Jen Tsi Yang

A new approach to the calculation of secondary structures of globular proteins by optical rotatory dispersion and circular dichroism

Summary The rotatory contributions can be written as X = ΣfiXi, where the subscripts i = H, β and R represent the fractions of the helix, β-form and unordered form. X can be the bo of the Moffitt equation, [m]233, [θ]222 or other parameters at any chosen wavelength. With fis from the X-ray diffraction studies, the reference values, X′s, are solved by a least-square method. The helical content of a protein can be determined by: (1) bo = −580fH, (2) [m]233 = −12700fH −2520, and (3) [θ]222 = −30300fH − 2340. The estimation of the β-form is still uncertain.

Optical rotatory dispersion and circular dichroism of nucleic acids.

Publisher Summary Optical Rotatory Dispersion and Circular Dichroism of Nucleic Acids 1 1This work was supported by grunts from the United States Public Health Service (GM-K3-3441, GM-10880, and HE-06285) This chapter discusses the optical rotatory dispersion (ORD) and circular dichroism (CD) of nucleic acids. It discusses the principles of ORD and CD. It presents the optical activity of mononucleosides and mononucleotides, which are in a class by themselves, and discusses the similarities between ORD and CD between oligonucleotides and polynucleotides and the differences between DNA and RNA. The ORD and CD of dinucleoside phosphates and trinucleoside diphosphates are sequence-dependent; the positions of the extremes and their magnitudes vary among the isomers, even though the general spectra are very similar. Thus, optical activity can become a new means for determining the base sequence in oligonucleotides.

Effect of chain length and concentration of anionic surfactants on the conformational transitions of poly(L-ornithine) and poly(L-lysine) in aqueous solution☆

Abstract The conformation of poly(L-ornithine) (PLO) and poly(L-lysine) (PLL) in solutions of sodium alkyl sulfates, CH3(CH2)nSO4Na with n = 7, 9, 11, 13 and 15 was studied by circular dichroism. PLO adopts a helical conformation in all 5 homologs and PLL a β-form in only 4 of the homologs. With octyl sulfate PLL has a helical conformation instead. These conformations were observed in solution of surfactants both below and above the critical micelle concentration.

Conformation of naturally-occurring peptides in surfactant solution: its relation to the structure-forming potential of amino acid sequence.

Abstract Short polypeptides are unordered in aqueous solution. Surfactants provide a proteinaceous environment in which ordered conformation of the peptides can be induced if they have a structure-forming potential. Ten peptides studied are classified into four types: (I) helix-forming, (II) β-forming, (III) either helix- or β-forming, and (IV) neither structure-forming. At low molar ratio, R, of sodium dodecyl sulfate to peptide (residue) (R

Reconstitution of alkaline-denatured catalase☆

Abstract Bovine liver catalase at pH 12 dissociates into four physically indistinguishable sub-units and loses its enzymatic activity. The dissociation is accompanied by (1) changes in the hydrodynamic properties (its intrinsic viscosity increases from 0.039 to 0.23 dl/g and sedimentation coefficient decreases from 11.4 to 2.85 S), (2) the disappearance of the Soret band at 405 mμ characteristic of heme proteins and the appearance of a small broad peak at 420 mμ, (3) the disappearance of the Cotton effects in the Soret band, and (4) the reduction of the 233-mμ trough of the Cotton effects in the ultraviolet region to about one half in magnitude, suggesting the reduction of helical content. However, the reconstituted catalase molecule has the same physical and chemical properties as the native protein. The subunits can be reconstituted by rapidly diluting the alkaline solution with a pH 7 phosphate buffer. The percentage of enzymatic activity recovered depends on the time required for denaturation and renaturation, concentration of the protein, dilution volume, and temperature of the solution.

Conformation of abortifacient proteins: trichosanthin, α-momorcharin and β-momorcharin

The conformations of three abortifacient proteins, trichosanthin from the Chinese herb Tianhuafen and alpha- and beta-momorcharin from the Chinese herb Kuguazi, were studied by circular dichroism. Their spectra in the ultraviolet region (188-250 nm) were similar to each other and also pH-independent (between pH 5 and 9). All three proteins contained about 30% helix, as compared with 39% for trichosanthin by X-ray diffraction study, and 40-60% beta-sheets, but had no beta-turns, suggesting a structural homology among the proteins. The addition of 20 mM sodium dodecyl sulfate nearly doubled the helicity of beta-momorcharin at the expense of beta-sheets but had a small effect on the conformation of alpha-momorcharin, whereas the corresponding change in conformation for trichosanthin was in between the two momorcharins. This implies a marked difference in stability of the three proteins against the surfactant.

