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

The pentapeptide Arg-Lys-Asp-Val-Tyr (TP5) is a biologically active fragment of thymopoietin, the thymic hormone that induces selective T-cell differentiation. The formation of lanthanide(III) complexes of TP5 is demonstrated through the observation of Tb³⁺ fluorescence enhancement. The equilibria, stoichiometry and solution conformation of the La³⁺, Pr³⁺ and Yb³⁺ complexes of TP5 have been investigated using NMR spectroscopy. In addition, the dissociation constants of two methyl ester analogs of TP5 have been studied. Evidence is presented supporting an interaction between the arginine guanidino NϵH and the aspartate carboxylate of TP5. Binding of Ln³⁺ appears to be accompanied by a disruption (or weakening) of this interaction and a concomitant increase in the 180° rotamer population for the aspartate carboxylate group. The observed trends in the magnitudes of the dissociation constants and the rotamer populations appear to suggest that, although a significant amount of monodentate complexes may also exist, the metal ion binds predominantly to both carboxylates in a bidentate fashion.

Further evidence is presented for the existence in teleost fish of proteins homologous with mammalian C-reactive protein. The amino acid composition is given for a C-reactive protein isolated from the eggs of a marine teleost, Cyclopterus lumpus, by extraction with lecithin in the presence of Ca²⁺, followed by electrofocusing. A molecular weight of 150000 was calculated from gel filtration and electrophoresis at different polyacrylamide gel concentrations, while the $\text{s}{}_{\mathrm{<mtext>20,</mtext><mtext>w</mtext>}}$ was 7.4 S. The 1.5-S subunits had an apparent $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of 20 000 by SDS-polyacrylamide gel electrophoresis and 21 000 by computer analysis based on amino acid composition. Comparison is made with the physicochemical properties of mammalian C-reactive protein.

Hybrids of lactate dehydrogenases from pig heart and muscle and from chicken heart and pig heart were obtained by the freeze-thaw method [1,2]. Ion-exchange chromatography of the resulting mixtures of hybrids yielded unusual elution patterns, i.e., the 2 + 2 hybrids (H₂^PM₂^P and H₂^CH₂^P) were eluted in two separate peaks. These subforms were concluded to result from partial resolution of the three geometric isomers. The hybrids of chicken heart and pig heart lactate dehydrogenase showed three distinct levels of stability. The characteristic temperatures of denaturation were 61.5°C for H₄^P, H^CH₃^P and H₂^CH₂^PI; 71°C for H₂^CH₂^PII and H₃^CH^P and 76.5°C for H₄^C. The resistance towards thermal denaturation thus seemed to be governed by the least stable dimer within the tetrameric enzyme. The arrangement of stabilities of the dimers was in excellent agreement with the number of additional ion pairs between Arg₂₄₁ (chicken) and Asp₅₇ (chicken and pig) [3] within the Q-contact areas. The rate-determining step of thermal denaturation of lactate dehydrogenase was concluded to comprise the distortion or dissociation of one of two Q-contacts of the tetramer.

Evidence that a protease is responsible for the proposal of differing models for the structure of the haemoglobin of the brine shrimp Artemia salina is presented. The protease, a minor contaminant in highly purified haemoglobin preparations, is activated by SDS of the SDS-polyacrylamide gel system and readily degrades the haemoglobin subunit. Removal of the protease by chromatography on Con A-Sepharose 4B has shown the haemoglobin ($\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{260 000}$) to comprise two subunits of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}\text{130 000}$.

The self-assembly of bovine brain tubulin into microtubules is inhibited by low molar ratios of 1-fluoro-2,4-dinitrobezene (FDNB). Binding studies using [¹⁴C]FDNB indicate that the incorporation of between one and two dinitrophenyl groups is sufficient to inhibit assembly completely, although more dinitrophenyl groups can be incorporated using higher FDNB/tubulin ratios. Dinitrophenyltubulin, under assembly conditions, tends to assemble into amorphous aggregates. Thiolysis by 2-mercaptoethanol removes the dinitrophenyl moieties. Paper chromatography and high-voltage electrophoresis of acid-hydrolyzed modified tubulin containing one dinitrophenyl group identified the residue as $\text{S-}\text{dinitrophenylcysteine}$. The β-monomer of tubulin is preferentially modified at low FDNB/tubulin ratios. The reaction with FDNB is greatly reduced when tubulin is in polymerized form. FDNB reduces the colchicine binding activity but does not affect the Mg(II) content of the protein.

The conformational energy surface of a small protein, basic pancreatic trypsin inhibitor, is studied to characterize small-amplitude thermal fluctuations in the protein molecule. In order to see the shape of the conformational energy surface near the energy minimum point, the thermal equilibrium of the molecule is simulated by the Monte Carlo method of Metropolis et al. From the sample of the equilibrium population, which reflects the shape of the energy surface, orthogonal directions are generated in the conformational space, and the conformational energy is actually calculated along these directions. All energy profiles along these directions are found to be approximately a parabola within the range of thermal fluctuations, which suggests the possibility of harmonic approximation to the conformational energy surface of the globular protein.

