Time-resolved absorption, circular dichroism, and emission of tRNA. Evidence that the photo-cross-linking of 4-thiouridine in tRNA occurs from the triplet state

The time-resolved optical density (TROD) and time-resolved circular dichroism (TRCD) spectra of the lowest triplet state of 4-thiouridine (4t-Urd) in aqueous solutions of tRNA are reported. The TROD spectrum is consistent with the triplet state being primarily in the thione tautomer. The intersystem crossing yield to the triplet is 0.35 and 0.27 (+/- 10%), respectively, with and without 10(-2) Mg2+ added to the solution. Upon addition of increasing amounts of I- to solutions of tRNA, the initial triplet yield decreases, the rate of the observed triplet decay increases, and the quantum yield of internal photo-cross-linking decreases for the 4t-Urd chromophore. The results show quantitatively that the near-UV-induced photo-cross-linking reaction in tRNA occurs from the triplet state of 4t-Urd. From the TRCD spectrum the dissymmetry factor (delta epsilon/epsilon) of some of the triplet-triplet absorption bands is shown to be significantly larger than for any of the ground-state absorption bands. Two CD transitions are seen in the triplet-triplet spectrum which are obscured in the TROD spectrum by the strong ground-state bleaching signal near 335 nm. This shows that TRCD may be useful, in some cases, in locating electronic transitions that are not observed in TROD spectra.     

Conformation of poly-L-tyrosine in trimethyl phosphate solution

Absorption, circular dichroism (CD), and optical rotatory dispersion (ORD) measurements were carried out on poly-L -tyrosine in trimethyl phosphate solution over the spectral range 185–600 mμ. There is evidence in the CD spectrum for side chain-side chain interactions in poly-L -tyrosine. ORD and CD data in dimethylformamide and pyridine closely parallel those in trimethyl phosphate, indicating a similarity in conformation of the polymer in all three solvents. In the polarized infrared spectrum both position and polarization of amide A, I, and II bands are characteristic of α-helical polypeptides. Bands corresponding to side chain also exhibit dichroism, suggesting that the side chains are not randomly oriented. Viscosity and light-scattering studies are consistent with α-helical structure for the polymer that, remains rigid over a temperature range of 15–50°C and becomes somewhat flexible at higher temperatures. Optical rotatory properties were found to vary gradually and continuously with temperature over the range of ?30 to +100°C. This suggested that all three electronic transitions of tyrosyl side chain are optically active, and that the side chains have some freedom of motion that decreases with decreasing temperature, disappearing only at about ?30°C.     

Effect of counterions on the conformation of hyaluronic acid.

To study the effect of metal ions on the conformation of hyaluronic acid, circular dichroism (CD) and optical rotatory dispersion (ORD), along with viscosity measurements of the Na, Li, Ca, and Mg salts of the polymer, were carried out. With divalent cations, the results show a decrease in CD minima at 210 nm and an increase in ORD troughs at 220 nm, as compared to monovalent ions. To account for this behavior, the ORD in the visible range corresponding to the observed CD bands was directly calculated from the Moscowitz equation using Kronig-Kramer's transform. The background rotation was found to be more levorotatory in bivalent than in monovalent cations. The ORD spectra of various metal hyaluronates differ significantly from each other in the far ultraviolet region, especially at lower pH values. The values of intrinsic viscosities of these hyaluronates, on the other hand, are almost the same in the pH range of 1–3. These results indicate a local conformation variation rather than any appreciable change in the chain conformation of the molecule in the presence of different counterions.     