Conformation similarities of the globular and tailed forms of acetylcholinesterase from Torpedo california

The conformation of the globular dimer (G2), the tailed asymmetric dodecamer (A12, also containing some tailed octamer A8) and the globular tetramer (G4, prepared by removing the collagen-like tail from A12) of acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) was studied by circular dichroism (CD) in the ultraviolet region. The G2 and G4 forms had similar conformation with about 40% alpha-helix, 35% beta-sheets and 4% beta-turns; the tailed form had a lower helicity (about 34%) and beta-form (about 25%) content probably because of the presence of the tail whose CD spectrum resembles that of an unordered form, but it had about the same amount of beta-turns as the other two forms. All three forms also had similar CD spectra in the near-ultraviolet region due to their non-peptide chromophores. The pH, thermal and urea denaturation of the three acetylcholinesterase forms was also similar to each other. The pH-dependency of both the enzymatic activity and CD intensity of the three forms showed bell-shaped curves with a plateau at pH 7-8. The activity was completely lost at pH below 5 or above 10, but the corresponding CD spectra retained 70-80% of the original magnitudes. Thermal denaturation of the three forms at pH 7.5 showed a conformational transition and loss of activity between 30 and 40 degrees C, but the CD intensity of the helical band at 222 nm was reduced by only 20-30%. Urea denaturation of the three forms began at 1 M urea; it was protein concentration- and time-dependent. Again, the activity disappeared faster than the decreasing CD intensity. Thus, the overall conformation of the three acetylcholinesterase forms appears to be relatively stable, but their active site is easily perturbed by changing the environment. The loss of activity correlated well with the disappearance of the CD band of tryptophan(s) in the near-ultraviolet region, suggesting that the Trp residue(s) might be at or near the active center of the enzyme.

Helical conformation of myoglobin and its cyanogen bromide-treated fragments in sodium dodecyl sulfate solution

Abstract Circular dichroic spectra of metmyoglobin and apomyoglobin were measured in neutral and acidic solution. Addition of sodium dodecyl sulfate (NaDodSO 4 ) 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 NaDodSO 4 . The middle fragment (76 residues) retains a considerable amount of helicity in water alone, which further increases in the presence of NaDodSO 4 . The NH 2 -terminal fragment (55 residues) also has some helical conformation in water, which is enhanced by the addition of NaDodSO 4 . The circular dichroic spectrum of an equimolar mixture of the three peptides in NaDodSO 4 solution is the same as that calculated from the spectra of isolated peptides under similar conditions.

Intermolecular contacts of deoxyhemoblogin S: a hypothesis and search for possible anti-sickling agents.

Summary A single replacement of β6 glutamic acid in each of the β chains of hemoglobin (Hb) A by valine in Hb S should have only a local conformational change near the N-terminals. It is hypothesized that the first turn of helix A (β4–6) is unwound and residues β1–6 of one β chain of a Hb S molecule contacts the nonhelix EF region of residues β77–84 (between helices E and F) of another Hb S molecule. The aggregation proceeds in both directions of the two β chains leading to single filaments, followed by lateral aggregation into 6-stranded fibers and gelation of sickle-cell hemoglobins. We propose that small peptides which mimic portions of the binding sites might interfere with the gelation, thus becoming possible anti-sickling agents.

Conformation of Bilirubin Oxidase in Native and Denatured States

The conformation of bilirubin oxidase (EC 1.3.3.5) fromMyrothecium verrucaria was studied by circular dichroism (CD). The far-UV CD spectrum showed a single minimum at 215 nm and a maximum near 198 nm, suggesting the dominance ofβ-sheets. There was another negative band at 187 nm that is absent from the spectra of modelα-helix orβ-sheet. CD analysis by the method of Changet al. agreed well with the estimates based on the Chou and Fasman sequence-predictive method, but the Provencher-Glöckner method of CD analysis agreed well with the sequence-predictive method of Garnieret al. AtpH 12 the 215- and 187-nm bands completely disappeared and the protein was denatured. This denaturation was accompanied by the appearance of a large positive band at 250 nm, probably due to ionization of tyrosine residues. In 20 mM sodium dodecyl sulfate the magnitude of the 215-nm band increased, but the spectrum transformed to that of partial helices after heating at 100°C. In 6 M guanidine hydrochloride the far-UV CD spectrum was monotonic and became more negative at the lower wavelength limit (near 212 nm), suggesting that the secondary structure of the protein was disrupted. However, the near-UV CD spectrum retained residual aromatic bands even after heating at 100°C. Thus, our denaturation studies suggest that bilirubin oxidase has a rigid tertiary structure.