Conformational characteristics of legume 7S globulins as revealed by circular dichroic., derivative u. v. absorption and fluorescence techniques

The 7S globulin storage proteins, phaseolin, vicilin, and β-conglycinin of, respectively, dry bean, field pea, and soybean, are highly homologous, have similar predicted protein structures, and yet exhibit considerable differences in their susceptibility to various proteinases [Nielsen, S.S., Deshpande, S.S., Hermodson, M.A. & Scott, M.P. (1988) J. Agric. Food Chem. 36 , 896-902]. These differences in their proteolytic behavior were studied in relation to their solution conformational states. The secondary structures of these three proteins determined by far u. v. circular dichroism were characterized by predominantly β-sheet and β-turn parameters. However, characterization of tertiary and quaternary structures using second derivative u. v. absorption spectroscopy, surface hydrophobicity using cis-parinaric acid as hydrophobic probe, and fluorescence quenching studies of intrinsic Trp fluorescence using an ionic (iodide) and a neutral (acrylamide) quencher indicated sharp differences in the conformation of these proteins. About 9.6 and 10.2 out of 13 and 15 tyrosyls/subunit of phaseolin and β-conglycinin, respectively, were exposed to polar solvent, while the surface hydrophobicity varied β-conglycinin > vicilin > phaseolin. The Trp residues in phaseolin were not accessible to iodide, while half those of vicilin and β-conglycinin were quenched. The order of Trp accessibility to acrylamide was vicilin > β-conglycinin > phaseolin. The relative compactness of these three proteins based on these studies was related to the observed differences in their susceptibility to various proteinases.     

Cellobiohydrolase from Trichoderma reesei.

The secondary structure pattern of a cellobiohydrolase from Trichoderma reesei was predicted using three different algorithms. The relative amounts of the different secondary structure classes derived by this procedure were in good agreement with the values determined by circular dichroism measurements. A twofold internal sequence homology with a repeat distance of approximately 200 residues is observed and possibly also a third, partial, repetition in the C-terminal region. The predicted secondary structure and hydrophobicity pattern show a similar repeat.     

重组HPV病毒样颗粒二级结构的圆二色谱法预测

目的 分析重组HPV病毒样颗粒（virus-like particle,VLP）的二级结构。方法 取重组HPV16、18、52、58型VLP各1批,均调整蛋白浓度至250〜500 μg/ml,采用圆二色谱远紫外扫描,预测重组HPV VLP的二级结构。结果 重组HPV16 VLP二级结构的预测结果为α螺旋11.7%,反平行β折叠37.1%,平行β折叠4.5%,β转角19.2%,不规则卷曲30.3%;重组HPV18 VLP二级结构的预测结果为α螺旋11.9%,反平行β折叠36.9%,平行β折叠4.6%,β转角19.0%,不规则卷曲30.7%;重组HPV52 VLP二级结构的预测结果为α螺旋12.3%,反平行β折叠37.1%,平行β折叠4.7%,β转角19.0%,不规则卷曲30.2%;重组HPV58 VLP二级结构的预测结果为α螺旋11.7%,反平行β折叠35.3%,平行β折叠4.4%,β转角20.4%,不规则卷曲31.3%。结论 圆二色谱法预测了重组HPV16/18/52/58型VLP的二级结构,该技术可用于重组HPV VLP的结构确证研究。     

Conformation and stability of two recombinant human interferon-α analogs

Structural features of a recombinant E. coli derived interferon-α analog, interferon consensus₁, was studied by circular dichroism and fluorescence spectroscopy. Circular dichroic spectra of the purified protein showed that it has about 70% α-helix and a distinct tertiary structure. These structural features are similar to those for a natural interferon-α subtype, interferon-α₂, indicating that the amino acid substitutions in interferon consensus₁ apparently did not alter the protein structure. Another analog, interferon consensus₅, which has Ser instead of Cys at residues 1 and 99 but is otherwise identical to interferon consensus₁, was prepared to study the role of the disulfide bond between Cys 1 and 99. Circular dichroic and fluorescence spectra indicated similarity in the structure of these two analogs. However, interferon consensus₁ was significantly more stable than interferon consensus₅ against denaturation. pH unfolding experiments indicated that the former protein is more stable in the transition region by about 1.6 kcal/mol, which was interpreted in terms of the increased free energy of the denatured state due to an extra disulfide bond in interferon consensus₁.     