Kramers-Kronig transformation of experimental electronic circular dichroism: application to the analysis of optical rotatory dispersion in dimethyl-L-tartrate

When a limited region of the experimental electronic circular dichroism (ECD) spectrum is subjected to Kramers-Kronig (KK) transformation, the resulting optical rotatory dispersion (ORD) may or may not reproduce the experimentally measured ORD in the long-wavelength nonresonant region. If the KK transform of experimentally measured ECD in a limited wavelength region reproduces the experimentally measured ORD in the long-wavelength nonresonant region, then that observation indicates that the ORD in the long-wavelength nonresonant region should be satisfactorily predicted from the correspondingly limited number of electronic transitions in a reliable quantum mechanical calculation. On the other hand, if the KK transform of experimentally measured ECD in a limited region does not reproduce the experimentally measured ORD in the long-wavelength nonresonant region, then it should be possible to identify the ECD bands in the shorter wavelength region that are responsible for the differences between experimentally observed ORD and KK-transformed ECD. This approach helps to identify the role of ECD associated with higher energy-excited states in the nature of ORD in the long-wavelength nonresonant region. These concepts are demonstrated here by measuring the experimental ECD and ORD for dimethyl-L-tartrate in different solvents. While ECD spectra of dimethyl-L-tartrate in different solvents show little variation, ORD spectra in the long-wavelength nonresonant region show marked solvent dependence. These observations are explained using the difference between experimental ORD and KK-transformed ECD. Quantum mechanical predictions of ECD and ORD are also presented for isolated (R, R)-dimethyl tartrate at the B3LYP/aug-cc-pVDZ level.     

Free will and evolution

The circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of a suspension of arbitrarily shaped optically active particles may be calculated by using the Rayleigh-Debye theory. Subject to restrictions on the size of the particles and the refractive index of the suspending medium the CD and ORD spectra of the suspension are the same as the intrinsic molecular spectra. The size of the particles can be comparable to or larger than the wavelength of the incident light provided that the optical properties of particles and medium are sufficiently similar. Therefore, it should be possible to experimentally reduce scattering artefacts in CD and ORD spectra of suspensions by suitably choosing a medium in which the particles are suspended.     

Dynamics of protein and chromophore structural changes in the photocycle of photoactive yellow protein monitored by time-resolved optical rotatory dispersion

The dynamics of the PYP photocycle have been studied using time-resolved optical rotatory dispersion (TRORD) spectroscopy in the visible and far-UV spectral regions to probe the changes in the chromophore configuration and the protein secondary structure, respectively. The changes in the secondary structure in PYP upon photoisomerization of the chromophore can be described by two exponential lifetimes of 2 +/- 0.8 and 650 +/- 100 ms that correspond to unfolding and refolding processes, respectively. The TRORD experiments that follow the dynamics of the chromophore report three exponential components, with lifetimes of 10 +/- 3 micros, 1.5 +/- 0.5 ms, and 515 +/- 110 ms. A comparison of the kinetic behaviors of the chromophore and protein shows that during the decay of pR(465) an initial relaxation that is localized to the chromophore hydrophobic pocket precedes the formation of the chromophore and protein structures found in pB(355). In contrast, the protein and chromophore processes occur with similar time constants during inactivation of the signaling state.     

Optical studies on the structure of hemoglobin messenger ribonucleic acid

The secondary structure of hemoglobin mRNA (HbmRNA has been investigated by optical rotatory dispersion (ORD), circular dichroism (CD) and ultraviolet absorbance spectrophotometry. The dependence on temperature or reaction with formaldehyde of the CD and absorbance are characteristic of a structure with substantial base pairings at 20°C. The presence of Cotton effects in one of the major ultraviolet absorption bands indicates a highly ordered secondary structure. The UV hyperchromism on thermal denaturation is consistent with a value of 58% double helical content.     

