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.     

Calorimetric,density and circular dichroism studies of the reversible structural transition in Pf1 filamentous bacterial virus

The macromolecular structural transition of Pf1 filamentous bacterial virus detected by X-ray diffraction analysis has been studied in virus solutions by density, circular dichroism, and microcalorimetric measurements. The reversible structural change occurring between 5 °C and 25 °C has a calorimetrically determined transition enthalpy ΔH_t,cal of 14·5 ± 1.5 kJ (mol protein)^?1. The transition curves resulting from the density, circular dichroism, and calorimetric measurements have been analysed in terms of a two-state process to extract the van't Hoff enthalpy. Comparison of the effective transition enthalpy and the calorimetric ΔH_t,cal values gives about 26 protein subunits as the size of the co-operative unit. Parallel heat capacity and density measurements on fd virus show no such transition, in agreement with X-ray diffraction studies.     

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.     

Low-frequency spectroscopy of four-photon scattering in aqueous solutions of biopolymers

The novel method of nonlinear laser spectroscopy — low frequency spectroscopy of four-photon scattering of laser radiation was applied to detect a considerable growth of ortho-H₂O spin isomer and also H₂O₂ molecule concentration in a hydrate layer at the interface between water and DNA, denatured DNA molecules and α-chymotrypsin. Spectra of rotational resonances of ortho/para-H₂O spin isomers were observed in aqueous solutions of different biopolymers and also in distilled water in the range from zero to 100 cm^?1 with the spectral resolution of 0.05–0.1 cm^?1. The fitting of four-wave mixing spectra shows notable growth of the H₂O₂ concentration and rotational line’s amplitude by a factor of ～3 in DNA solutions due to denaturizing. Besides, we studied the four-photon scattering spectra of α-chymotrypsin aqueous solutions at protein concentrations between 0 and 20 mg/cm³ in the range of ±7 cm^?1. We found that the velocity of sound in the protein aqueous solution measured by the shift of the Mandelstam-Brillouin scattering spectrum components was a cubic dependence on the protein concentration and reached the value of about 3000 m/s at 20 mg/cm³.     

Mass,length, composition and structure of the filamentous bacterial virus fd

Determinations by three independent methods gave an average of (14.6 ± 0.6) × 10⁶ daltons for the anhydrous mass of the filamentous bacterial virus fd; a determination of the mass per unit length by light scattering of the virus in solution gave 1560 ±60 daltons/Å; and three independent methods show that 12.0±O.2% of the virus mass is from the single-stranded circular DNA molecule. The data give an average axial distance of 3.82 ±0.15 Å between major coat protein subunits (5240 daltons each) for virus in solution. The DNA has an up strand and a down strand within the filament, and an average axial distance of 3.29 ± 0.14 Å between neighbouring nucleotides in a given strand is obtained from the data. There are 2.32 ±0.07 nucleotides per major coat protein subunit and hence each of the nucleotides cannot interact in the same way with subunits of coat protein. The results provide a basis for the interpretation of X-ray diffraction patterns of oriented fibers of the virus. The uncertainties cited above are 95% confidence limits.     

Reductive Methylation of the Lysyl Residues in the fd Gene 5 DNA-Binding Protein: CD and 13C NMR Results on the Modified Protein

Abstract

The ∈-amino groups of the six lysyl residues of the fd gene 5 DNA-binding protein have been modified by reductive methylation to form N^∈, N^∈-dimethyl lysyl derivatives containing ¹³C-labeled methyl groups. The α-amino terminus of the protein was not accessible to methylation. Circular dichroism studies show that the modified protein binds to fd DNA, but with a slightly reduced affinity compared with that of unmodified gene 5 protein. We also find that both the modified and unmodified proteins bind to an oligodeoxynucleotide, d(A)₇, but in neither case does binding cause a decrease in the 228 nm CD band of the protein as occurs when the protein binds to long DNA polymers. ¹³C NMR spectra at 50.1 MHz of [¹³C]methylated gene 5 protein show five distinct resonances between 43.30 and 42.76 ppm originating from the six N^∈, N^∈-dimethyl lysyl residues. We attribute one of the resonances to two solvated lysyl residues and the other four to individual lysyl residues in different microenvironments. All four of these latter resonances are affected by the binding of d(A)₇. However, since two of these resonances are similarly affected by the presence of salt in the absence of DNA, only two are uniquely affected by DNA binding.     

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.     

