of the amide I band agrees closely with the normal-mode calculations of Lagant et al. (1984b) on model peptides and is indicative of the presence of type III beta-turns (or 3(10)-helical segments) in MT-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Secondary structure determination in proteins from deep (192-223-nm) ultraviolet Raman spectroscopy

Raman intensities obtained with UV laser excitation at 223, 218, 204, 200, and 192 nm are reported for the amide I, II, III, and II' bands of random-coil polylysine. The excitation profiles show enhancement via the pi-pi electronic transition, at approximately 190 nm. Enhancement for amide I is weak, however, and most of the intensity can be accounted for by preresonance with a deeper UV transition at approximately 165 nm. The amide II' band dominates the spectrum in D2O, consistent with the suggestion that the main distortion coordinate in the pi-pi excited state is the stretching of the C-N peptide bond. Amide II intensities with 200- and 192-nm excitation are reported for several proteins. The previously reported negative linear correlation with alpha-helix content (due to Raman hypochromism in the alpha-helices) is found not to apply to proteins with high beta-sheet content when the excitation wavelength is 200 nm. Much higher intensities are seen for these proteins and are attributed to a red shift of the pi-pi absorption for the beta-structure. A linear correlation with alpha-helix content is found for excitation of 192 nm, which corresponds to an isosbestic point of the beta-sheet and random-coil absorption bands. Characteristic amide II Raman cross sections are derived for alpha-helical, beta-sheet, and random-coil elements and are used to determine secondary structure for alpha 1- and beta-purothionin, by use of amide II intensities with 200- and 192-nm excitation. The results are in good agreement with a previous determination based on amide I band deconvolution in off-resonance Raman spectra.     

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.     

Studies of myosin and its proteolytic fragments by laser Raman spectroscopy.

Two bands in the Raman spectrum of myosin, at 1,304 cm-1 and 1,270 cm-1, are attributable to alpha-helical structure. The first of these, also present in the spectrum of light meromyosin (LMM) but not in that of subfragment-1 (S-1), is assigned to the coiled-coil tail region of myosin; the second, seen in spectra of S-1 or heavy meromyosin (HMM), is largely absent from the spectrum of light meromyosin and is likely to correspond to the alpha-helical segments of the head region. When myosin or LMM aggregates, spectral bands attributable to backbone and sidechain groups sharpen suggesting a reduction in motional freedom. This sharpening is particularly apparent in the 902 cm-1 C--C stretching mode. Mg2+ broadens and shifts the peak at 1,244 cm-1 to 1,237 cm-1 and diminishes the intensity from 1,230 to 1,240 cm-1, changes which appear to be associated the S-1 region. MgPPi produces changes in the 1,300 cm-1 region attributable to alpha-helical regions in coiled-coil structures suggesting that MgPPi affects not only S-1, but also some part of the myosin rod.     

Determination of beta-turn conformation by laser Raman spectroscopy.

To correlate the Raman frequencies of the amide I and III bands to beta-turn structures, three peptides shown to contain beta-turn structure by x-ray diffraction and NMR were examined. The compounds examined were tertiary (formula: see text). The amide I band of these compounds is seen at 1,668, 1,665, and 1,677 cm-1, and the amide III band appears at 1,267, 1,265, and 1,286 cm-1, respectively. Thus, it is concluded that the amide I band for type III beta-turn structure appears in the range between 1,665 and 1,677 cm-1 and the amide III band between 1,265 and 1,286 cm-1.     

Laser Raman spectroscopy of calf bone Gla protein

The Raman spectra of solid calf bone Gla protein in its native state, decarboxylated, with reduced disulfide bond, and as the calcium salt have been obtained. The amide I and III bands are consistent with the presence of alpha-helical, antiparallel beta-sheet, and random-coil regions in all four forms of bone Gla protein. Random coil appears to be the prevailing conformation. The protein conformation in the calcium salt exhibits an increased alpha-helix character compared to the native protein. No significant differences in the backbone conformation are observed among the native, decarboxylated, and reduced forms of bone Gla protein. The Raman band at 504 cm-1, due to the disulfide stretching vibration in native bone Gla protein, is unchanged upon decarboxylation and binding of Ca2+ to the protein, indicating the absence of any changes in the conformation around the disulfide bond in these protein species. The tryptophan and most of the tyrosine residues appear to be 'exposed' rather than 'buried' in the native protein. The environment of at least one of the phenylalanine residues changes when Ca2+ is bound to bone Gla protein. A small change also appears to take place in the environment of at least one of the tyrosine residues upon Ca2+-binding or reduction of the disulfide bond.     