Oligonucleotide studies: optical rotatory dispersion of normal and 2'-O-methylated diribonucleoside monophosphates

The optical rotatory dispersion (ORD) of several diribonucleoside monophosphates (NpN) and the corresponding 2′-O-methyl substituted dinucleoside monophosphates containing 2′-O-methyl ribosyl 3′-nucleotide and a 5′-nucleoside (NmpN) were measured at pH 1, 7, and 11.2, at 0.1 ionic strength in order to examine the role of the 2′-hydroxyl group of the ribose in the conformation of the oligoribonucleotides. The optical measurements are reported from 210 to 340 mμ. The pH effect on the ORD spectra of NpN as well as NmpN are large. No dramatic changes are seen in the shapes of the ORD spectra of the NmpN to the corresponding NpN at pH 7. However, a decrease in the amplitude is seen in most of the NmpN over that of the corresponding NpN ranging from 7 percent in the case of UmpG to 46 percent in AmpA. The differences seen in the NpN and the corresponding NmpN ORD results are best explained as a consequence of a change in the ribosyl conformation on 2′-O-methylation, rather than the involvement of the 2′-hydroxyl group in intramolecular hydrogen bonding in the ribo dimmer. The NmpN behave like NpN and not dNpdN, suggesting that the geometry of the stack in NpmN and NpN depends on the oxygen at the 2′-carbon and not on what is attached to it.     

Critical comparison of the experimental optical activity of helical polypeptides and the predictions of the molecular exciton model

The predictions of the presently accepted molecular exciton model for the optical activity of helical polypeptides are in reasonable agreement with experimental spectra in the accessible wavelength range. However, crucial verification requires the detect of a significant negative rotatory hand just below the accessible range. A computer-oriented method is utilized to obtain information concerning the inaccessible range. Optical rotatory dispersion computed by evaluation of the Kronig-Kramers integral transform from the experimentally determined circular dichroism of several helical homopolypeptides in solution are compared with the experimentally determined optical rotatory dispersion. Computed and experimental curves are congruent within an uncertainty approaching that of the experimental technique, whatever the polypeptide sample lot, side chains, and solvent. It is shown that t his agreement is not a computational or experimental artifact. These results can be interpreted in two ways: (1) that the predicted band does not exist, and (2) that the perturbation of the predicted band is being negated by other inaccessible bands in the vacuum ultraviolet. Arguments are presented to show that the first of these two possibilities is more probable.     

Solution studies on heme proteins. Circular dichroism and optical rotation of Glycera dibranchiata hemoglobins

Circular dichroism (CD) and optical rotatory dispersion (ORD) spectra of several liganded derivatives of the monomer and polymer hemoglobin components of the marine annelid, Glycera dibranchiata were measured over the wavelength range 650--195 nm. The differences observed between the monomer and polymer components for the heme dichroic bands in the visible, Soret and ultraviolet wavelength regions seem to result from changes in the heme environment, geometry and coordination state of the central heme iron in these proteins. Within the Soret region, the liganded derivatives of the monomer hemoglobin exhibit predominantly negative circular dichroic bands. The heme band at 260 nm is also absent for the monomer hemoglobin. The ORD and CD spectra in the far-ultraviolet, peptide absorbing region suggest also differences in the alpha-helix content of the monomer and polymer hemoglobins. The values for the single-chain G. dibranchiata hemoglobin are in the expected range (about 70% alpha-helix) as predicted by the X-ray structure of this protein. The lower estimates of the alpha-helix content for the polymer hemoglobin (approx. 50%), may reflect the differences in amino acid composition, primary structure and polypeptide chain foldings. Changes in oxidation state and ligand binding appears to have no pronounced effect on the helicity of either the monomer or polymer hemoglobins. The removal of the heme moiety from the monomer hemoglobin did result in a major decrease in its helix content similar to the loss of heme from myoglobin.     

