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.     

A study of subtilisin types Novo and Carlsberg by circular polarization of fluorescence.

The circular polarization of the luminescence of a chromophore, in addition to its circular dichroism and optical rotatory dispersion, is a manifestation of its asymmetry. In the study of proteins, the circular polarization of luminescence yields more specific information than circular dichroism or optical rotatory dispersion since nonfluorescent chromophores do not contribute, and the spectra of the tyrosine and the tryptophan residues are much better resolved in emission than in absorption. The circular polarization of the fluorescence of the tyrosine and tryptophan residues in derivatives of subtilisin Carlsberg and subtilisin Novo were indeed resolved in this study. The tyrosine residues in the Carlsberg protein, and both tyrosine and tryptophan residues in the Novo protein, were found to be heterogeneous with respect to their optical activity and emission spectra. Changes in the environment of the emitting tyrosine residues in both proteins and in the tryptophan residues in the Novo protein were found on changing the pH from 5.0 to 8.3. The pH dependence of the enzymatic activity of these proteins may thus be due, at least in part, to conformational changes in the molecules. Fluorescence circular polarization also revealed that covalently bound inhibitors at the active site of subtilisin Novo affect the environment of the emitting aromatic side chains, presumably via changes in conformation.     

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.     

Rotatory dispersion and circular dichroism of low-molecular-weight poly- -benzyl-L-glutamate

Rotatory dispersion and circular dichroism of low-molecular-weight poly-γ-benzyl-L -glutamate, which was prepared from the N-carboxyanhydride by n-hexylamine initiation at [A]/[I] 3, 4, and 8, have been measured in ethylene dichloride and dioxane at different concentrations. The rotatory properties of the polypeptides are all characterized by a trough at 233 mμ of a negative Cotton effect or by a negative circular dichroic band at 223 mμ. With increasing A/I value or concentration, dextrorotation increases and the negative dichroic band becomes deeper. Both the trough magnitude and the negative ellipticity are linearly dependent on the content of β-structure, and the rotatory parameters for the pure β-structure are estimated by extrapolation of the linear relations. Circular dichroism and infrared spectra of the polypeptides have also been measured in trifluoroethanol, and the effect of solvents on the polypeptide conformation is discussed.     

Letter: Fragments of polyproline-like structure from a structural protein, cuticlin

Cuticlin is a proline-rich protein isolated from the cuticle of Ascaris lumbricoides. The protein was digested by pronase and fragments very rich in proline were isolated from the digest. Proline accounted for about three-quarters of the total amino acids in the fragments. The number average molecular weight of the fragments was about 3200. The optical rotatory dispersion and circular dichroism measurements indicated that the fragments formed a conformation similar to poly-l-proline II in aqueous solution.     

Chiroptical properties of phospholipids

The chiroptical properties of phospholipids were investigated by optical rotatory dispersion and circular dichroism. The spectra of phosphatidylcholine varied with the solvent used. The sign of the circular dichroism effects differed in some cases from previously reported. The results show that it is possible to distinguish different types of phospholipids from each other. The results also show that optical rotatory dispersion and circular dichroism might be a very useful tool for the determination of the configuration of various phospholipids.     

Purification properties and subunit composition of pig heart lipoate acetyltransferase.

Lipoate acetyltransferase [acetyl-CoA: dihydrolipoate S-acetyl-transferase, EC 2.3.1.12], the core enzyme of the pyruvate dehydrogenase complex, has been highly purified by gel chromatography on Sepharose 6B and sucrose density gradient centrifugation in the presence of potassium iodide. The native enzyme has a sedimentation coefficient (S020,W) of 26.7S and a diffusion coefficient (D020,W) of 1.25 x 10(-7) cm2.-sec-1. The weight-average molecular weight was estimated to be 1.8 million from the sedimentation equilibrium data. The content of right-handed alpha helix in the enzyme molecule was estimated to be about 25% by optical rotatory dispersion and about 22% from the circular dichroism spectra. The enzyme was found to contain about 23 moles of protein-bound lipoic acid per mole of enzyme; some other properties are also reported. Lipoate acetyltransferase dissociated to yield a single subunit with a molecular weight of 74,000 as estimated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and by gel filtration on Bio-Gel in 6 M guanidine-HCl. The molecular weight was also estimated to be 74,000 from sedimentation equilibrium data in 6 M guanidine-HCl] containing 0.1 M 2-mercaptoethanol. Evidence is presented that 1 molecule of lipoate acetyltransferase apparently consists of 24 very similar subunits, each of which contains NH2-terminal alanine. Each subunit contains 1 molecule of covalently bound lipoic acid.     

