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.     

Raman spectra of D and L amino acid copolymers. Poly-DL-alanine, poly-DL-leucine, and poly-DL-lysine.

The Raman spectrum of poly-DL -alanine (PDLA) in the solid state is interpreted in terms of the disordered chain conformation, in analogy with the spectrum of mechanically deformed poly-L -alanine. The polymer is largely disordered with only a small α-helical content in the solid state. When PDLA is dissolved in water, the spectra suggest that short α-helical segments are formed upon dissolution. These helical regions might be stabilized by hydrophobic bonds between side-chain methyl groups. Addition of methanol to the aqueous PDLA solutions results in a Raman spectrum resembling that of solid PDLA. This result suggests that the methanol disrupts the helical regions by breaking the hydrophobic bonds. The Raman spectra of poly-DL -leucine (PDLL) and poly-L -leucine (PLL) are compared and only slight differences are observed in the amide I and III regions, indicating that PDLL does not have an appreciable disordered chain content. Significant differences are observed in the skeletal regions. The 931-cm^?1 lines in the PLL and PDLL spectra are assigned to residues in α-helical segments of the preferred screw sense, i.e., L -residues in right-handed segments and D -residues in left-handed segments (in PDLL). On the other hand, the 890-cm^?1 line in the spectrum of PDLL is assigned to residues not in the preferred helical sence, i.e., L -residues in left-handed segments and D -residues in right-handed ones. The Raman spectra of poly-DL -lysine and poly-L -lysine in salt-free water at pH 7.0 are compared. The Raman spectra of the two polymers are very similar. However, this does not negate the hypothesis of local order in poly-L -lysine because the distribution of the residues in poly-DL -lysine probably tends towards blocks, and the individual blocks may take up the 3₁ helix.     

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.     

Low-frequency vibrational spectra of some homopolypeptides in the solid state

Low-frequency Raman and far-infrared spectra of polyglycine, poly-L -alanine, and poly-L -valine have been measured. The Raman spectra exhibit an intense band near 100 cm^?1 for these homopolypeptides. Lattice calculations of the polyglycines are used to assign the intense Raman band to a rotary lattice mode. For homopolypeptides in the β conformation, an infrared band is observed whose frequency varies inversely with the square root of the mass of the peptide repeating unit. This infrared band is assigned to the hydrogen bond stretching lattice vibration.     

Raman scattering of some synthetic polypeptides: poly(gamma-benzyl L-glutamate), poly-L-leucine, poly-L-valine, and poly-L-serine

The Raman spectra of poly-γ-benzyl-L -glutamate, poly-L -leucine, poly-L -valine, and poly-L -serine are reported. For the α-helical polymers, the conformationally sensitive amide I, II, and III modes are observed in the Raman as, well as the infrared. For the β form, the Raman effect, supplies the infrared inactive inphase motion which is useful for the determination of a parallel or antiparallel chain alignment. Modes characteristic of the specific polypeptide are also observed which are insensitive to conformation.     

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.     

Far-infrared spectra of poly(-α-amino acids) with basic alkyl group side chains

Far-infrared spectra in the region from 700 to 60 cm^?1 have been measured for the α-helix structures of poly(L -α-amino-n-butyric acid), poly-L -norvaline, poly-L -norleucine, and poly-L -leucine and for the β-form structures of poly(L -α-amino-n-butyric acid), poly-L -valine, poly(DL -amino-n-butyric acid), poly-DL -norvaline, and poly-DL -norleucine. The changes of the spectra on N-deuteration have been measured in the region between 700 and 400 cm^?1. It is concluded that, the α-helix has characteristic bauds near 690, 650, 610, 380, 150, and 100 cm^?1, and that the β-form has characteristic bands near 700, 240, and 120 cm^?1. The main-chain vibrations in the region from 600 to 200 cm^?1 are strongly coupled with the side-chain deformation vibrations.     

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 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.     

A Raman difference spectroscopic investigation of ovalbumin and S-ovalbumin

Raman spectra from 800 to 1850 cm^?1 of aqueous solutions of ovalbumin and its more heat-stable form, S-ovalbumin, are presented. A Raman difference spectrum (ovalbumin minus S-ovalbumin) shows differences in intensity in the amide I and III regions. These intensity differences lead us to postulate that the conversion of ovalbumin to S-ovalbumin involves a conformation change of a small part (～3–4%) of the protein from α-helix to antiparallel β-sheet geometry. This small difference in the three-dimensional arrangement of the peptide chain may contribute to the large difference in the thermodynamic stability between ovalbumin and S-ovalbumin.     

