Observation of small conformational changes in the sperm-whale myoglobin by the far-infrared spectra

Small conformational changes in a molecule of sperm-whale myoglobin in its native solid state for different pH values at room temperature as well as during heat denaturation in alkali medium at different stages of unfolding of the globule were observed by using far-infrared spectroscopy in the region from 30 to 600 cm^?1. The changes appeared in the absorption bands near 420 and 470 cm^?1 ascribed to the side-chain vibrations of helical segments of the myoglobin molecule. For the first time the high structural sensitivity of the far-infrared region of the skeletal vibrations has been confirmed experimentally and the applicability of this technique to globular proteins demonstrated.     

Far-infrared absorption of nucleotides and poly(I)·poly(C) RNA

We have measured the ir absorption of 5′CMP, 5′IMP, and poly(I)·poly(C) from ～25 to ～500 cm^?1. From a comparison of the data with the previously measured absorption of the corresponding nucleosides and bases we can identify several “lines” associated with the deformation of the ribose ring. Out-of-plane deformation of the bases contributes strongly to vibrations near 200 cm^?1. The same ribose vibrations observed in the nucleotides are found in poly(I)·poly(C). They sharpen with increasing water absorption. A study of the spectra of poly(I)·poly(C) as a function of the adsorbed water indicates that water does not contribute in a purely additive fashion to the polynucleotide spectrum but depends on the conformation of the helix. However, the only spectral feature that shifts drastically with conformation is near 45 cm^?1. Measurements at cryogenic temperatures indicate some sharpening of the spectrum of poly(I)·poly(C). Instead, no sharpening is observed in the spectrum of the nucleotides. Shear degradation of poly(I)·poly(C) produces significant spectral changes in the 200-cm^?1 region and sharpening of the features assigned to the low-frequency ribose-ring vibrations.     

Protein folding: evaluation of some simple rules for the assembly of helices into tertiary structures with myoglobin as an example

A computer program designed to fold a peptide chain consisting solely of helical segments and connecting links of known length is described and evaluated. This study is a detailed extension of certain aspects of the earlier work of Ptitsyn &; Rashin (1975). Possible interaction sites on the helices are sequence dependent and are calculated as described by Richmond &; Richards (1978) using probable changes in solvent contact area as a guide. The helices are then paired according to the list of potential sites, with each helix being paired at least once. The lists of pairings are then examined geometrically, each site having a defined dihedral helix axis angle, a specified inter-helix axis distance, and defined rotations, when required, about each helix axis. Two simplified filters are used: (1) lengths of connecting links must be equal to or greater than the end-to-end distances of the helices; and (2) non-paired helices must not collide. With myoglobin as a test example and only six of the eight helices being considered, a conformation space consisting of more than 3 × 10⁸ structures was surveyed. The two filters reduced the acceptable structure list to 121. Slight readjustment of the parameters in the filters would have reduced this to 20 structures. Of these 20, one closely resembles the actual distribution of helices in myoglobin. The possible utility and pitfalls of this approach as part of an overall protein folding program are discussed.     

Structural investigation of poly d(BrU-A) by ultraviolet resonance raman spectroscopy

The resonance Raman spectra of a DNA containing bromodeoxy-uridine (BrdUrd), the poly d(BrU-A), are reported, using U.V. laser as a source of excitation. The conformational change from the ordered, base paired form of poly d(BrU-A) (at 25°C) to the melted form at high temperature (63°C) is reflected in a pronounced hyperchromism of Raman bands at 1627 cm^?1, 1352 cm^?1 and 1230 cm^?1. Particularly the band at 1627 cm^?1 assigned to the vibrations of C4 carbonyl which is hydrogen bonded to adenine increases strongly its intensity upon melting. This represents a new approach for a detection of base unpairing and of modifications in geometry of selective molecules (BrdUrd) in a DNA chain in dilute solutions (10^?4 M).     

Changes in Raman vibrational bands of calf thymus DNA during the B-to-A transition

The B -to-A conformational transition of calf thymus DNA fibers was followed employing Raman spectroscopy. The transition was induced by soaking DNA fibers in water/ethanol mixtures increasing from 60 to 85% ethanol (v/v). Intensity changes of 17 Raman vibrational bands were quantified in the region from 400 to 860 cm^?1. Two bands at 500 and 784 cm^?1 were employed as internal standards. These bands do not appear to change in intensity with ethanol concentration. Large intensity changes relative to these two bands are observed between 70 and 74% ethanol for backbone vibrations at 708, 808, and 835 cm^?1, and base vibrations at 682, 730, and 750 cm^?1. These results indicate that a highly cooperative conformational change takes place between different portions of DNA in the B -to-A transition. Relative intensity changes preceding the onset of the major transition are observed in only two bands; at 835 cm^?1, assigned to a ribose–phosphate vibration, and at 750 cm^?1, assigned to thymine. The implications of these pretransition changes are discussed.     