Demonstration by ultraviolet resonance Raman spectroscopy of differences in DNA organization and interactions in filamentous viruses Pf1 and fd

Pf1, a class II filamentous virus, has been investigated by ultraviolet resonance Raman (UVRR) spectroscopy with excitation wavelengths of 257, 244, 238, and 229 nm. The 257-nm UVRR spectrum is rich in Raman bands of the packaged single-stranded DNA (ssDNA) genome, despite the low DNA mass (6%) of the virion. Conversely, the 229-nm UVRR spectrum is dominated by tyrosines (Tyr 25 and Tyr 40) of the 46-residue alpha-helical coat subunit. UVRR spectra excited at 244 and 238 nm exhibit Raman bands diagnostic of both viral DNA and coat protein tyrosines. Raman markers of packaged Pf1 DNA contrast sharply with those of the DNA packaged in the class I filamentous virus fd [Wen, Z. Q., Overman, S. A., and Thomas, G. J., Jr. (1997) Biochemistry 36, 7810-7820]. Interestingly, deoxynucleotides of Pf1 DNA exhibit sugars in the C2'-endo/anti conformation and bases that are largely unstacked, compared with C3'-endo/anti conformers and very strong base stacking in fd DNA; hydrogen-bonding interactions of thymine carbonyls are also different in Pf1 and fd. On the other hand, coat protein tyrosines of Pf1 exhibit Raman markers of ring environment identical to those of fd, including an anomalous singlet at 853 cm-1 in lieu of the canonical Fermi doublet (850/830 cm-1) found in globular proteins. The results indicate markedly different modes of organization of ssDNA in Pf1 and fd virions, despite similar environments for coat protein tyrosines, and suggest strong hydrogen-bonding interactions between DNA bases and coat subunits of Pf1 but not between those of fd. We propose that structural relationships between the protein coat and encapsidated ssDNA genome are also fundamentally different in the two assemblies.     

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.     

Ultraviolet circular dichroism of polyproline and oriented collagen

The ultraviolet absorption, linear dichroism, circular dichroism, and oriented circular dichroism of collagen are reported and the spectra are resolved into a self-consistent set of bands in accord with exciton theory. The parallel band at 200 nm has 40% of the π → π* intensity; the perpendicular band is placed at 189 nm yielding a splitting of 2700 cm^?1. The circular dichroism is resolved into two Gaussians at λ_∥ and λ_τ (rotational strengths +14 × 10^?40 and ?32 × 10^?40 esu². cm²) plus a large non-Gaussian (“helix”) band with ampplitude ?25,000° at 201 nm. These data appear to be in reasonably good accord with recent calculations. Measurements of the absorption, linear dichroism and circular dichroism of polyproline I and II are also reported and are resolved into their component bands. Polyproline I is in good accord with exciton theory, whereas polyproline II remains unsatisfactory.     

Filamentous Bacterial viruses

The filamentous bacterial virus is a simple and well-characterized model system for studying how genetic information is transformed into molecular machines. The viral DNA is a single-stranded circle coding for about 10 proteins. The major viral coat protein is largely α-helical, with about 46 amino acid residues. Several thousand identical copies of this protein in a helical array form a hollow cylindrical tube 1–2μ long, of outer diameter 60 Å and inner diameter 20 Å, with the twisted circular DNA extending down the core of the tube. Before assembly, the viral coat protein spans the cell membrane, and assembly involves extrusion of the coat from the membrane. X-ray fibre diffraction patterns of the Pf 1 species of virus at 4°C, oriented in a strong magnetic field, give three-dimensional data to 4 Å resolution. An electron density map calculated from native virus and a single iodine derivative, using the maximum entropy technique, shows a helix pitch of 5.9 Å. This may indicate a stretched A-helix, or it may indicate a partially 3₁₀ helix conformation, resulting from the fact that the coat protein is an integral membrane protein before assembly, and is still in the hydrophobic environment of other coat proteins after assembly.     

Hydrodynamic properties and structure of fd virus

A length of 8950 ± 200 Å and a diameter of 90 ± 10 Å have been obtained for fd virus from a simultaneous solution of the Broersma equations relating the length and diameter of a rod-like particle to its rotational, D_R, and translational, D_T, diffusion coefficients. Measurements of D_R were by transient electric birefringence, and of D_T by low-angle intensity fluctuation spectroscopy. A mass of (16.4 ± 0.6) × 10⁶ daltons was calculated from the Svedberg equation using our measured values of D_T, the sedimentation coefficient and the density increment. These results, together with the molecular weight of fd DNA, give a total number of major coat protein subunits of 2710 ± 110 and a ratio of nucleotides to protein subunits which is definitely non-integral, 2.30 ± 0.11. These measurements help delineate significant structural differences between fd and other filamentous viruses. Also included in this paper is an Appendix (by L. A. Day & S. A. Berkowitz) concerning the number of nucleotides, 6370 ± 140, and the density and refractive index increments of fd DNA.     

The measurement of transmembrane helices by the deconvolution of CD spectra of membrane proteins: A review

The interpretation of the CD spectra of proteins to date requires additional secondary structural information of the proteins to be analyzed, such as x-ray or nmr data. Therefore, these methods are inappropriate for a CD data base whose secondary structures are unknown, as in the case of the membrane proteins. The Convex Constraint Analysis algorithm [A. Perczel, M. Hollósi, G. Tusnády, and G. D. Fasman (1991) Protein Engineering, Vol. 4, 669–679], on the other hand, operates only on a collection of spectral data to extract the common spectral components with their spectral weights. The linear combinations of these derived “pure” CD curves can reconstruct the original data set with great accuracy. For a membrane protein data set, the five-component spectra so obtained from the deconvolution consisted of two different types of α-helices (the α-helix in the soluble domain and the α_T-helix, for the transmembrane α-helix), a β-pleated sheet, a class C-like spectrum related to β-turns, and a spectrum correlated with the unordered conformation. The deconvoluted CD spectrum for the α_T-helix was characterized by a positive red-shifted band in the range 195–200 nm (+95,000 deg cm² dmol^?l), with the intensity of the negative band at 208 nm being slightly less negative than that of the 222 nm band (?50,000 and ?60,000 deg cm² dmol^?1, respectively) in comparison with the regular α-helix, with a positive band at 190 nm and two negative bands at 208 and 222 nm with magnitudes of + 70,000, ?30,000, and ?30,000 deg cm² dmol^?1, respectively. © 1994 John Wiley & Sons, Inc.     