Conformation of poly-L-tyrosine in aqueous solution

The conformational transition of poly-L -tyrosine in 0.1M KCl was investigated by ORD and infrared spectroscopy, potentiometric titration, and sedimentation velocity experiments. It is shown that the fully ordered conformer is obtained by slow titration of the random coil with 0.1N HCl at 25°C. The charge-induced transition, at variance with other poly-α-amino acids, is completed in a narrow range of α. An aggregation process was detected both by potentiometric titration and sedimentation velocity. The polyamino acid aggregates around α = 0.7 at 25°C when the conformational transition is almost complete. Infrared spectra, in the region of the amide I band (1650 cm^?1) showed that the transition is a random coil → antiparallel β one. Evidence exists that the form is of the intramolecular type. The foregoing interpretations of ORD and CD spectra in terms of the α-helix conformation are discussed.     

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 studies of the crystalline, solution, and alkaline-denatured states of beta-lactoglobulin.

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.     

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 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.     

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.     

Laser Raman scattering of cobramine B, a basic protein from cobra venom

Cobramine B, a small basic protein from cobra venom, is selected as a model for studying the scattering intensity of tyrosyl ring vibrations in the Raman spectra of proteins. All three tyrosines in this protein appear to be “buried” in the interior of the molecule and probably involved in interactions which are similar to those of the three “buried” tyrosines in RNase A when it is dissolved in water. Spectral evidence is presented and discussed. The Raman spectra in the 300–1800 cm^?1 region of cobramine B in the solid and solution are compared quantitatively. Several differences exist between the two spectra and may be interpreted in terms of difference in conformation. In the amide I region, a strong single line was observed at 1672 cm^?1 both in the solid and solution spectra, suggesting that this protein may contain a large fraction of antiparallel-β structure. This is supported by the presence of a line at 1235 cm^?1 in the amide III region, which is also characteristic of β-structure. The resolved peaks at 1254 and 1270 cm^?1 indicate the coexistence of some hydrogen-bonded random-coil and some α-helix with the β-structure.     

Raman spectroscopy of the milk globule membrane and triglycerides

The acyl chain mobilities of the lipids of bovine milk fat globules and the component triglycerides have been determined by Raman spectroscopy as a function of temperature from -100°C to 80°C. A broad transition is observed centered about 2–6°C. The C-H and C-C stretching bands in the spectra of liquid and crystalline triglycerides are used comparatively to show that the lipids of the milk globule membrane are 30–40% more ordered than the lipids of the intact milk fat globules at 20°C. Synthetic triglyceride melts, quenched rapidly, are used to illustrate the effect of the thermal history of a sample on lipid order as determined spectroscopically.Strong infrared amide I and amide II bands at 1646 and 1543 cm^?1, respectively, indicate that the protein conformation of the globule membrane is not characterized by extensive regions of beta-sheet structure. Raman spectra of the globule triglycerides indicate cis unsaturation of $\text{39 ± 5\%}$ by comparison to triolein and trielaidin.     

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.     

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.     

Conformational and kinetic studies of synthetic polypeptides by NMR

The α-helix–coil transition of poly-L -leucine, poly-L -alanine and poly-L -methionine in chloroform–trifluoroacetic acid system was studied by nuclear magnetic resonance (NMR) and optical rotatory dispersion (ORD). The kinetics of the hydrogen–deuterium exchange in the peptide was also followed in these polymers by means of NMR. Two types of the NMR spectra and the hydrogen–deuterium exchange reaction were found, corresponding to the high and low molecular weight polypeptides. In high molecular weights, the NH and α-CH resonance lines gave single peaks and the hydrogen–deuterium exchange was expressed as a single first order reaction. In low molecular weights, the NH and α-CH lines were separated into two peaks, corresponding to helical and random-coiled states, respectively, and the exchange react ion was expressed as super-position of a very rapid exchange reaction in the random-coiled part and another slow exchange reaction of the first order in the helical part. These results suggest that the helix–coil interconversion of low molecular weight polypeptides has a longer relaxation time (? 4.5 × 10^?3 sec) than that of high molecular weight polypeptides.