The Structure of Tobacco Mosaic Virus and Its Components: Ultraviolet Optical Rotatory Dispersion

An investigation has been made of the optical rotatory dispersion in the region 226 to 366 mμ of tobacco mosaic virus (TMV), the protein subunits isolated therefrom, the rods synthesized from the protein subunits, and the ribonucleic acid (RNA) isolated from TMV. Both TMV and the protein rods show anomalous rotatory dispersion. The RNA shows a Cotton effect with an inflection point around 260 mμ, which is shifted to 272 mμ in concentrated urea solution. A suggested interpretation of the RNA rotatory dispersion is given. The rotatory dispersion of the protein subunits shows an incipient Cotton effect with an inflection point around 293 mμ and the beginnings of a large negative Cotton effect with a trough at 232 mμ. The dispersion data from the protein subunits can be interpreted to indicate that they contain between 25 and 35 per cent α-helix. On the basis of recent sequence investigations and the relationship between amino acid composition and polypeptide structure, the helical portion of the protein subunits can be located in the central section of the protein chain.     

The Effects of Solvent Environment on the Optical Rotatory Dispersion Parameters of Polypeptides: II. Studies on Poly-l-Glutamic Acid

The Moffitt b₀ parameter of poly-L-glutamic acid in the presumed helical state varied with solvent composition, ranging in magnitude from less than 600° in aqueous solution to 800° in methanol. b₀ was also dependent on temperature throughout the excessable temperature range. The value in aqueous solution is at least 100° smaller than the values for a number of polypeptides in organic solvents, when compared at the same refractive index. Therefore the optical rotatory dispersion data do not provide evidence that the molecule is completely helical in aqueous solution. Since other types of evidence for helical content are not sufficient to establish that PLGA is a complete helix, the helical content of proteins and polypeptides determined by rotatory dispersion measurements should be regarded as uncertain by about 20 per cent.     

Rotatory dispersion and circular dichroism of brain "proteolipid" protein

Abstract— The proteolipid and its partially delipidated protein from brain tissue have been studied by optical rotatory dispersion and circular dichroism in various solvents. The protein exhibits a helix content of 60–70 per cent in chloroform-methanol and 2-chloroethanol and is dextrorotatory in the visible region. In 1,1,1-trinuoroethanol, a higher helix content of 90 per cent is observed but the protein is laevorotatory. The helix content decreases in water and methanol with a much greater negative specific rotation in methanol. No random coil form has been observed.     

Secondary structural changes of metmyoglobin and apomyoglobin in anionic and cationic surfactant solutions: Effect of the hydrophobic chain length of the surfactants on the structural changes

Secondary structural changes of metmyoglobin and apomyoglobin were examined in solutions of sodium alkylsulfates with hydrocarbon numbers of 8 and 12, and alkyltrimethylammonium bromides with hydrocarbon numbers of 10, 12, 14, and 16. The relative proportion ofa-helical structure was estimated by the curve-fitting method of circular dichroic spectrum. The helical proportions of metmyoglobin and apomyoglobin were 82 and 63%, respectively. The shorter the hydrocarbon chain the surfactant had, the higher the concentration necessary to disrupt the secondary structures of these proteins. However, the helical proportion had a tendency to decrease down to lower values in solutions of the cationic surfactants with short hydrophobic groups. On the other hand, thea-helical structure of apomyoglobin was disrupted in lower concentrations of each cationic surfactant than that of metmyoglobin, although the disruptions of the same structures in both the proteins occurred in the same concentration range of each anionic surfactant. It appeared likely that the removal of the heme group unstabilized the myoglobin conformation only in the cationic surfactant solutions.     

The myoglobin protein radical. Coupling of Tyr-103 to Tyr-151 in the H2O2-mediated cross-linking of sperm whale myoglobin