Spectroscopic studies of random chain and -helical polypeptides in hexafluoroisopropanol

The infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra of five synthetic polypeptides dissolved in hexafluoroisopropanol are reported. This solvent is useful because it dissolves most proteins and non-ionic polypeptides and also is transparent in spectral regions critical for polypeptide conformational diagnoses. Poly-γ-morpholinylethyl-L -glutamamide has random chain type spectra in this solvent, whereas the spectra of poly-γ-methyl-L -glutamate, poly-L -methionine, poly-ε, N -Carbo-benzoxy-L -lysine, and poly-L -homoserine indicate that these four polypeptides are α-helical. Small but significant variability between the different α-helical polypeptides is seen in their circular dichroism spectra and optical rotatory dispersions. An argument is presented that these differences may be due to slight geometry differences between different α-helices.     

The conformation of poly-L-alanine in hexafluoroisopropanol

High-molecular-weight poly-L -alanine dissolved in hexafluoroisopropanol exhibits infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra which are unique and unlike any other previously reported polypeptide spectra. Strong evidence that a helical conformation is present is shown by the high degree of hypochromism in the 187mμ absorption peak and by the positions of the amide infrared bands. The CD and ORD spectra are also similar to those of α-helical polypeptides, though important qualitative and qualitative differences are observed. To explain the novel spectra, which are not mixtures of the spectra of previously reported polypeptide conformations, a new α-helix-like conformation is proposed. The postulated conformation (a doubly hydrogen-bonded helix) is a distorted α-helix in which the peptide carbonyl groups point slightly out from the helix axis and are hydrogen bonded simul taneously both to the NH of the fourth peptide residue to the carboxyl terminal side (as in the classical α-helix), as well as to a solvent molecule's hydroxyl hydrogen.     

Evaluation of RNA conformation from circular dichroism and optical rotatory dispersion data

An analysis of optical rotatory dispersion and circular dichroism of RNA is described which leads to an estimate of the degree of base pairing. By the use of new standards for the double-helical parts of the molecule, based on data for two-stranded viral RNA species, a good fit between calculated and observed curves can be achieved. Where data are available the results of analyses of optical rotatory dispersion and circular dichroism in general show satisfactory consistency.     

Conformational aspects of polypeptides. XXV. Solvent and temperature effects on the conformations of copolymers of benzyl and methyl L-aspartate with nitrobenzyl L-aspartate

A series of copolymers of β-p-nitrobenzyl L -aspartate with β-benzyl L -aspartate and with β-mcthyl L -aspartatc in helix-supporting and helix-breaking conditions have been reexamined by using ultraviolet isotropic, absorption, optical rotatory dispersion, and circular dichroism techniques. Many different conformations are apparent, depending on solvent and temperature. Chloroform, trifluoroethanol, and methylene dichloride support the left-handed helical conformation of the copolymers containing less than about 20 mole-% nitroaromatic residues and the right-handed helical conformation of the copolymers containing more than approximately 30 mole-% nitroaromatic residues. In trifluoroacetic acid all the copolymers are in a random-coil conformation. In hexa-fluoroacetone trihydrate and in trimethyl phosphate, the copolypeptides with low nitroaromatic residues content are predominantly in a disordered conformation, while those with high nitroaromatic residues content show a right-handed helical array. Reversible helix-ramlom-coil transitions are observed with increasing temperature in trimethyl phosphate. An example of right-handed-left-handed helix reversible transition with temperature is reported in a chloroform-trimethyl phosphate (2:1) mixture. Nitrobenzyl-nilrobenzyl side-chain interactions in chloroform, but not in trifluoroacetic acid or in trimethyl phosphate, have been confirmed. For the first time we report the circular dichroism spectra in which the n-π* peptide band of a left-handed helical conformation is almost completely evident.     

Double-helical character of ribonucleic acid from virus-like particles found in Penicillium chrysogenum

1. RNA was isolated from virus-like particles found in Penicillium chrysogenum and resolved into two fractions by gel filtration through agarose columns. 2. Fraction 1 was excluded and had the following properties: 50.9% G+C [AMP 0.246, UMP 0.246, CMP 0.252, GMP 0.255 (mole fraction)]; mol.wt. about 1.2x10(6) daltons; s(20,w) 12.3S and ;melting' temperature about 100 degrees C (solvent 0.15m-sodium chloride-0.015m-sodium citrate pH7.2); optical rotation [alpha](max.) 6000 degrees at 278nm; circular dichroism (epsilon(L)-epsilon(R))(max.)=8.181mol(-1) cm(-1) at 260nm. 3. Properties of fraction 2 include 37.8% G+C [AMP 0.313, UMP 0.312, CMP 0.186, GMP 0.189 (mole fraction)]; mol.wt. about 140000 daltons; s(20,w) 7.3S, T(m) about 85 degrees C (solvent 0.15m-sodium chloride-0.015m-sodium citrate, pH7.2); optical rotation [alpha](max.) 6000 degrees at 278nm; circular dichroism (epsilon(L)-epsilon(R))(max.)=8.241mol(-1) cm(-1) at 260nm. 4. The properties of both fractions were consistent with a double-helical conformation.     

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.     