Attempt of Microbial Mutagen Screening with Rec-assay; Isolation of Chartreusin

A strong Cotton effect, which practically govern the sign of the optical rotation at 589 nm ([M]_d), was studied in phenyl 1-thio-α (and β)-d-glycopyranosides with our new chiroptical technique. The proposal optical rotatory dispersion (ORD) method, with calculations based on a one term Drude equation, showed the presence of a strong Cotton effect at 200–210 nm. Circular dichroism (CD), with accumulation technique, also gave the same Cotton effect. Agreement in these two methods suggests the usefulness of the proposed ORD calculation method. The rotational strengths and the signs were shown to reflect the anomeric configurations and conformations (α-anomer gave positive and β-anomer gave negative signs; axial gave strong and equatorial gave weak bands). This result is an extension of the ring oxygen helicity rule of alkyl and alkyl thioglycosides to phenyl 1-thioglycopyranosides, and probably to other aromatic glycosides.     

A LOV story: the signaling state of the phot1 LOV2 photocycle involves chromophore-triggered protein structure relaxation, as probed by far-UV time-resolved optical rotatory dispersion spectroscopy

Light-, oxygen-, or voltage-regulated (LOV1 and LOV2) domains bind flavin mononucleotide (FMN) and activate the phototropism photoreceptors phototropin 1 (phot1) and phototropin 2 (phot2) by using energy from absorbed blue light. Upon absorption of blue light, chromophore and protein conformational changes trigger the kinase domain for subsequent autophosphorylation and presumed downstream signal transduction. To date, the light-induced photocycle of the phot1 LOV2 protein is known to involve formation of a triplet flavin mononucleotide (FMN) chromophore followed by the appearance of a FMN adduct within 4 micros [Swartz, T. E., Corchnoy, S. B., Christie, J. M., Lewis, J. W., Szundi, I., Briggs, W. R., and Bogomolni, R. A. (2001) J. Biol. Chem. 276, 36493-36500] before thermal decay back to the dark state. To probe the mechanism by which the blue light information is relayed from the chromophore to the protein, nanosecond time-resolved optical rotatory dispersion (TRORD) spectroscopy, which is a direct probe of global secondary structure, was used to study the phot1 LOV2 protein in the far-UV region. These TRORD experiments reveal a previously unobserved intermediate species (tau approximately 90 micros) that is characterized by a FMN adduct chromophore and partially unfolded secondary structure (LOV390(S2)). This intermediate appears shortly after the formation of the FMN adduct. For LOV2, formation of a long-lived species that is ready to interact with a receptor domain for downstream signaling is much faster by comparison with formation of a similar species in other light-sensing proteins.     

Conformational aspects of polypeptides. XXIX. Conformational assignments for some aromatic polypeptides by far-UV Cotton effects. New results

Recent improvements in apparatus permit the examination of circular dichroism (CD) and optical rotatory dispersion (ORD) spectra to 185 mμ. In addition, new solvents which are transparent to 185 mμ have become available for synthetic polypeptides. The spectral region 185–250 mμ is extremely important for the amide (peptide) chromophore, because of the presence at these wavelengths of the n–π* and π–π* bands,¹ and of another transition, the assignment of which remains unsettled.²     

Interaction of palladium (II) with DNA

The nature of interaction of palladium (II) with calf thymus DNA was studied using viscometry, ultraviolet, visible and infrared spectrophotometry and optical rotatory disperison and circular dichroism measurements. The results indicate that Pd(II) interacts with both the phosphate and bases of DNA. The ORD/CD data indicate that the binding of Pd(II) to DNA brings about considerable conformational changes in DNA.     

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.     