SPECTRAL STUDIES ON TWO GENETIC FORMS OF THE HUMAN SERUM PROTEINASE INHIBITOR,ALPHA-1-ANTITRYPSIN*

Alpha-1-antitrypsin, the major inhibitor of proteolytic enzymes in human serum, was isolated from normal individuals (protease inhibitor type MM) and from those with an inherited deficiency (protease inhibitor type ZZ) of circulatory protein. The two proteins were compared by circular dichroism spectroscopy, and by fluorescence quenching experiments using anionic (I^-), and neutral (acrylamide) probes. Both proteins share a similar secondary structure, i.e. ?45–50%α-helix and 15–20%β-structure. Evidence was accumulated to show that the micro-environment in the vicinity of the three tryptophanyl residues is altered in Z form as compared to the M form as shown by (a) the absence of the positive dichroic band in the region 290–300nm of the circular dichroism spectra, (b) a > 50% increase in quantum yield in the tryptophanyl fluorescence emission spectra, (c) an increased accessibility of tryptophan to quenching by iodide, and (d) acrylamide quenching experiments which indicate that all tryptophanyl residues in the Z protein are quenched equally or that quenching is dominated by a single residue, while in the M protein, heterogeneous quenching occurs. The potential significance of these findings in terms of alpha-1-antitrypsin deficiency state are discussed.     

EFFECT OF TEMPERATURE UPON THE CIRCULAR DICHROISM OF BRADYKININ

Analysis of the effect of temperature on the circular dichroism spectrum of bradykinin has led to a more precise understanding of the solution conformation of the peptide. Circular dichroism and ¹³C n.m.r. have been used in a complementary fashion to support the picture that bradykinin spends a maximum of about 20% of its time in a partially ordered conformation featuring a γ-turn with Pro⁷ as the second residue. Since the γ-turn probability is insensitive to temperature, some other conformational effect dominated by the structure of water presumably produces the pronounced change in the circular dichroism spectrum with increasing temperature.     

SECONDARY STRUCTURE PREDICTION OF ANTERIOR PITUITARY HORMONES Lack of Correlation between Predicted Values and Circular Dichroism Data

The secondary structures of human somatotropin, human choriomammotropin, ovine and porcine prolactin, human, ovine and porcine β-lipotropin, human and ovine lutropin, human thyrotropin, human corticotropin, α-melanotropin and human β-melanotropin have been predicted by the method of Chou & Fasman. Predicted contents of α-helix and β-sheet do not correspond well with values estimated from circular dichroism spectra.     

Circular dichroic and immunological properties of human choriogonadotropin-β carboxyl terminal peptides

Circular dichroic spectra have been obtained in aqueous solution and in trifluoroethanol for several synthetic (non-glycosylated) human choriogonadotropin carboxyl terminal peptides of the β-subunit ranging in size from 10 residues to 40 residues. There was no evidence for formation of α-helicity or β-structure, but the spectra in 90% (v/v) trifluoroethanol were consistent with the occurrence of β-turns. The Chou-Fasman predictive rules also suggest a high probability of β-turns in these peptides which could result in the occurrence of repeating kinks. Disulfide-linked dimers were also investigated by circular dichroism, and there was evidence of stabilization of particular skewness of the disulfide dihedral angle depending upon the location of the disulfide bond. The single phenylalanyl residue at position 115 in the β-subunit also contributed to the circular dichroic spectra above 250 nm. Antibodies raised to a peptide consisting of residues 111–145 have been shown to contain two immunological determinants, but the sum of antibodies raised to separate determinant sequences do not equal those raised to the full length peptide. These data could reflect the existence of a conformation-related determinant on the 111–145 peptide or stearic hindrance of immunoglobulin binding of two antibodies to the same peptide.     