Sperm whale metmyoglobin, which has tyrosine residues at positions 103, 146, and 151, dimerizes in the presence of H2O2. Equine metmyoglobin, which lacks Tyr-151, and red kangaroo metmyoglobin, which lacks Tyr-103 and Tyr-151, do not dimerize in the presence of H2O2. The dityrosine content of the sperm whale myoglobin dimer shows that it is primarily held together by dityrosine cross-links, although more tyrosine residues are lost than are accounted for by dityrosine formation. Digestion of the myoglobin dimer with chymotrypsin yields a peptide with the fluorescence spectrum of dityrosine. The amino acid composition, amino acid sequence, and mass spectrum of the peptide show that cross-linking involves covalent bond formation between Tyr-103 of one myoglobin chain and Tyr-151 of the other. Replacement of the prosthetic group of sperm whale myoglobin with zinc protoporphyrin IX prevents H2O2-induced dimerization even when intact horse metmyoglobin is present in the incubation. This suggests that the tyrosine radicals required for the dimerization reaction are generated by intra- rather than intermolecular electron transfer to the ferryl heme. Rapid electron transfer from Tyr-103 to the ferryl heme followed by slower electron transfer from Tyr-151 to Tyr-103 is most consistent with the present results.     

High-resolution study of the three-dimensional structure of horse heart metmyoglobin

The three-dimensional structure of horse heart metmyoglobin has been refined to a final R-factor of 15.5% for all observed data in the 6.0 to 1.9 A resolution range. The final model consists of 1242 non-hydrogen protein atoms, 154 water molecules and one sulfate ion. This structure has nearly ideal bonding and bond angle geometry. A Luzzati plot of the variation in R-factor with resolution yields an estimated mean co-ordinate error of 0.18 A. An extensive analysis of the pattern of hydrogen bonds formed in horse heart metmyoglobin has been completed. Over 80% of the polypeptide chain is involved in eight helical segments, of which seven are composed mainly of alpha-helical (3.6(13))-type hydrogen bonds; the remaining helix is composed entirely of 3(10) hydrogen bonds. Altogether, of 102 hydrogen bonds between main-chain atoms only six are not involved in helical structures, and four of these six occur within beta-turns. The majority of water molecules in horse heart metmyoglobin are found in solvent networks that range in size from two to 35 members. The size of water molecule networks can be rationalized on the basis of three factors: the number of hydrogen bonds to the protein surface, the presence of charged side-chain atoms, and the ability to bridge to neighboring molecules in the crystal lattice. Bridging water networks form the dominant intermolecular interactions. The backbone conformation of horse heart metmyoglobin is very similar to sperm whale metmyoglobin, with significant differences in secondary structure occurring only near residues 119 and 120, where residues 120 to 123 in sperm whale form a distorted type I reverse turn and the horse heart protein has a type II turn at residues 119 to 122. Nearly all of the hydrogen bonds between main-chain atoms (occurring mainly in helical regions) are common to both proteins, and more than half of the hydrogen bonds involving side-chain atoms observed in horse heart are also found in sperm whale metmyoglobin. Unlike sperm whale metmyoglobin, the heme iron atom in horse heart metmyoglobin is not significantly displaced from the plane of the heme group.     

Structural Stability of Myoglobin in Organic Media

The structural stability of metmyoglobin in organic solvents and cosolvents was investigated aiming the choice of a suitable medium to perform its dissolution with maintenance of the native folding. The spectroscopic behavior of metmyoglobin solution in UV–Visible and circular dichroism was used to evaluate the solubility and the secondary structure. The results were dependable of the chemical structure of the organic compounds, their polarity and content, in the case of cosolvents. Protic solvents showed better ability than the aprotic ones for the biomolecule dissolution, since they are able to establish hydrogen bonds. Solvents with high polarity usually damage the secondary structure of the protein. Myoglobin was dissolved in pure methanol, ethylene glycol and glycerol. The secondary structure was retained in some extent. The controlled addition of sodium dodecyl sulfate to myoglobin aqueous solution changed the surface moiety of the protein. The complex was extracted to hexane with efficiency of 77%.     

The optical rotatory properties of poly- -D-glutamic acid

Measurement of the optical rotatory dispersion spectra of poly-γ-D -glutamic acid (obtained from Bacillus anthracis) dawn to 200 nm wavelength reveal difference between the unionized and ionized froms. The profile of the unionized polyacid shows similarities to those obtained for α-helical polypeptides, although with displaced frequencies of the respective maxima and minima. It is suggested that the relative position and magnitudes of the Cotton effect are consistent with a helical structure such as proposed by Rydon (J. Chem. Soc., 1964 , 1328). The optical rotatory dispersion spectrum of the ionized from resembles those obtained from the β-chain from of α-L polypeptides. From model-building studies an extended chain similer to the β-from would seen the most reasonable structure for the ionized poly-γ-acid to adopt, since the charged groups in such a conformation would be at their maximal distances from each other. Such an ordered structure for a polymer is consistent with the hypotheses put forward in the recent literature that charged polypeptides adopt ordered rather than random-coiled conformations.     