Conformation of oxytocin studied by laser Raman spectroscopy

The peptide backbone conformation and salient structural details of oxytocin were examined by laser Raman spectroscopy. Spectra were obtained in the solid phase, water, 2H2O, and dimethyl sulfoxide solutions. A distinct Amide I band was obtained at 1663 cm-1 for aqueous and deuterated samples and 1666 cm-1 for the solid sample. A relatively high frequency Amide III band at 1260 cm-1 was obtained. It is concluded that these Amide I and III bands arise from the "beta-turn"-like conformation of oxytocin. The tyrosine side chain, according to the I850 cm-1/I830 cm-1 intensity ratio, is exposed to the solvent. The S-S stretching vibration at 512 cm-1 indicates the conformation of C-C-S-S-C-C in the disulfide bridge of oxytocin in the ring is gauche-gauche-gauche.     

Application of Raman spectroscopy to biological macromolecules.

The Raman spectra of biological macromolecules arise from molecular vibrations of either the backbone chains or the side chains. The frequencies of the Raman bands lie in a region between 200 cm-1 and 3000 cm-1. From certain frequencies of the vibrations of the backbone chains one can determine the conformation or secondary structure of a macromolecule. Thus for polypeptides and proteins the frequencies of the Amide I and Amide III vibrations allow one to determine the averge conformation of their backbone chain. In polynucleotides and nucleic acids, the frequency of the phosphate diester stretch of the phosphate furanose chain varies between 814 cm-1 for A conformation and 790 cm-1 for B conformation. Raman spectra of the bases in nucleic acids can be used to determine base stacking and hydrogen bonding interactions. Thus Raman spectroscopy is an important tool for determining the conformation structure of proteins and nucleic acids.     

Conformational states of the bacteriophage P22 capsid subunit in relation to self-assembly

The formation of closed icosahedral capsids from a single species of coat protein subunit requires that the subunits assume different conformations at different lattice positions. In the double-stranded DNA bacteriophage P22, formation of correctly dimensioned capsids is mediated by interaction between coat protein subunits and scaffolding protein. Raman spectroscopy has been employed to compare the conformations of coat protein subunits which have been polymerized to form capsids in the presence and absence of the of scaffolding protein display a Raman spectrum characterized by a broad amide I band centered at 1665 cm-1 with a discernible shoulder near 1653 cm-1, and a broad amide III profile centered at 1238 cm-1 but asymmetrically skewed to higher frequency. These spectral features indicate that the protein conformation in procapsid shells is rich in beta-sheet secondary structure but contains also a significant distribution of alpha-helix. When biologically active, purified subunits assemble in the absence of scaffolding protein, they form polydisperse multimers lacking the proper dimensions of procapsid closed shells. We designate these multimers as "associated subunits" (AS). The Raman spectrum of associated subunits indicates a narrower distribution of secondary structure. The associated subunits are characterized by a sharper and more intense Raman amide I band at 1666 cm-1, with no prominent amide I shoulder of lower frequency. An analogous narrowing of the Raman amide III profile is also observed for AS particles, with an accompanying shift of the amide III band center to 1235 cm-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Beta-sheet and associated turn signatures in vibrational Raman optical activity spectra of proteins.