Infrared absorption and raman scattering of sulfate groups of heparin and related glycosaminoglycans in aqueous solution

The i.r. spectra for aqueous solutions of sulfated glycosaminoglycans and model compounds in the transmittance “window” region of the solvent (1400-950 cm^?1) are dominated by the strong and complex absorption centered at ～1230 cm^?1 and associated with the antisymmetric stretching vibrations of the SO groups. Primary and secondary O-sulfate groups absorb at somewhat higher frequencies (1260-1200 cm^?1) than N-sulfates (～1185 cm^?1). Each sulfate band lends itself to quantitative applications, especially within a given class of sulfated polysaccharide. Laser-Raman spectra of heparin and model compounds have been obtained in aqueous solution and in the solid state. The most-prominent Raman peak (at ～1060 cm^?1) is attributable to the symmetrical vibration of the SO groups, with N-sulfates emitting at somewhat lower frequencies (～1040 cm^?1) than O-sulfates. The Raman pattern in the 950-800 cm^?1 region (currently used in the i.r. for distinguishing between types of sulfate groups) also involves vibrations that are not localized only in the COS bonds.     

A vibrational spectroscopic study of the self-association of 5′-GMP in aqueous solution

The Raman spectra of highly concentrated solutions of 5′-GMP at neutral and acid pH were recorded in order to better characterize the structure of the self-aggregates formed in these solutions and their melting behavior. Vibrational coupling of the C?O stretching vibrations in tetrameric units at neutral pH is shown to yield a characteristic pattern of two Raman bands at ca. 1730 and 1680 cm^?1 (1708 and 1664 cm^?1 in D₂O), and an iractive mode at 1678 cm^?1 in D₂O. From the intensity of the 1730-cm^?1 band, proportional to tetramer concentration, and that at 1485 cm^?1, which reflects the stacking of the bases, the thermal stability of the self-associates formed at neutral pH is shown to be higher for stacked tetramers. At acid pH, the melting of the helical aggregates responsible for the formation of a gel is preceded by the freeing of the hydrogen-bonded phosphate groups, accompanied by a change of conformation from C3′-endo to C2′-endo in some of the associated ribose units. Previous spectroscopic results suggesting the formation of tetramers as an intermediate step in the melting of the gel were not reproduced in this study.     

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.     

A new approach to the characterization of the B and A forms of DNA by I.R. spectroscopy

The RNA conformational changes of B, A and C forms are reflected in the infrared absorption spectra in the region of 800 cm^?1 to 900 cm^?1 and allow one to investigate unoriented samples. The transition to the A form is characterized by the appearence of bands at about 870 cm^?1 and at 813 cm^?1 whereas the B and the C forms exhibit a band at 837 cm^?1, these bands undoubtedly arise from phosphate diester stretching vibrations and yield information about backbone conformation. The presence of these infrared bands provides a criterion for testing the simultaneous presence of two coexisting forms of DNA. It represents a useful method for structural studies of nucleic acid complexes such as protein-DNA for which it is difficult to obtain orientation.     

Resonance Raman scattering and optical absorption of adrenodoxin and selena-adrenodoxin

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.     

Orientational oscillations of the peptide groups of an α-helix

The low-energy orientational oscillations of the peptide groups of an -helix are considered and the value of the frequency is estimated to be in agreement with experiments. Approximate formulae are derived for the projection of a dipole moment on the helix axis and for the helix parameters. Within the framework of a three-chain model, the asymptotics of the soliton solution is obtained using a discrete approach.The analysis of -helix geometry exhibits two types of low-frequency oscillations of the -helix. The first one is connected with atom movements along the helix axis with the peptide groups twisting around the helix axis. Accordingly, it changes the hydrogen bond lengths between neighbouring peptide groups. In the second case, the slopes of the peptide groups to the helix axis oscillate without the helix parameters changing. Here, the energy of interactions between peptide-group dipoles is changed and, as a result, the oscillations have an optical nature. The frequency of the optical orientational oscillations is approximately 100 cm^-1.     

A vitrational study of poly (L-proline) I and II in the far infrared.

The far infrared spectra of poly(L -proline) I (190–35 cm^?1) and II (400–35 cm^?1) were obtained in the solid state at both 300° and 110°K. A significant difference in the region below 100 cm^?1 was observed. A very intense band located at 60 cm^?1 in the infrared spectrum of form II has no counterpart in form I. This indicates the sensitivity of low-frequency vibrations to the difference in conformation assumed by both forms in the solid state. Additional bands observed in this study are correlated with ir and Raman data previously reported and tentative assignments are made using the results of normal mode calculations (in the single-chain approximation) which have been reported.     