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.     

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.     

The interaction of Trolox C, a water-soluble vitamin E analog, with ferrylmyoglobin: reduction of the oxoferryl moiety.

The oxidation of the heme iron of metmyoglobin by H2O2 yields an oxo ferryl complex (FeIV = O), similar to Compound II of peroxidases, as well as a protein radical; this high oxidation state of myoglobin is known as ferrylmyoglobin. The interaction of Trolox, a water-soluble vitamin E analog, with ferrylmyoglobin entailed two sequential one-electron oxidations of the phenolic antioxidant with intermediate formation of a phenoxyl radical and accumulation of a quinone end product. These oxidation reactions were linked to individual reductions of ferrylmyoglobin to metmyoglobin, as indicated by the value of the relationship [metmyoglobin]formed/[Trolox]consumed: 1.92 +/- 0.28. The Trolox-mediated reduction of ferrylmyoglobin to metmyoglobin could proceed directly, i.e., electron transfer from the phenolic-OH group in Trolox to the oxoferryl moiety, or indirectly, i.e., sequential electron transfer from Trolox to a protein radical to the oxoferryl moiety. The former mechanism is supported by the finding that the high oxidation heme iron is reduced under conditions where the tyrosyl residues are blocked by o-acetylation and when hemin is substituted for myoglobin. The latter mechanism is consistent with the following observations: (a) the EPR signal ascribed to the protein radical is suppressed by Trolox, with the concomitant appearance of the EPR spectrum of the Trolox phenoxyl radical and (b) the rate of ferrylmyoglobin reduction by Trolox is decreased with increasing number of tyrosyl residues in the proteins of horse myoglobin (titrated by o-acetylation) and sperm whale myoglobin. The apparent discrepancy between these observations can be reconciled by considering that both electrophilic centers in ferrylmyoglobin--the oxoferryl heme moiety and the protein radical--function independently of each other and that recovery of ferrylmyoglobin by Trolox could be effected through the tyrosyl residues, albeit at slower rates. The mechanistic aspects of these results are discussed in terms of the two main redox transitions in the myoglobin molecule encompassing valence changes of the heme iron and electron transfer of the tyrosyl residue in the protein and linked to the two sequential one-electron oxidations of Trolox.     

Encapsulation of myoglobin with a mesoporous silicate results in new capabilities

A metmyoglobin (Fe3+), an oxidized form of myoglobin (Fe2+), was confined in nanospaces of about 4 nm in diameter in mesoporous silica (FSM; folded-sheet mesoporous material), forming a metmyoglobin (Fe3+)-FSM nanoconjugate. The spectral characteristics of metmyoglobin (Fe3+)- and myoglobin (Fe2+)-FSM show an absorption curve quite similar to that of native metmyoglobin, indicating that myoglobin retains its higher-order structure in the pores of FSM. The metmyoglobin (Fe3+)-FSM conjugate had not only a peroxidase-like activity in the presence of hydrogen peroxide (a hydrogen acceptor) and 2,2-azino-bis(3-ethylbenzothiazoline)-6-sulfomic acid (ABTS) or guaiacol (a hydrogen donor) but also an advanced molecular recognition ability enabling it to distinguish between ABTS and guaiacol. Furthermore, the metmyoglobin (Fe3+)-FSM showed the peroxidase-like activity even in an organic media using benzoyl peroxide as the hydrogen acceptor and leucocrystal violet as the hydrogen donor. The simple immobilization of metmyoglobin (Fe3+) into FSM results in enhanced catalytic activity in organic media compared to that of native metmyoglobin (Fe3+).     