Secondary structure of a dynamic type I’β‐hairpin peptide

Abstract: A spontaneously folding β‐hairpin peptide (Lys‐Lys‐Tyr‐Thr‐Val‐Ser‐Ile‐Asn‐Gly‐Lys‐Lys‐Ile‐Thr‐Val‐Ser‐Ile) and related cyclic (cyclo‐Gly‐Lys‐Tyr‐Ile‐Asn‐Gly‐Lys‐Ile‐Ile‐Asn) and linear (Ser‐Ile‐Asn‐Gly‐Lys) controls were studied to determine the effects of various factors on secondary structure. Secondary structure was evaluated using circular dichroism (CD) and 1D and 2D ¹H nuclear magnetic resonance (NMR). The effects of chemical modifications in the peptide and various solution conditions were investigated to determine their impact on peptide structure. The β‐hairpin peptide displayed a CD minimum at 216 nm and a TOCSY i + 1 ? i + 2 and i + 2 ?i + 3 interaction, confirming the expected structure. Using NMR α‐proton (H) chemical shifts, the extents of folding of the β‐hairpin and linear control were estimated to be 51 and 25% of the cyclic control (pH 4, 37 °C), which was taken to be maximally folded. Substitution of iso‐aspartic acid for Asn reduced the secondary structure dramatically; substitution of aspartic acid for Asn also disrupted the structure. This result suggests that deamidation in unconstrained β‐turns may have adverse effects on secondary structure. N‐terminal acetylation and extreme pH conditions also reduced structure, while the addition of methanol increased structure.     

A new B‐chain mutant of insulin: comparison with the insulin crystal structure and role of sulfonate groups in the B‐chain structure

Abstract: The solution structure of a new B‐chain mutant of bovine insulin, in which the cysteines B7 and B19 are replaced by two serines, has been determined by circular dichroism, 2D‐NMR and molecular modeling. This structure is compared with that of the oxidized B‐chain of bovine insulin [Hawkins et al. (1995) Int. J. Peptide Protein Res. 46 , 424–433]. Circular dichroism spectroscopy showed in particular that a higher percentage of helical secondary structure for the B‐chain mutant is estimated in trifluoroethanol solution in comparison with the oxidized B‐chain. 2D‐NMR experiments confirmed, among multiple conformations, that the B‐chain mutant presents defined secondary structures such as a α‐helix between residues B9 and B19, and a β‐turn between amino acids B20 and B23 in aqueous trifluoroethanol. The 3D structures, which are consistent with NMR data and were obtained using a simulated annealing protocol, showed that the tertiary structure of the B‐chain mutant is better resolved and is more in agreement with the insulin crystal structure than the oxidized B‐chain structure described by Hawkins et al. An explanation could be the presence of two sulfonate groups in the oxidized insulin B‐chain. Either by their charges and/or their size, such chemical groups could play a destructuring effect and thus could favor peptide flexibility and conformational averaging. Thus, this study provides new insights on the folding of isolated B‐chains.     

1H-NMR signal assignments and secondary structure analysis of martentoxin

Martentoxin is a peptide of 37 amino acid residues purified from the venom of the Chinese scorpion Buthus martensi Karch, which has been demonstrated to be an inhibitor of voltage-dependent sodium channel and voltage-dependent delayed rectifier potassium channel. To elucidate the molecular mechanism of this interaction, the structure of martentoxin was studied by 2D-NMR. The secondary structure of martentoxin consists of a triple-stranded β-sheet connected to a α-helical structure. This helix encompasses 10 residues from Ser11 to Lys20. The three strands of β-sheet probably comprise residues Gly2-Asp5, Q27-N30 and Glu33-Cys36, Cys30-Asn33 with a type I′β turn centered on Asn31-Asn32. The results indicate that martentoxin possesses the conserved β α β β structure of all the potassium channel toxins.     

Investigation of cis/trans proline isomerism in a multiply occurring peptide fragment from human salivary proline-rich glycoprotein

The solution-state conformations of eight proline-containing peptide fragments found in human salivary proline-rich glycoprotein (PRG) were investigated in 2 × distilled water (treated with metal ion chelating resin) using ¹³C-nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. The peptide sequences and acronyms were as follows: PRG9-2 = NH₂-G(I)-P(2)-CONH₂, PRG9-3 = NH₂-G(1)-P(2)-P(3)-CONH₂,PRG9-4 = NH₂-G(1)-P(2)-P(3)-P(4)-CONH₂, PRG9-5 = NH₂-G(1)-P(2)-P(3)-P(4)-H(5)-CONH₂,PRG9-6 = NH₂-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-CONH₂, PRG9-7 = NH₂-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-CONH₂, PRG9-8 = NH₂-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-CONH₂ and PRG9-9 = NH₂-G(1)-P(2)-P(3)-P(4)-H(5)-P(6)-G(7)-K(8)-P(9)-CONH₂. Sequence-specific resonance assignments from the ¹³C-NMR spectra indicated that the trans proline isomer dominated the conformations of the peptides. CD results clearly showed the presence of the poly-l -proline II helix as the major conformation in PRG9-3 → PRG9-5, supplemented by β- and/or γ-turns in PRG9-6 → PRG9-9. These data suggest that in “metal free” water, native PRG could contain several small poly-l -proline II helices along with β- and/or γ-turns. Since proline is the major amino acid present in native PRG, these localized conformations may contribute to PRG's global conformation and act as a primary force in determining its biological activities.     