Optical Rotatory Dispersion and Circular Dichroism of Amino Acid Hydantoins

Optical rotatory dispersion (ORD) and circular dichroism (CD) of 17 amino acid hydantoins were measured between 190 and 600 nm. Most of hydantoins exhibited the negative Cotton effect which showed the trough between 238 and 245 nm. The negative trough of CD was also observed between 212 and 236 nm. The Cotton effect of hydantoins was attributable to n→π^* transition of carbonyl group at C-4 of hydantoin ring.     

Further studies on the binding of divalent cations to the phosphoglycoprotein phosvitin.

Dialysis equilibrium measurements at 25 degrees indicate that, at pH 6.8 and at a concentration of 1.0 times 10(-10) 3 M MnC12 or CoC12, phosvitin binds 113 Mn2+ and 120 Co2+. The binding is cooperative at low cation concentrations. The number of Mg2+, Ca2+, Mn2+, and Co2+ bound is not affected by temperatures of up to 60 degrees; however, the cooperactivity is enhanced. Optical rotatory dispersion and circular dichroism studies indicate that a conformational change occurs on binding of Mn2+ and Co2+ which parallels the one produced by Ca2+ and reported elsewhere [Grizzuti, K., and Perlmann, G.E. (1973), Biochemistry 12, 4399]. The conformational changes induced by Mg2+ and Mn2+ follow different paths. Upon binding of Mn2+ and Co2+ the intrinsic viscosity, [eta], of phosvitin decreases from about 0.5 to 0.03 dl/g, while Mg2+ and Ca2+ decrease [eta] to 0.048 dl/g. The ultraviolet absorption spectrum of phosvitin is altered upon binding of Ca2+, Mn2+, and Co2+, but not upon binding of Mg2+; an increase of the temperature to 60% has no further effect on the spectra.     

On the possibility of determining the secondary structure of proteins in solution

A method to determine the contribution of various side chains to the optical rotatory dispersion and circular dichroism or proteins is presented. This method assumes that the side chain of any given amino acid in a similar situation in different proteins contributes the same to the optical rotatory dispersion and circular dichroism of those proteins. The method has a great deal of flexibility in allowing the investigator to postulate any hypothesis about the contributions of such side chains and ultimately to use statistical tests to test that hypothesis. If attained, knowledge of the contribution of side chains will enable investigators to determine the secondary structure of proteins in terms of certain reference conformations. The current use of polyamino acids as standard reference conformations is not entirely satisfactory. Some of the questions raised by their use are discussed and possible solutions proposed.     

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.     

Conformation of gonadotropin releasing hormone.

The conformation of the gonadotropin releasing hormone (Gn-RH), whose primary sequence is pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-GlyNH2, and of several of its structural analogues has been studied by circular dichroism, optical rotatory dispersion, and fluorescence spectroscopy. The effects of pH, guanidine, and temperature on fluorescence emission have also been examined. Titration data demonstrate that the histidine and tyrosine residues are free of any mutual interactions. The similarity of emission spectra in water and in guanidine hydrochloride solutions precludes significant interactions between the fluorescent groups and other residues. Neither the temperature nor the pH profiles of the emission intensities of either tyrosine or tryptophan reveal any fixed secondary structure in Gn-RH. Both the extent of alkaline quenching and the distance of 10-11 A calculated from F?rster energy transfer theory are in accord with a randomly coiled structure with only one residue between tyrosine and tryptophan. Furthermore, the circular dichroism spectrum and optical rotatory dispersion do not exhibit any contributions from peptide bonds in an ordered structure, although there is a perturbation of the peptide absorption region due to overlapping bands from side-chain chromophores. Gn-RH, therefore, appears to behave as a random coil polypeptide in water devoid of any intrachain residue interactions. This nonordered structure in Gn-RH and the lack of any significant differences in the physical-chemical properties of the hormone analogues indicate that a predetermined solution conformation is not required for biological activity. In contrast to its behavior in water, Gn-RH in trifluoroethanol exhibits a conformational transition, with the formation of a beta structure. Differences in conformational changes exhibited by several analogues in trifluoroethanol may be relevant to their relative biological activities at the receptor site.     

Conformational changes accompanying the binding of antithrombin III to thrombin.

The conformational aspects of the binding of antithrombin III to thrombin were investigated by difference spectroscopy, circular dichroism, and optical rotatory dispersion. The CD and ORD studies indicate an increase of 6--8% in alpha-helix content at the expense of the beta structure, while the results from difference spectroscopy showed an increased exposure of approximately seven tyrosine residues. In the presence of heparin there is a slightly greater increase in helicity which is accompanied by exposure of an average of two tryptophan and one tyrosine residues. These spectral results indicate that the thrombin-antithrombin III complex formed in the presence of heparin differs in its conformation from that produced in its absence.     

Interaction of manganese (II) with valinomycin: observation of mixed complexes

The interaction of antibiotic valinomycin with manganese (II) has been studied using circular dichroism, electron spin resonance and infrared techniques. Results show that Mn(II) forms complexes with valinomycin in both 2:1 (valinomycin-ion-valinomycin sandwich) and 1:1 (equimolar) stoichiometries. The 1:1 type observed here is very different from the well known K⁺-valinomycin bracelet conformation.