Biochimica et Biophysica Acta (BBA) - Protein Structure最新文献

The magnetic circular dichroism (MCD) spectra of the dithionite-reduced and thionine-oxidized forms of the molybdenum-iron protein, Kp1, from the nitrogenase of Klebsiella pneumoniae have been recorded between 300–1000 nm at temperatures from 1.5 to 120 K and at magnetic fields up to 5.1 T. At these temperatures the MCD spectrum is dominated by the contributions from the paramagnetic centres present. Therefore, the low-temperature MCD spectrum of dithionite-reduced Kp1 arises from the EPR-active molybdenum-iron centre known to be the cofactor extractable with $\text{N-}\text{methylformamide}$. The MCD spectra of thionine-oxidized Kp1 reveal the form of the optical spectrum of the other major iron-containing components in the protein, which are believed to be in iron-sulphur clusters. MCD magnetization curves are quite distinctive for the two types of paramagnet. The curve for the oxidized form of Kp1 shows the unusual magnetic properties of the EPR-silent paramagnetic centre. It has an electronic doublet as the lowest component of a complex ground state. At 1.54 K this centre magnetizes steeply with increase in magnetic field such that it is more than 90% magnetized at 1.5 T. The spin of the cluster is estimated to be either $\text{7}\text{2}$ or $\text{5}\text{2}$. The forms of the MCD magnetization curves and of the MCD spectra provide an excellent set of spectroscopic fingerprints for the two predominant types of metal centre. Comparison of these properties with those of model compounds, with extracted cofactor and with apoprotein should provide criteria for assessing the validity of structural models for the complex metal centres in nitrogenase.

Infrared absorptions of heavy meromyosin solutions were studied in the frequency range of 2600 cm⁻¹ to 1800 cm⁻¹ with a Fourier transform infrared spectrophotometer. An absorption band characteristic of the stretching vibration of sulfhydryl groups was found at about 2565 cm⁻¹. By comparison with the infrared absorption spectrum of a cysteine solution, the absorption band of sulfhydryl groups in heavy meromyosin showed that the absorption intensity is much stronger, the absorption peak shifts to a lower wavenumber and the width of the absorption band is much broadened. These results indicate that the sulfhydryl groups in heavy meromyosin are strongly hydrogen-bonded. The additions of ATP and ADP increased the absorption intensity of the absorption band, suggesting that the hydrogen-bonded structure involving the sulfhydryl groups becomes more strengthened on the binding of ATP and ADP. This indicates that myosin heads change conformation around the sulfhydryl groups during ATP hydrolysis.

Autocorrelation functions of the intensity fluctuations of light quasielastically scattered from solutions of synthetic myosin filaments were measured by means of single photon counting. The angular distribution of the total scattering intensity was measured simultaneously, and a supplementary investigation of depolarized light scattering was also performed. The light scattering data were analyzed based on the length distributions of synthetic filaments observed under an electron microscope. The results showed that synthetic filaments in a solution behave essentially as rods. The hydrodynamic frictional coefficient of synthetic filaments, however, was shown to be about twice as high as would be expected if the filaments took a ‘compact’ conformation. This strongly suggests the existence of certain projections which are widely spread from the filament backbone in a solution. The molecular weight of the synthetic filament was calculated using the observed values of the translational diffusion coefficient and the sedimentation coefficient. The number of myosin molecules per 14.3 nm repeat was shown to be six or greater, which suggests that synthetic filaments lack an effective width-limiting mechanism which is presumed in the native thick filament. Additions of Ca²⁺, Mg²⁺ and ATP, alone or in combination, have been shown to cause no notable changes in the light scattering profile of synthetic filaments.

Human haemoglobin was immobilized by cross-linking with glutaraldehyde as soluble polymers and artificial membranes. Effects of pH and 2,3-diphosphoglycerate on oxygen binding and cross-linking were studied with haemoglobin immobilized in both the oxy and deoxy states. The cooperativity is suppressed and the affinity is increased when compared with native haemoglobin. Haemoglobin immobilized in the oxy state exhibited a higher oxygen affinity than that immobilized in the deoxy state. The alkaline Bohr effect is not significantly different from that of native haemoglobin. The 2,3-diphosphoglycerate influence on oxygen binding was reduced by one third with immobilization. In order to separate the chemical and the ‘conformation freezing’ effects on the properties of immobilized haemoglobin, glutaraldehyde-modified haemoglobin in oxy and deoxy states was produced. Oxygen binding was studied and chemical modifications were checked by electrophoresis and gel filtration. This chemically modified haemoglobin without polymerization and without intra-chain bridging exhibits a behaviour similar to that of cross-linked soluble polymers or membranes of haemoglobin.