We have measured the aqueous solution vibrational Raman optical activity (ROA) spectra of concanavalin A, alpha-chymotrypsin, and beta-lactoglobulin, all of which are rich in beta-sheet, together with that of the model beta-turn peptide L-pro-L-leu-gly-NH2. Possible ROA signatures of antiparallel beta-sheet include a strong sharp positive band at approximately 1,313 cm-1 associated with backbone amide III C alpha H and NH deformations, and an amide I couplet, negative at low wavenumber and positive at high, centered at approximately 1,658 cm-1. Negative ROA bands in the range approximately 1,340-1,380 cm-1, which might originate in glycine CH2 deformations, appear to be characteristic of beta-turns. Our results provide further evidence that ROA is a more incisive probe of protein conformation than conventional vibrational spectroscopy, infrared, or Raman, because only those few vibrational coordinates within a given normal mode that sample the skeletal chirality directly contribute to the corresponding ROA band intensity.     

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.     

Role of proteins in protection against radiation-induced damage in membranes

The effect of gamma irradiation on liposomes in the presence of a large number of commercially available proteins has been studied. Experiments were designed to demonstrate that the configuration of both acyl chain and cis C = C bonds created by lipid-protein associations are crucial in autocatalyzed radiation-induced lipid peroxidation. Raman spectroscopy was used to characterize these states. Raman spectra in the C-C stretching region show three prominent bands at 1064, 1090, and 1125 cm-1, assigned to trans, gauche, and trans C-C bonds, respectively. A single symmetrical C = C stretching band assigned to the cis isomer occurs at 1660 cm-1. The intensity ratios (I1064/I1090) and (I1660/I1440) are used as Raman probes to define the conformational states of acyl chains and C = C bonds, respectively. Our data show that the ratio (I1064/I1090) decreases in the presence of proteins, indicating that these proteins induce more gauche structures. Upon irradiation, the ratio (I1064/I1090) increases by about 30% in the absence of proteins and by about 15% in the presence of proteins. This shows that proteins retain the gauche structures in irradiated samples. The ratio (I1660/I1440) decreases in liposomes containing proteins, showing that proteins modify the configuration of cis C = C bonds. Upon irradiation, this ratio decreases by about 45-50% in samples without proteins and by about 10% in samples with proteins. These data show that proteins inhibit the radiation-induced configurational changes in the cis C = C bonds. The determination of radiation-induced peroxides (as malondialdehyde equivalents) in liposomes reveals that proteins inhibit the formation of peroxide products at low molar ratio and that the preventive capacity of different proteins is different. We conclude that proteins alter the conformation of both acyl chains and cis C = C bonds in liposomes and that these altered states are less sensitive to radiation-induced peroxidation.     

Comparison of various molecular forms of bovine trypsin: correlation of infrared spectra with X-ray crystal structures

Fourier-transform infrared spectroscopy is a valuable method for the study of protein conformation in solution primarily because of the sensitivity to conformation of the amide I band (1700-1620 cm-1) which arises from the backbone C = O stretching vibration. Combined with resolution-enhancement techniques such as derivative spectroscopy and self-deconvolution, plus the application of iterative curve-fitting techniques, this method provides a wealth of information concerning protein secondary structure. Further extraction of conformational information from the amide I band is dependent upon discerning the correlations between specific conformational types and component bands in the amide I region. In this paper, we report spectra-structure correlations derived from conformational perturbations in bovine trypsin which arise from autolytic processing, zymogen activation, and active-site inhibition. IR spectra were collected for the single-chain (beta-trypsin) and once-cleaved, double-chain (alpha-trypsin) forms as well as at various times during the course of autolysis and also for zymogen, trypsinogen, and beta-trypsin inhibited with diisopropyl fluorophosphate. Spectral differences among the various molecular forms were interpreted in light of previous biochemical studies of autolysis and the known three-dimensional structures of the zymogen, the active enzyme, and the DIP-inhibited form. Our spectroscopic results from these proteins in D2O imply that certain loop structures may absorb in the region of 1655 cm-1. Previously, amide I' infrared bands near 1655 cm-1 have been interpreted as arising solely from alpha-helices. These new data suggest caution in interpreting this band. We have also proposed that regions of protein molecules which are known from crystallographic experiments to be disordered absorb in the 1645 cm-1 region and that type II beta-turns absorb in the region of 1672-1685 cm-1. Our results also corroborate assignment of the low-frequency component of extended strands to bands below 1636 cm-1. Additionally, the results of multiple measurements have allowed us to estimate the variability present in component band areas calculated by curve fitting the resolution-enhanced IR spectra. We estimate that this approach to data analysis and interpretation is sensitive to changes of 0.01 unit or less in the relative integrated intensities of component bands in spectra whose peaks are well resolved.     