Shape of the conformational energy surface near the global minimum and low-frequency vibrations in the native conformation of globular proteins

Based on the assumption that the conformational energy surface of a protein molecule can be approximated near the global minimum point by a multidimensional parabola, conformational fluctuations in the native state are discussed. In this approximation the conformational fluctuations can be viewed as excitations of coupled harmonic oscillations of dihedral angles. For the purpose of estimating the range of frequencies vibrations, globular proteins are assumed to made of homogeneous continuous elastic material. The number of vibrational modes in such an elastic body, with the wavelength no less than the characteristic length of an amino acid residue, are estimated roughly to be three times the number of amino acid residues in a protein, which is slightly less than the number of variable dihedral angles in a protein. Their frequencies, when converted to the wavenumber of corresponding light, are found to range from 1.8 × 10 cm^?1 to 2.1 × 10²cm^?1 for a protein with the diameter d = 40 Å, when Young's E = 10¹¹ dyne/cm² is assumed. A significant fraction of the coupled vibrations of dihedral angles in real globular proteins are collective ones, i.e., those involving the whole protein molecules. Based on these results, it concluded that the depth of the global minimum s at least 150 Kcal/mol.     

Computer analyses of characteristic infrared bands of globular proteins.

Infrared spectra of myoglobin, ribonuclease, lysozyme, α-chymotrypsin, α-lactalbumin, and β-lactoglobulin A were obtained in deuterium oxide solution in units of absorbance versus wavenumber from 1340 to 1750 cm^?1. The spectra were resolved into Gaussian components by means of an iterative computer program. Resolved characteristic absorption peaks for the two infrared active amide I′ components of antiparallel chain-pleated sheets (β-structure) were obtained. The characteristic amide I′ peaks of α-helical regions and apparently unordered regions overlap in D₂O solution. Absorptivity values for the resolved β-structure peak around 1630 cm^?1 were estimated on the basis of the known structure of ribonuclease, lysozyme, and β-chymotrypsin. The β-structure content of β-lactoglobulin was estimated to be ca. 48% of α-lactalbumin ca. 18%, and of α_s-casein close to zero. The results are in general agreement with conclusions drawn from circular dichroism and optical rotatory dispersion studies.     

Infrared study of synthetic peptide analogues of the calcium‐binding site III of troponin C: The role of helix F of an EF‐hand motif

The EF‐hand motif (helix–loop–helix) is a Ca²⁺‐binding domain that is common among many intracellular Ca²⁺‐binding proteins. We applied Fourier‐transform infrared spectroscopy to study the synthetic peptide analogues of site III of rabbit skeletal muscle troponin C (helix E–loop–helix F). The 17‐residue peptides corresponding to loop–helix F (DRDADGYIDAEELAEIF), where one residue is substituted by the D ‐type amino acid, were investigated to disturb the α‐helical conformation of helix F systematically. These D ‐type‐substituted peptides showed no band at about 1555 cm^?1 even in the Ca²⁺‐loaded state although the native peptide (L ‐type only) showed a band at about 1555 cm^?1 in the Ca²⁺‐loaded state, which is assigned to the side‐chain COO^? group of Glu at the 12th position, serving as the ligand for Ca²⁺ in the bidentate coordination mode. Therefore, helix F is vital to the interaction between the Ca²⁺ and the side‐chain COO^? group of Glu at the 12th position. Implications of the COO^? antisymmetric stretch and the amide‐I′ of the synthetic peptide analogues of the Ca²⁺‐binding sites are discussed. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 342–347, 2013.     

Infrared spectrum of a DNA-RNA hybrid

The infrared absorption spectrum in the 4000–400 cm^? region of an oriented film of a DNA–RNA hybrid in its undeuterated and deuterated states was observed with the polarized radiation. Most of the stronger bands found in the double-helical DNA's and double-helical RNA's are identified in the spectrum of the hybrid. The absorption band at 1225 cm^?1 shows a perpendicular dichroism and that at 1085 cm^?1 shows almost no dichroism. These facts indicate that the orientation of the group with respect to the helix axis in the hybrid structure is not entirely the same as that in the double-helical Na DNA at, 75% RH., although the x-ray diffraction pattern of the hybrid is quite similar to that of the DNA A form. The PO₂^? orientation is not the same as that in the double-helical RNA either. The observed dichroism is explained, however, by considering that the PO₂^? group in the RNA moiety takes nearly the same orientation as that in the double-helical RNA, and the PO₂^? group in the DNA moiety takes nearly the same as that in the double-helical DNA.     