The effect of the trivalent cations scandium (Sc³⁺) and yttrium (Y³⁺) on the conformation of G-actin was examined using ultraviolet difference and high resolution ¹H-NMR spectroscopy. A comparison was made with data obtained previously with the trivalent lanthanide cations (Ln³⁺). These results indicate that the first and subsequent Y ions (ionic radius 101.9 pm) behave exactly like Ln³⁺. Sc³⁺ is a smaller ion (87 pm) than any of the Ln³⁺. The first Sc³⁺ binds to a site on actin that is inaccessible to Mg²⁺, Y³⁺ and Ln³⁺. However, the second Sc³⁺ to bind behaves like an Ln³⁺. On replacing the native divalent cation (Mg²⁺), both Y³⁺ and Sc³⁺ mobilize the adenine ring of ATP bound to actin, thus exposing underlying residues to the solvent. When Y³⁺ and Sc³⁺ saturate their binding sites on actin, and when the ionic strength is raised to 0.1 M with KCI at pH 6.9, the actin aggregates. Y³⁺ binds to actin with a ratio of 6 : 1 and induces the aggregation of actin into crystalline actin tubes, whilstSc³⁺ Sc³⁺binds with a ratio of 8 : 1 and induces amorphous actin aggregates. These results are consistent with the suggestion that actin tubes are induced by trivalent cations, principally on the basis of their binding stoichiometry, which in turn is determined by ionic radius.

The unfolding of horse ferricytochrome c in the presence of several inorganic salts has been studied under a variety of denaturing conditions and followed by means of absorbance changes in the Soret region (390–450 nm) and visible region (450–750 nm), as well as by viscosity measurements. Changes in the Soret region, usually sensitive to the heme environment, were characterized by gradual increases in absorbance at 409 nm for low concentrations of NaClO₄ and LiClO₄. As denaturant concentrations were increased, the low-spin state of the ferric heme is altered, as seen by a maximum shift to shorter wavelengths (402–407 nm) accompanied by a further increase in absorbance in the Soret region. Unlike the effect of several organic denaturants and the above salts, denaturation in the presence of LiCl and CaCl₂ resulted in an overall decrease in the Soret region with a blue-shift to 401 and 400 nm, respectively. Visible spectra of ferricytochrome c exhibited new bands at 633 nm (9.0 M LiCl) and 636 nm (4.5 M CaCl₂) indicative of a change in the spin state of the iron upon displacement of methionine 80. LiCl and LiBr produced intermediate states of protein unfolding with midpoints ($\text{D}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) at 4.0 and 8.6, and 2.6 and 6.4, respectively. A determination of ΔG_U^H₂O and m, the free energy of unfolding in the absence of denaturant and the dependence of free energy of denaturation on denaturant concentration, was used to analyze the relative effectiveness of these denaturants.

The assembly of microtubules from tubulin prepared without glycerol was inhibited by blocking the two most reactive sulfhydryl groups of the eight free sulfhydryl groups present per tubulin dimer. The assembly was also inhibited by Cu²⁺ ions in a redox-reaction with the two most reactive sulfhydryl groups. These two sulfhydryl groups had almost the same reactivity towards $\text{N-}\text{ethylmaleimide}$ and $\text{p-}\text{chloromercuribenzoate}$, in spite of the fact that they are located on different subunits of tubulin. It was not possible to label just one single sulfhydryl group at a time by $\text{N-}\text{ethylmaleimide}$, and it was not possible to decide whether one or two free sulfhydryl group(s) are needed for assembly. The EPR technique based on the interaction of spin labels with transition metals was used for the study of the distance between the two most reactive sulfhydryl groups and the sites of exchangeable GTP and Mg²⁺, respectively. The sulfhydryl groups were spin labelled with a nitroxide derivative of $\text{N-}\text{ethylmaleimide}$. Cr(III)GTP was used as a paramagnetic substitute for GTP, and Mn²⁺ for Mg²⁺. It was found that: a. The exchange of GTP and the total content of GTP were not affected by modification of the sulfhydryl groups, b. The binding sites of the exchangeable GTP and Mg²⁺ are located 10 Å, at least, from the two most reactive sulfhydryl groups, c. The distance between the spin labels introduced on the two most reactive sulfhydryl groups was larger than 17 Å. The findings indicate that there is no direct interaction between exchangeable GTP and the two most reactive sulfhydryl groups.

The complete primary structure of an acidic ribosomal protein YPA1 from Saccharomyces cerevisiae has been determined. YPA1 is composed of 110 amino acid residues and has the composition: Asp₇, Asn₂, Thr₂, Ser₉, Glu₁₅, Gln₂, Pro₃, Gly₁₅, Ala₂₁, Val₆, Met₂, Ile₄, Leu₉, Tyr₂, Phe₃, Lys₇ and Arg₁. The molecular weight of YPA1 is 11 020. The amino acid sequence was determined by $\text{4-N,N-}\text{dimethylaminoazobenzene}$ 4′-isothiocyanate degradation of the peptides obtained by digestions with trypsin, chymotrypsin, thermolysin, pepsin and Staphylococcus aureus protease of intact protein. A comparison of protein YPA1 from yeast with eL12 from Artemia salina shows a high sequence similarity. A considerable similarity is also shown with HL20 from Halobacterium cutirubrum. On the other hand, there is very little apparent sequence similarity between YPA1 and the eubacterial acidic protein L12 either from E. coli or B. subtilis.