Laser-excited raman spectroscopy of biomolecules. XII. Thermally induced conformational changes in poly(L-glutamic acid)

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

Further studies of detergent-induced conformational transitions in proteins. Circular dichroism of ovalbumin, bacterial alpha-amylase, papain, and beta-lactoglobulin at various pH values.

The conformational transitions of ovalbumin, bacterial α-amylase, papain, and β-lactoglobulin were studied in the absence and presence of sodium dodecyl sulfate (SDS) between pH 2.75 and 12.0 by means of circular dichroism (CD) measurement. The weight ratios of SDS to protein in solutions were 14:1 in all experiments. The CD bands in the near-ultraviolet spectral region were strongly reduced by SDS, whereas those in the far-ultraviolet were enhanced. With the exception of the amylase, the mean residue ellipticities of the proteins at 222 nm were increased by SDS, especially in acidic solutions. At a pH of about 3.0, the [θ]₂₂₂ values approached ?17 (±2) · 10³ deg · cm² · dmol^?1. It is assumed that at a sufficiently low pH value the proteins which are complexed with SDS have a similar backbone conformation of moderate helical content. In alkaline solutions, the detergent effect was largely reduced due to electrostatic repulsion between the negatively charged protein and dodecyl ions. The near-ultraviolet spectra of ovalbumin, papain, and β-lactoglobulin at pH 6.4 were analyzed. Assignment of the resolved bands to the appropriate chromophores was also attempted.     

An FT-IR Study of DNA and RNA Conformational Transitions at Low Temperatures

Abstract

Fourier Transform Infrared (FT-IR) spectra of solid samples of DNA and RNA obtained from freeze-drying at solid CO₂ and liquid nitrogen temperatures, have been recorded and correlation between the conformational transitions and spectral changes is proposed. It is concluded that an equilibrium exists between A, B and Z conformations at low temperatures for the DNA molecule, which is temperature dependent, whereas the RNA molecule exhibits only the A conformation. The results have been compared with the metal-adducts of DNA and RNA, where one of the conformations is predominant.

Marker infrared bands for the B conformer have been found to be the strong band at 825 cm^?1 (sugar conformer mode) and a band with medium intensity at 690 cm^?1 (guanine breathing mode). The A conformation showed characteristic bands at 810 and 675 cm^?1. The B to Z conformational transition was characterized by the strong absorption bands near 820-810 cm^?1 and at 665-600 cm^?1.     

Polarized Fourier transform infrared (FTIR) difference spectroscopy of the M412 intermediate in the bacteriorhodopsin photocycle

The possibility that light-induced protein conformational changes accompany the formation of the M₄₁₂ species in the bacteriorhodopsin photocycle is investigated by polarized Fourier transform infrared (FTIR) spectroscopy on oriented films of purple membrane. From the light-induced FTIR dichroism changes, it is estimated that: (i) the CO stretching vibration at 1762 cm⁻¹, which has been assigned to a protonated Asp carboxyl group in M₄₁₂ [(1985) Biochemistry 24, 400-407], is oriented at (θ = 35 ± 5° from the normal to the membrane plane; (ii) the limit for the change in the average tilt angle of the α-helices after photoconversion is less than 2°. The latter observation excludes the large variations in the protein conformation during the M⁴¹² formation proposed by Draheim and Cassim [(1985) Biophys. J. 47, 497-507].     

Structure of Bovine αs-Casein

The secondary structure of bovine α_s-casein and chemically modified α_s-casein in various solvents was investigated by infrared absorption spectrum and optical rotatory dispersion measurements. Amino groups of α_s-casein were either succinylated or acetylated, and carboxyl groups were either methylated or ethylated. Acetylated- and ethylated-α_s-caseins are insoluble in water. Water-soluble samples have unordered structure in water. In organic solvents, such as 2-chloroethanol and ethylene glycol, they have about 50% α-helical fraction. On the other hand, it was found that methylated-α_s-casein had two infrared absorption peaks centered at 1625 and 1643 cm^?1 in D₂O-CH₃OD mixed solvent. This fact may be connected with the presence of β-structure. In the case of solid film of this sample, cast from solution containing CH₃OH, the presence of β-structure was indicated, too. The authors attempted to explain the formation of β-structure in methylated-α_s-casein in terms of the electrostatic interactions due to the differences in the net charge between methylated and unmodified α_s-caseins.     

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.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.