N.m.r. analyses of the histidine microenvironments in a human salivary proline-rich glycoprotein

The pK_a's of the three histidine residues in a proline-rich glycoprotein from human parotid saliva (PRG) were determined by 360 MHz proton n.m.r. spectroscopy. The addition of calcium (0.64 mm ) caused drops in the pK_a's of all three histidines by ～0.25 units. When imidazole and cyclo(l -histidine-l -proline) were used as model compounds, corresponding concentrations of calcium had no effect on their pK_a's. Also, the model compounds gave absolute pK_a values in good agreement with similar chemical species reported in the literature. Exchange lifetime data and previously reported hydrogen → deuterium exchange experiments suggest that the PRG histidine N^tH protons are not involved in hydrogen-bonds. Collectively, these data imply that changes in PRG conformation occur upon the addition of calcium.     

Conformation similarities of ricin A-chain and trichosanthin

The conformation of ricin A-chain from castor bean was studied by circular dichroism at pH 4.7, 7 and 9 and compared with that of trichosanthin from the Chinese herb Tianhuafen. The CD spectra of ricin A-chain and trichosanthin were nearly identical at each of the three pHs. Analysis of the data indicated that, like trichosanthin, ricin A-chain had about 29%α-helix and 42%β-sheet but no β-turn. However, there was a subtle difference in the CD spectra in 20 mm sodium dodecyl sulfate, the addition of which at pH 7 slightly increased the helicity and decreased the content of β-sheet of ricin A-chain in contrast to a larger increase in helicity at the expense of β-sheet for trichosanthin, thus indicating a different stability against the surfactant. Native ricin A-chain and trichosanthin had about the same amount of secondary structure, which supports the belief that a high degree of sequence homology of the two proteins [Zhang & Wang (1986) Nature 321, 477–478] may lead to a conformational similarity between them, even though the two proteins are not taxonomically related.     

A spectroscopic study of the interaction of allylisothiocyanate with mustard 12S protein

The intraction of allylisothiocyanate (AIT) with mustard 12S protein resulted in a change in the absorption spectrum of the protein below 280nm without any shift in the ^γmax. The spectrophotometric titration of mustard 12S protein after interaction with AIT suggested the involvement of phenolic groups. Upon interaction with AIT, a new band at 250nm appeared in the near ultraviolet circular dichroism spectrum of the protein. However, no change occurred in the far ultraviolet circular dichroism spectrum. Circular dichroism measurements with amino acids suggest the possible involvement of tyrosine residues in the interaction of AIT with mustard 12S protein.     

Circular dichroism studies of human big-endothelin-1 (Big ET-1)

The circular dichroism (CD) spectra of human endothelin (ET-1) and its precursor (Big ET-1) have been compared in order to provide information on the secondary structure of Big ET-1. It appears that the secondary structures of the common parts of the two molecules are very similar and that the additional C-terminal residues in Big ET-1 may contain both helical and sheet components, information which may be of use in modeling studies of the Big ET-1 structure. In studies of the pH dependence of the conformation of Big ET-1, it was found that Big ET-1 adopts similar structures at pH 6.0 and 7.0, but has a subtly different conformation at pH 8.0 that may result in a reduced susceptibility to proteolysis at this pH. This difference may be in the conformation of a turn-type structure, producing a less accessible peptide bond in the molecule at the site of cleavage. © Munksgaard 1997.     