Keratin orientation in wool and feathers by polarized raman spectroscopy

Good quality polarized Raman spectra of a single wool fiber and an intact feather barbule are presented. The intensity ratio of the alpha-helix component of the amide I band measured parallel and perpendicular to the wool fiber axis was 0.39 +/- 0.05. This is consistent with theoretical predictions based on orientational calculations using the normal Raman polarizability tensor for an alpha-helical amide I band where the protein strands are aligned roughly parallel with the fiber axis. However, the depolarized spectral intensity of the alpha-helix mode was greater than expected. For the feather barbule, despite high quality spectra, a unique orientation of the beta-sheet structure could not be determined using the Raman intensity ratios of the amide I band alone. Using previously developed methods, the protein chains were found to be oriented between 60 and 90 degrees from the long axis of the barbule compared to an angle of 51 degrees calculated from polarized IR spectra of the same barbule. The Raman tensor methods for the determination of protein orientation in these fibers was found to be constrained by the complexity of the materials and the limitations of the band fitting methods used to apportion the intensity among the various vibrational modes of their spectra. Other advantages and limitations of polarized Raman microscopic methods of structural determination are discussed.     

Peptide-chain secondary structure of bacteriorhodopsin.

Ultraviolet circular dichroism spectroscopy in the interval from 190 to 240 nm and infrared spectroscopy in the region of the amide I band (1,600 cm-1 to 1,700 cm-1) has been used to estimate the alpha-helix content and the beta-sheet content of bacteriorhodopsin. Circular dichroism spectroscopy strongly suggests that the alpha-helix content is sufficient for only five helices, if each helix is composed of 20 or more residues. It also suggests that there is substantial beta-sheet conformation in bacteriorhodopsin. The presence of beta-sheet secondary structure is further suggested by the presence of a 1,639 cm-1 shoulder on the amide I band in the infrared spectrum. Although a structural model consisting of seven alpha-helical rods has been generally accepted up to this point, the spectroscopic data are more consistent with a model consisting of five alpha-helices and four strands of beta-sheet. We note that the primary amino acid sequence can be assigned to segments of alpha-helix and beta-sheet in a way that does not require burying more than two charged groups in the hydrophobic membrane interior, contrary to the situation for any seven-helix model.     

Study of calcium binding to calmodulin using laser raman spectroscopy

Laser Raman spectroscopy has been used to study calcium binding to calmodulin, Ca2+-dependent regulator protein. Cation binding accompanied by spectral changes of tyrosine residues in the regions of Fermi-resonance doublet and 1600-1620 cm-1, of some carboxylate-containing residues, amide I, III and C-C(N) skeletal vibrations. Amide III contour analysis and calculations of Amide I contours show that complexation causes peptide backbone conformational changes characterized mainly by increased alpha-helical content.     

Raman and resonance-Raman scattering by erythrocyte ghosts.

1. We present the laser-Raman spectra of human erythrocyte ghosts, isolated by standard conditions and compare these with the spectra of lecithin liposomes and fat-free serum albumin. 2. The hydrocarbon stretching modes of membrane lipids are temperature sensitive and may serve as a index of hydrocarbon chain motion. 3. The Amide I and Amide III bands of ghosts in H-2O and 2-H-2O, indicate a mixture of alpha-helical and unordered conformation, but do not allow a quantitative estimate of secondary structure. 4. Strong, scattering bands at 1530 and 1165 cm-1 are attributable to conjugated double bond systems, probably of membrane-associated carotenoids. Their high intensity is due to resonance enhancement.