pH dependence of the circular dichroic bands of phenylmethanesulfonyl-mesentericopeptidase.

The effect of pH on the circular dichroism spectra of phenylmethanesulfonyl-mesentericopeptidase (peptidyl peptide hydrolase, EC 3.4.21) was studied. The ellipticity of the bands below 250 nm, which reflects the backbone conformation of the protein molecule, remains almost unchanged in the pH range 6.2--10.4. However, below pH 6.2 and above pH 10.4 a conformational transition occurs. The pH-dependent changes above 250 nm were also studied. The titration of the CD band at 296 nm reflects the ionization of the "exposed" tyrosines, which phenolic groups are fully accessible to the solvent. An apparent pK of 9.9 is calculated from the titration curve. It is concluded that ionization of the tyrosyl residues with normal pK's is complete before conformational changes in the protein molecule occur.     

Conformational changes in human serum albumin around the neutral pH from circular dichroic measurements.

The molar ellipticity of the warfarin-albumin complex at 310 nm increases with pH from 6 to 9. This pH dependence runs parallel with that of the molar ellipticity of the albumin alone at 292 nm. The change in molar ellipicity with pH occurs in a smaller pH interval after addition of the physiological concentration of calcium ions. These findings give support to the assumption that the binding site for warfarin on the albumin molecule is affected by the neutral-to-base transition in the protein.     

On the analysis of circular dichroic spectra of proteins.

A new method is presented for analyzing circular dichroism spectra. The method employs integrals over the data and calculates the alpha-helical, beta-sheet, and random coil content of the proteins from such integrals. It is shown that the analyzed alpha-helical content is usually reliable to within 5%, beta-sheet values are somewhat less reliable, and random coil values are least reliable. Curve fitting techniques are shown to be misleading. The method has a number of advantages over existing procedures.     

Mammalian mitochondrial ribosomes. Studies on the exchangeability of polypeptide chain elongation factors from bacterial and mitochondrial systems

Mammalian mitochondrial ribosomes from rat liver synthesised poly(phenylalanine) from [14C]-Phe-tRNA in the presence of a homologous 10(5) X gav supernatent fraction. The activity depended on the addition of synthetic template and was resistant to cycloheximide. The polyanion spermidine had a stimulatory effect on peptide synthesis in vitro. In contrast to Escherichia coli ribosomes, which also functioned with heterologous supernatant fractions, 55-S mitochondrial ribosomes were inactive when supplemented with heterologous supernatant fractions from E. coli or with purified bacterial elongation factors. EF-T slightly stimulated polyphenylalanine synthesis when added in combination with mitochondrial supernatant fractions. Two-dimensional electrophoretic analysis of the protein content of both supernatant fractions revealed considerable differences in the distribution of the species-specific proteins according to their isoelectric points. The mitochondrial supernatant proteins were in general more basic, and the few acidic proteins did not co-migrate with EF-Tu or EF-G from E. coli.     

Studies on ribosomes from barley leaves. Changes during senescence

The effect of sucrose, Mg²⁺ and deoxycholate on the yield of ribosomes from barley leaves was determined and the changes in the amount and the composition of ribosomes during senescence of intact and excised first seedling leaves were examined.     

Absorption and circular dichroic spectral studies on the self-association of daunorubicin

The self-association of daunorubicin in aqueous solution has been examined using visible absorption, fluorescence, and CD measurements. Spectral changes in the concentration range 10^?6 to 1.5 × 10^?3M have been interpreted in terms of a monomer–dimer equilibrium for daunorubicin. The data have been analyzed using a nonlinear curve-fitting technique. The results obtained in this study differ markedly from previously published results, and possible reasons for this difference are discussed. The effect of solvent, temperature, and ionic strength on the dimerization process is investigated.     

Role of bacterial ribosomes in barotolerance.

The effects of high hydrostatic pressures on protein synthesis by whole cells and cell free preparations of Escherichia coli, Pseudomonas fluorescens, and Pseudomonas bathycetes were determined. Actively growing cells of P. bathycetes and P. fluorescens were less sensitive than were E. coli cells. Protein synthesis by cell free preparations of E. coli and P. fluorescens showed the same extent of inhibition as their respective whole cell preparations, whereas cell free preparations of P. bathycetes showed a marked increase in pressure sensitivity over whole cells. Protein synthesis by hybrid protein synthesizing cell free preparations (the ribosomes from one organism and the S-100 supernatant fraction from another) demonstrated that response to high pressure is dependent on the source of the ribosome employed. A hybrid system containing E. coli ribosomes and P. fluorescens S-100 shows the same sensitivity to pressure as a homologous E. coli system, whereas a hybrid containing P. fluorescens ribosomes and E. coli S-100 shows the greater pressure tolerance characteristic of the P. fluorescens homologous system.     