Conformation of allantoicase in aqueous solution

1. The separation of 0.9-S and 10.8-S allantoicase with the aid of a 2H2O-H2O gradient was described. The resulting preparations were subjected to sedimentation equilibrium, optical rotatory dispersion (ORD), circular dichroism (CD) and infrared studies. 2. The molecular weight of 0.9-S allantoicase was determined to be about 1.1 x 10(4) g/mole in studies on the sedimentation behavior, the metal content and amino acid composition. The molecular weight of 10.8-S allantoicase was about 15.4 x 10(4) g/mole. 3. Optical rotatory dispersion, circular dichroism and infrared studies indicated that both molecules contain alpha-helix, beta conformation and random coil. A Cotton effect at 418 nm was ascribed to the asymmetric binding of Mn2+ to the enzyme. Competitive inhibitors decreased the absorption and circular dichroism bands at about 280 nm and 418 nm. These phenomena suggested that the aromatic groups may play an essential role in the binding of substrates and inhibitors by the Mn(2+)-enzyme complex. 4. Comparison of alpha-helical contents of metalloallantoicases showed that the enzymes with low helical contents exhibited high enzymic activities. 5. The nearly identical physicochemical behavior and specific enzymic activity of 0.9-S and 10.8-S allantoicase indicated that they are very similar in structure and conformation.     

Conformational changes in the progesterone binding globulin-progesterone complex.

An improved purification procedure for the progesterone-binding globulin (PBG) of the pregnant guinea pig has been developed utilizing sulfopropyl Sephadex, a strong cation exchanger, in the first step. The method exploits the low pI (2.8) and favorable acid stability of the glycoprotein. Subsequent chromatographies on DEAE-cellulose and Sephadex G-200 afford a highly purified PBG that exhibits the previously observed polydispersity (R.M. Burton et al. (1974), Biochemistry 13, 3554-3561). Circular dichroism, optical rotatory dispersion, and difference uv spectra all indicate the purified protein to undergo a conformational transition upon forming a complex with a steroid ligand. The CD and ORD spectra cannot be interpreted in terms of tertiary structure probably due to carbohydrate contributions. However, the difference spectra indicate strong perturbation of both a tryptophan residue and the steroid chromophore in the complex.     

Conformational studies on a modified poly-L-tryptophan: Circular dichroism and optical rotatory dispersion of poly-2-(2-nitrophenylsulfenyl)-L-tryptophan and of random copolymers of L-tryptophan and 2-(2-nitrophenylsulfenyl)-L-tryptophan

Optical rotatory dispersion (ORD) and circular dichroism (CD) measurements were carried out on a block copolymer, (γ-ethyl DL -glutamate)₁₆₀ (L -Trp)₃₂, in which the tryptophan sequence has been modified to various extents by using 2-nitrophenylsulfenyl chloride. The CD spectrum of the completely modified copolymer exhibits bands in some of the regions of maximum absorption of the sidechain chromophores. In the peptide absorption region the spectrum is similar to that reported in the literature for polypeptides in the α-helical conformation. When the extent of modification of the tryptophan sequence is progressively reduced, there is a gradual change in the ORD spectra of the copolymers. On the basis of these data the assumption was made that no conformational change occurs on proceeding from the pure unmodified tryptophan sequence to the completely modified sequence. The results are discussed in connection with the study of possible conformational effects arising from selective chemical modification of tryptophan residues in proteins.     

Extension of the Ring Oxygen Helicity Rule to Phenyl and p-Nitrophenyl Glycopyranosides

The Cotton effect below 200 nm was first studied in aromatic glycopyranosides with our new technique of optical rotatory dispersion (ORD). The phenyl and p-nitrophenyl α- and β-d-glycopyranosides showed a strong Cotton effect at around 180 nm which practically governed the optical rotations in the visible wavelength region. The rotational strength and sign of this Cotton effect were shown to reflect the anomeric configurations and conformations, which led to an extension of the ring oxygen helicity rule for alkyl and alkyl 1-thioglycosides to aromatic glycopyranosides.     

Conformational changes in the reassembly of MS2 protein subunits

Conformatioiial changes during the reassembly of bacteriophage MS2 coat proteiii from guanidiiie hydrochloride have been investigated by optical rotatory dispersion. Optical rotatory dispersion aid circular dichroism data of the reassembled particles are interpreted as those of β structure. Refolding of the subunit with increasing temperature demonstrated in 2 M guauidine hydrochloride. The stabilization of this structure by hydrophobic bonds and its role in reassembly is discussed.