In contradistinction to previous reports, the lectin of soybeans (Glycine soja) has been shown to retain its erythroagglutinating activity after complete removal of manganese from its molecule. The applied demetallization procedures (dialysis against 0.1 M HCl or 1 M acetic acid and against 0.1 M EDTA followed by dialysis against 1 M acetic acid) had no effect on the stability of the lectin subunit structure, as proved by ultracentrifugation analysis or thin-layer chromatography on Sephadex G-200 Superfine. Affinity electrophoresis' on polyacrylamide gel containing immobilized α-d-galactosyl residues and affinity chromatography on α-d-galactosyl derivative of Separon have shown that the interaction of the demetallized lectin preparations does not differ from that of the native lectin in alkaline media but is decreased in acidic media. Demetallized preparations of soybean lectin can be fully reactivated by dialysis against solutions containing Mn²⁺, Zn²⁺ or Cd²⁺.

The chemically synthesized proalbumin hexapeptide was coupled to rabbit albumin with carbodiimide. Subsequently rabbits were immunized by subcutaneous injection of the conjugate. After 5 weeks the rabbits had developed antibodies against the hexapeptide, which could be detected by immunodiffusion. A sensitive enzyme-linked immunosorbent assay was developed. Using the hexapeptide and other very similar peptides as antigens, a high specificity of the antibodies against the proalbumin hexapeptide was found.

MCD was applied to ferric and ferrous low-spin complexes of cytochrome $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$ to elucidate the electronic states and the nature of the axial ligands of the heme in cytochrome $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$. (1) Low-spin complexes of ferric cytochrome $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$, produced either by ligation of external ligands such as pyridine and imidazole derivatives or by being freed of (−)-camphor, showed sinusoidal Soret and α-MCD bands. The magnitude ratio of the Soret vs. α-MCD bands was quite sensitive to the nature of axial ligands of the ferric low-spin complexes. The ratio (2.7) for the camphor-free form of cytochrome $\text{P-450}{}_{\mathrm{<mtext>cam</mtext>}}$, thus, was the smallest among those (2.7–9.0) for low-spin forms of cytochrome P-450_cam and other corresponding low-spin hemoproteins (ratios 7.8–13.9). The ratio (4.2) for the α-picoline-bound form of cytochrome P-450_cam, however, was the closest to that (2.7) for the camphorfree form of cytochrome P-450_cam among those (4.2–9.0) for the external ligand-bound form of cytochrome P-450_cam. The ratio for the 2-methylimidazole-bound form of cytochrome P-450_cam was the smallest among those of cytochrome P-450_cam bound with imidizole derivatives. Thus, among the nitrogen-bound low-spin forms, the low-spin form with a sterically hindered nitrogen ligand trans to the thiolate anion (−S⁻) most reproduced spectral characteristics of the native low-spin ferric form. Low-temperature absorption studies offered the same results. (2) It was found that MCD magnitudes of α-bands of ferrous low-spin complexes are intimately related to the electronic character of axial ligands. Thus, the CO, O₂ and NO-bound forms of cytochrome P-450_cam, which have two π-type axial ligands, showed the smallest α-MCD bands ($\text{[θ]}{}_{\mathrm{M}}\text{= 5.2–7.5}$) among complexes, while ferrous cytochrome $\text{b}{}_5$ and cytochrome $\text{c}$, which have two σ-electron-donating axial ligands, showed the largest magnitude ($\text{[θ]}{}_{\mathrm{M}}\text{= 120–176}$). The data for the ferrous low-spin complexes of other hemoproteins so far available were well rationalized in consideration of the property of the axial ligands.

We have investigated the role of structural differences in collagen molecules and the effect of proteoglycan preparations on the control of collagen fibril formation. Collagen and proteoglycans were extracted, purified and characterized from two structurally and functionally different connective tissues, rabbit corneal stroma and sclera. Corneal collagen was found to form fibers 6- to 7-times more slowly than scleral type I collagen. Proteoglycans from both sources retard fibrillogenesis, with corneal proteoglycans having approximately 3-times the effect observed with scleral proteoglycans. The morphology of the fibers formed was normal in all cases. Therefore, the changes observed may reflect a true control mechanism related to the strict morphological arrangement associated with corneal transparency.

An analysis of the subunits of d-ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) of wheat (Triticum aestivum) L. cv. Falcon) by gel isoelectric focusing in 8 M urea revealed one type of large subunit and one type of small subunit. This observation contrasts with consistent reports in the literature that this enzyme has three types of large subunit. Carbamidomethylation of the enzyme before isoelectric focusing with a 300-fold molar excess of iodoacetamide over protein thiol groups resulted in three bands of large subunit. Preparative procedures involving aggregation of the enzyme also resulted in complex isoelectric focusing patterns. The simplest patterns, with one type of large subunit and one type of small subunit, were obtained when the enzyme was isolated rapidly and gently by immunoprecipitation or preparative polyacrylamide gel electrophoresis, and analysed by isoelectric focusing without alkylation of thiol groups. The native enzyme probably contains no charge diversity in the large subunit.