Variation of folded polypeptide surface area with probe size

Three types of polypeptide surface area (contact, accessible, and molecular) have been studied as a function of the radius of a probe sphere used to map the surface. The surfaces are: (1) three alpha-helices, the H-helix of myoglobin, the E-helix of leghemoglobin, and an artificial polyalanine helix, each with 26 residues; (2) two globins, myoglobin and leghemoglobin, each with 153 residues; and (3) a two-center model system for which the three types of surface area have been calculated analytically. The two globin helices have almost identical surface areas as a function of probe size as do the two globins. The polyalanine helix surface area is smaller but similar in shape to the globin helix areas. All three helix contact areas tend to the same limit as the probe size increases, and the globin contact areas behave similarly. Fractal dimensions were calculated for the helix and globin contact and molecular surfaces. All fractal dimensions showed strong dependence on probe size. The contact fractal dimension peaks at larger values for both the helices and globins. Most residues do not make contact with large probes (15 A).     

Li+ counterion self-diffusion in ordered DNA

The anisotropic self-diffusion coefficient of ⁷Li⁺ (I = 3/2) counterions has been studied in hydrated, macroscopically oriented Li-(B)DNA fibers at relatively high water contents, corresponding to approximate DNA-DNA helix axis distances of 22–35 Å, using the pulsed field gradient hmr spin-echo method. Self-diffusion coefficients parallel (D_∥) and perpendicular (D_?) to the DNA helix axis increase with increasing salt content and with increasing DNA-DNA helix axis distance. The observed anisotropy D_∥/D_? decreases from 1.6 to 1.2 with the DNA-DNA separation increasing from 22 to 35 Å in the salt-free sample. This result can be understood by the obstruction effect caused by the DNA molecules themselves. The values of the Li⁺ self-diffusion coefficients in the most water-rich system with no added salt (corresponding to an approximate distance of 35 Å between the DNA helix axes) were D_∥ ～ 1.15 × 10^?10 m² s^?1 and D_? ～ 0.98 × 10^?10 m² s^?1, compared to 9.14 × 10^?10 m² s^?1 for the diffusion of Li⁺ in an aqueous solution of LiCl (～ 2.1M). The possible occurrence of restriction effects in the DNA fibers have also been studied by determining the self-diffusion coefficient at different effective diffusion times. The self-diffusion coefficient of Li⁺ in the sample with the largest DNA-DNA helix axis distance seems to be independent of the effective diffusion time, which indicates that the lithium ions are not trapped within impermeable barriers. The possibility of diffusion through permeable barriers has also been investigated, and is discussed. © 1994 John Wiley & Sons, Inc.     

Studies on γ Globulin of Rice Embryo

The secondary structure of γ₁ globulin from rice embryo was investigated by means of optical rotatory dispersion, circular dichroism and infrared spectroscopy. The optical rotatory dispersion curve of the native γ₁ globulin gave a trough at 233mμ with a [m′]₂₃₃ value of ?2,100°, and the Moffitt-Yang plot gave the parameters of a₀= ?237 and b₀= ?20. These data suggest the presence of 3% helix and 38%β structure in the molecule. Circular dichroism exhibits a negative extremum at 218 mμ, giving a [θ]_R value of ?3,730, which suggests the presence of 16°β structure. Infrared spectrum of a thin film of γ₁ globulin showed absorption bands at 695 and 660 cm^?1 with a small hump at 615 cm^?1 characteristic of the β structure, random coil and α helix, respectively. The protein in heavy water exhibits the absorption maximum at 1,630cm^?1 which is also characteristic of the β structure.     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. I. A critical examination of the carbon-nitrogen vibrational modes

Resonance Raman spectra of bacteriorhodopsin are compared to the spectra of this protein modified in the following ways: (1) selective deuteration at the C-15 carbon atom of retinal, (2) full deuteration of the retinal, (3) the addition of a conjugated double bond in the β-ionone ring (3-dehydroretinal), (4) full deuteration of the protein and lipid components, (5) ¹⁵N enrichment of the entire membrane and (6) deuteration of the entire membrane (including the retinal). A detailed comparison of the ¹⁵N-enriched membrane and naturally occurring purple membrane from 800 cm^?1 to 1700 cm^?1 reveals that ¹⁵N enrichment affects the frequency of only two vibrational modes. These occur at 1642 cm^?1 and 1620 cm^?1 in naturally occurring purple membrane and at 1628 cm^?1 and 1615 cm^?1 in the ¹⁵N-enriched samples. Therefore, this pair of bands reflects the states of protonation of the Schiff base. However, our data also indicate that neither of these modes are simple, localized $\text{C}\text{=}\textcolor{red}{}\text{?}\text{H}$ or C=N stretching vibrations. In the case of the 1642 cm^?1 band motions of the retinal chain beyond C-15 are not significantly involved. On the other hand, in the 1620 cm^?1 band atomic motions in the isoprenoid chain beyond C-15 are involved.