Identification of neutral and anionic 8 alpha-substituted flavin semiquinones in flavoproteins by electron spin resonance spectroscopy

Circular dichroic spectra of metmyoglobin and apomyoglobin were measured in neutral and acidic solution. Addition of sodium dodecyl sulfate (NaDodSO₄) slightly reduces the helicity (based on the circular dichroic magnitude) of both proteins probably because of the loss of long-range interactions among helical segments. Lowering the pH of the protein-surfactant solution to 3 slightly enhances the helical conformation of myoglobin due to the protonation of acidic side groups and thereby the reduction of coulombic repulsion among negative charges. For BrCN-digested fragments the COOH-terminal peptide (22 residues) loses its helicity which can be restored by addition of NaDodSO₄. The middle fragment (76 residues) retains a considerable amount of helicity in water alone, which further increases in the presence of NaDodSO₄. The NH₂-terminal fragment (55 residues) also has some helical conformation in water, which is enhanced by the addition of NaDodSO₄. The circular dichroic spectrum of an equimolar mixture of the three peptides in NaDodSO₄ solution is the same as that calculated from the spectra of isolated peptides under similar conditions.     

Horse heart metmyoglobin. A 2.8-A resolution three-dimensional structure determination

The structure of horse heart metmyoglobin has been determined with a molecular replacement approach and subsequently refined using rigid body and restrained-parameter least squares methods to a conventional crystallographic R-factor of 0.16 for all observed reflections in the 6.0-2.8-A resolution range. The polypeptide chain of this protein is found to be organized into eight helical regions (labeled A-H) which collectively form a hydrophobic pocket in which the heme prosthetic group is bound. Our results show that the overall thermal motions of individual residues of horse heart metmyoglobin are correlated with their mean distances from the heme group. In comparisons with the structure of sperm whale metmyoglobin it has been found that horse heart metmyoglobin has unique polypeptide chain conformations in four regions. These include residues in the immediate vicinity of the amino and carboxyl termini, residues about Lys-16, and residues 117-124 which are in the interhelical region between helices G and H. Many of these conformational changes appear to occur as a consequence of a different pattern of salt-bridging interactions between charged residues on the surface of horse heart metmyoglobin. The overall average positional deviation observed between corresponding alpha-carbons in the polypeptide chains of horse heart and sperm whale metmyoglobin is 0.50 A. This value for atoms of the porphyrin core of the central heme group is 0.39 A. A total of 12 well defined water molecules and 1 sulfate ion are included in the current structural model of horse heart metmyoglobin. One of these water molecules is found to be coordinated to the heme iron atom and hydrogen bonded to the side chain of His-64. The sulfate ion is hydrogen bonded to amide groups at the amino-terminal end of the E-helix and, as well, forms similar interactions with the amino-terminal end of the D-helix of an adjacent protein molecule in the crystalline lattice.     

Redox cycling of myoglobin and ascorbate: a potential protective mechanism against oxidative reperfusion injury in muscle

Metmyoglobin catalyzes the decomposition of H2O2 as well as other hydroperoxides by using ascorbic acid as a substrate. The ratio of H2O2 reduced to ascorbate oxidized is close to one, whereas the rate of oxidation is directly proportional to both H2O2 and metmyoglobin concentrations. Ascorbate also prevents the protein modifications and the O2 evolution that accompany the reaction of metmyoglobin with hydroperoxides. In the absence of ascorbate, myoglobin and H2O2 promote the peroxidation of unsaturated fatty acids and, thus, may cause damage to cellular constituents. However, lipid peroxidation is inhibited in the presence of ascorbate and, for this reason, it is suggested that this heme protein functions in the opposite manner. The redox cycling of myoglobin by ascorbate may act as an important electron "sink" and defense mechanism against peroxides during oxidative challenge to muscle.     

Copper(II) protoporphyrin IX as a reporter group for the heme environment in myoglobin.

Copper(II) protoporphyrin IX has been introduced into apomyoglobin, and its utility as a reporter group of the heme environment has been examined. The Soret and visible absorption bands and electron spin resonance spectrum show that the Cu(II) is five coordinate, probably through coordination to the F-8 proximal histidine. The resonance Raman spectrum does not indicate any appreciable distortion from the solution conformation of copper(II) protoporphyrin IX dimethyl ester in CS2. The ultraviolet circular dichroism shows no alteration of the helical content of the globin from that of metmyoglobin. The circular dichroism of the porphyrin transitions suggests that the packing of the amino acid side chains around the porphyrin is different than that in the native metmyoglobin.     