The hydrophobic pocket of 24p3 protein from mouse uterine luminal fluid: Fatty acid and retinol binding activity and predicted structural similarity to lipocalins

Abstract: The conformation of 24p3 protein purified from mouse uterine luminal fluid was studied by circular dichroism spectroscopy in 200–300 nm. At pH 7.4, the spectrum in the UV region appears as one negative band with a minimum mean residue ellipticity of -3,600 deg.cm².dmole^?1 at 217 nm, suggesting a very low or no helical content, but a considerable amount of β-form, β-turn, and unordered form in the protein molecule. This agrees with the predicted secondary structures consisting of only one a-helical segment of residues 150–163 and nine segments of residues 28–35, 50–60, 67–72, 78–86, 94–97, 106–114, 119–125, 136–140 and 166–172 in β-forms, which would construct two orthonormal β-sheets to form a less polar β-barrel. The environments around Trp-31 and Trp-81 of this protein were studied by intrinsic fluorescence and solute quenching. They give an emission peak at 332 nm, and only about 21% of them are accessible to quenching by acrylamide. This together with their low accessibility to either CsCl or KI suggests that they are located in the less polar P-barrel. Hydrophobic compounds such as fatty acids, retinoids, and cholesteryl oleate in the protein solution diminish the protein fluorescence. Analysis of the fluorescence data suggests that the protein has a binding site for hydrophobic ligand. The association constants for the complex formation are 1.03 × 10⁶M^?1, 1.92 × 10⁵M^?1, 2.38 × 10⁵M^?1 or 1.25 × 10⁵M^?1for cholesteryl oleate, oleic acid, retinol, or retinoic acid at pH 7.4. Analysis of the equilibrium binding data from binding assay using [H³]-retinol and [H³]-retinoic acid reveals a singular type of retinoid-binding site in the protein with the association constant of 4.92 × 10⁵M^?1 and 1.17 × 10⁵M^?1 for retinol and retinoic acid, respectively. Trp-31 or/ and Trp-81 is in or very near the binding site and the gross conformation of protein changes considerably as the formation of protein-ligand complex.     

Modification of arginine residues in subtilisins Novo and Carlsberg

The modification of arginine 186 and arginine 247 in subtilisin Novo as well as the four guanidino groups in subtilisin Carlsberg decreased the catalytic activity. The inactivation proceeded by 60–70% toward casein and by 80% toward p-nitrophenyl acetate during 4 h of incubation with glyoxal. No decrease in the lysyl content was found. The modification had little effect on the fluorescence and circular dichroism properties of the two subtilisins. It was deduced that the inactivation of subtilisins was due to changes in the catalytically active conformation of the active sites, induced by the modification of the arginyl residues. The role of guanidino groups in structure and function of the subtilisins Novo, Carlsberg, DY and mesentericopeptidase is quite similar.     

Structure-toxicity relationships of neurotoxin RTX-III from the sea anemone Radianthus macrodactylus: modification of amino groups.

The effect of modification of amino groups on RTX-III induced lethality in mice has been studied. The toxicity was not affected by guanidination of one or two lysine residues with O-methylisourea, but guanidination of three or four lysine residues decreased lethality two-fold. Acetylation of the N-terminal amino group with [3H]acetic anhydride caused a 12-fold decrease of lethality. The toxin containing acetylated Lys-4 or one of three C-terminal lysine residues had half the lethal potency of the native RTX-III. Diacetylated derivatives were 30- to 35-fold less toxic than the native toxin. By circular dichroism, it was shown that modification of one or two amino groups did not affect the secondary structure of the toxin. We conclude that protonated amino groups are essential for neurotoxicity.     

Effect of divalent cations on structure-function relationships of the antitumor protein α-sarcin

α-Sarcin binds one Zn(II) cation per protein molecule, with a K_d value of 0.9 mM, determined by equilibrium dialysis experiments. Ca(II), Mg(II), and Mn(II) do not bind to α-sarcin. Cd(II) and Co(II) also behave as Zn(II). The binding produces local modifications on the protein conformation affecting the microenvironment of tryptophan residues. The three cations modify the fluorescence emission of the protein. The near-u. v. circular dichroism spectrum of the protein is also altered. The binding of Zn(II) and related cations does not modify the secondary structure of the protein. The ribonucleolytic activity of a-sarcin is inhibited upon Zn(II) binding, but no alteration of the ability of the protein to aggregate phospholipid vesicles has been observed.