Equilibrium dialysis and circular dichroic analysis of the novobiocin-bovine serum albumin complex.

The binding of the drug novobiocin by bovine serum albumin has been analyzed by Scatchard plots at two temperatures and three ionic strengths. In all cases the nonlinearity of the plots indicates heterogeneity of the combining sites on the protein, and an analysis of the changes observed in the circular dichroic spectra of the bound drug supports this observation. By use of a three-constant model the best fit for all of the parameters is achieved by assuming the initial site on the protein to be unique, the next five sites to be homogeneous, and subsequent binding to be described by a simple distribution between two phases. The temperature variation studies reveal that the first site derives its free energy of binding totally from a favorable change in entropy while the second class of five sites derives their free energy of binding from a favorable enthalpy change. Comparisons are made between the binding of novobiocin by bovine albumin and by human albumin.     

Effect of B-ring substituents on absorption and circular dichroic spectra of colchicine analogues.

Near-ultraviolet absorption and circular dichroic spectra of several B-ring derivatives of colchicine have been obtained in a variety of solvents. The spectra of the molecules in solvent were analyzed and compared with spectra of the molecules bound to tubulin. Absorption spectra of deacetamidocolchicine, deacetylcolchicine, demecolcine, and N-methyldemecolcine [B-ring substituents = H, NH2, NHCH3, and N(CH3)2, respectively] were analyzed by multiple differentiation of the spectrum. It was found that an amine substituent at the C-7 position on the B-ring of the colchicinoid affected the higher energy transition of the near-ultraviolet spectra of the colchicinoid in the absence of tubulin in a manner consistent with a hyperconjugative alteration of this transition. The fourth derivatives of the absorption spectra of all four molecules bound to tubulin were similar to each other and to colchicine. As was true in the case of colchicine, the negative near-ultraviolet circular dichroic band of the aminoclochicinoids was relatively unaffected by solvent, but the molar ellipticity of the band was greatly reduced with tubulin binding. It is concluded that the binding site environments of the B-ring analogues of colchicine, as probed by absorption and circular dichroic spectroscopy, are equivalent.     

Studies on the binding of bacterial elongation factors EF Tu and EF G to ribosomes

When EF G² from Escherichia coli or Pseudomonas fluorescens is pre-bound to ribosomes in the presence of GMD, or GTP and fusidic acid, a differential effect is observed on the subsequent EF Tu-catalyzed binding of aminoacyl-tRNA to ribosomes. The EF G from E. coli nearly completely prevents the binding reaction, whereas the corresponding factor from P. fluorescens displays a significantly lower inhibitory effect. Both EF G factors form stable complexes with ribosomes and are equally efficient in the polymerization reaction. The difference in inhibitory properties between the two factors persists over a wide range of NH₄Cl concentration.     

Interpretation of the absorption and circular dichroic spectra of oriented purple membrane films.

The absorption and circular dichroic (CD) spectra of purple membrane films in which the plane of the membranes is oriented perpendicular to the incident beam are compared with the solution spectra. This enables one to relate structural features of the purple membrane to a coordinate system as defined by a normal to the membrane plane and two mutually perpendicular in-plane axes. The film and solution absorption spectra were similar except for a relative depression in the 200 - 225-nm region of the film spectrum. However, the CD spectra showed significant differences in the visible region, where the biphasic band in the solution spectrum was replaced by a single positive band at 555 nm in the film spectrum and in the far ultraviolet region, where the 208-nm band was deleted from the film spectra of the native and regenerated membranes. Moreover, a small shoulder occurred at 208 nm in the film spectrum of the bleached membrane. The near ultraviolet spectra also showed differences, whereas the 317-nm band remained essentially the same for both spectra. Based on excitonic interpretations of the visible and far ultraviolet spectra the following conclusions were reached: (a) a relatively strong in-plane monomeric interaction occurs between te retinyl chromophore and apoprotein; (b) the helical axes of the native and regenerated membrane proteins are oriented primarily normal to the membrane plane; and (c) the helical axes of the bleached membrane proteins are tilted more in-plane than the axes of the native or regenerated membrane. Additional conclusions were that an interaction occurs between an in-plane magnetic dipole moment of the retinyl chromophore and probably an in-plane electric dipole moment of a nearby aromatic amino acid(s), and that although the membrane is anisotropic with respect to coupling between electric and magnetic moments of the aromatic amino acids, the transition dipole moments of the aromatic amino acids are not preferentially oriented in either direction.