Reversible Dissociation of Crystalline Yeast Phosphoglyceric Acid Mutase in Alkali

The structure of crystalline yeast phosphoglyceric acid mutase has been investigated by sedimentation-velocity and equilibrium measurements, optical rotatory dispersion measurements and viscometry. The data indicate that this enzyme is a globular, compact and highly organized protein with a low helix content. The native structure remains unchanged at pH 10.5. Dissociation of the enzyme into subunits has been observed at pH values of 11.5 and above. From optical rotatory dispersion measurements, it is found that the enzyme loses a large part of its organized conformation when it dissociates in alkaline solution. On neutralization, the alkali-treated enzyme regains its activity. The ability to regain the enzyme activity is gradually lowered with the increase of pH value to be incubated and with time of exposure. Inactivation at pH 13.0 is almost irreversible. However, the reversibility of the inactivation at pH 13.0 is appreciably enhanced by the presence of phosphate compounds in the reactivation system. Particulary, it is found that presence of substrates or the coenzyme is effective for considerable improvement of the reversibility. Molecular weight analyses by ultracentrifugation indicate that subunits have approximately equal molecular weights and that the native enzyme is consisted of four polypeptide chains.     

The isolation and properties of experimental allergic encephalitogenic protein

1. Experimental allergic encephalitogenic (EAE) protein was isolated from ox spinal cord by a modification of the method of Martenson & LeBaron (1966). 2. The protein was examined by acrylamide-gel electrophoresis and its amino acid composition determined. 3. Sedimentation-velocity runs in the ultracentrifuge indicate a molecular weight of about 15100 at pH7.8, and calculation suggests that approx. 137 amino acid residues are present per molecule. 4. Gel-filtration and diffusion studies suggest that the protein is non-globular. 5. Optical-rotatory-dispersion measurements show the protein to have no helical secondary structure even at pH values near to the isoelectric point. In the presence of triphosphoinositide, changes in the optical rotatory dispersion of the protein could be interpreted to mean that it develops a small degree of secondary structure. 6. On treatment with cyanogen bromide the experimental allergic encephalitogenic protein is split into at least two fragments, the larger of which is only about 12% smaller than the parent protein and is antigenically active, and the smaller of which is devoid of antigenic activity.     

Preparation and study of magnesium deuteroporphyrin myoglobin and hemoglobin species

The myoglobin and hemoglobin species containing magnesium deuteroporphyrin have been prepared and studied by electronic, circular dichroism and optical rotatory dispersion spectroscopy. The results are compared with those obtained for corresponding magnesium protoporphyrin and magnesium mesoporphyrin complexes. In all cases the magnesium-apomyoglobin species show additional band splittings. These may arise directly from differences in the protein environment or indirectly through water coordination to magnesium which is facilitated by features of the myoglobin heme pocket but inhibited in the hemoglobin complexes. The availability of results for three different porphyrins enables a red shift of spectral bands, observed in particular for MgPP-Mb^**, to be specifically associated with the presence of side-chain vinyl groups.     

Cyclic 3':5'-nucleotide phosphodiesterase. Ca2+ confers more helical conformation to the protein activator.

The ultraviolet spectrum of a protein activator of cyclic nucleotide phosphodiesterase and adenylate cyclase purified to homogeneity from bovine brain displayed absorption peaks at 252, 259, 265, 269, and 277 nm. The activator contained no phosphate and did not serve as a substrate for cyclic adenosine 3':5'-monophosphate- or cyclic guanosine 3':5'-monophosphate-dependent protein kinases. The activator binds Ca2+, and the active form appears to be a Ca2+ activator complex (Lin, Y.M., Liu, Y.P., and Cheung, W.Y. (1974) J. Biol. Chem. 249, 4943-4954). Optical rotatory dispersion measurement showed that the Ca2+-free activator exhibited a reduced mean residue rotation ([m']231) of -5700, corresponding to 39% of helical content. In the presence of Ca2+, the [m']231 was increased to -7500, corresponding to 57% of helical content. The Ca2+ -induced conformational change was corroborated by a chemical method. In the presence of Ca2+, the activator was more resistant to trypsin inactivation, presumably because proteins with more helical structures are more resistant to tryptic attack. The activator is rich in aspartate and glutamate. Chemical block of some of the carboxyl groups with glycine ethyl ester or methoxyamine diminished the [m']231 of the activator and its activity, suggesting that blockade of some of the carboxyl groups in the activator unfolded the molecule, leading to a loss of activity. We conclude that Ca2+, which confers more helical structure to the activator, converts the inactive, less helical structure to the active, more helical structure, and that chemical modification of the activator leading to unfolding of the molecule abolishes its biological activity.