Bruno Filippi

Protamines: II. Circular dichroism study of the three main components of clupeine☆

The three main components YI, YII, and Z of clupeine, a protamine from herring, have been purified and characterized. The conformational preferences of clupeines have been examined as a funciton of pH, temperature, added salts, and presence of structure-disrupting agents and helix-supporting solvents using circular dichroism. It was found that these small basic proteins assume predominantly an unordered conformation in aqueous solution. Addition of counter ions, in particular perchlorate, and 2-chloroethanol induces in various amounts the onset of the right-handed alpha-helical conformation. Urea favors the statistical coil state. It was also demonstrated that in the 0.1--4.0 . 10(-1) M range, in contrast to clupeines YI and Z, the circular dichroic properties of the YII component do not seem to be sensitive to the addition of mono- and diphosphate.

The influence of amino acid side-chains on α-helix stability: S-peptide analogues and related ribonucleases S′

Abstract In order to determine the influence of amino acid side-chains on α-helix stability, in relation to the protein folding process, the coil-helix transitions of some synthetic [Orn 10]-S-peptide analogues, containing, in position 8, Phe, Tyr, Ile, Ala, cpGly † and Gly, were investigated by the technique of circular dichroism under two different sets of conditions. First, the transitions of the Speptide analogues in water/trifluoroethanol mixtures were recorded. From the pattern of the transitions and from the ellipticity values in 97% trifluoroethanol, the following increasing order of amino acids as α-helix formers was found: Gly

Conformational changes of Carcinus maenas hemocyanin induced by urea

Abstract The effect of urea up to 9 m on the structure of hemocyanin from Carcinus maenas has been studied by different spectroscopic techniques. The renaturation of the protein before and after reduction of the disulphide bonds has also been investigated. Dissociation of the quaternary structure and drastic conformational modifications occur while the concentration of the denaturant increases. Some ordered structure, however, is still present in 8 m urea. In the absence of urea, evidence for tyrosine to tryptophan energy transfer is found (e = 0.6). No transfer occurs in 9 m urea. Different classes of tryptophan residues are evidenced by the shape of the emission spectra and by the fluorescence quenching with acrylamide. The renaturation of both apohemocyanin and reduced and methylated apohemocyanin does not reconstitute the original conformation. The unique -S-S- bridge seems to have some influence on the protein structure; however, also the reduced protein shows a refractory core to the denaturation.

Deflavination of flavo-oxidases by nucleophilic reagents

Using spectroscopic techniques we studied the effect of the nucleophilic reagents cyanide, cyanate and thiocyanate on three flavo-oxidases namely alcohol oxidase (AO), glucose oxidase (GOX) and D-amino acid oxidase (DAOX). All three ions, added at concentrations in the mM range, caused release of the flavin adenine dinucleotide (FAD) co-factors from the enzyme molecules. In the case of AO this was accompanied by significant conformational perturbations, which was not observed for GOX and DAOX. As suggested from fluorescence, absorption and circular dichroism spectral changes at least one phenolic hydroxyl group became ionized upon FAD release from AO and a new class of Trp residues, fluorescent only in apo-AO protein, was demasked.

Semisynthetic ribonucleases S′: The role of glutamine 11

Abstract The binding to the S-protein of synthetic [Orn10]-S-peptide analogues in which the invariant Gln11 residue was replaced by glutamate, leucine and lysine, respectively, was determined in the absence of substrates. The replacement of glutamine by glutamate produced an increase in binding energy, whereas a decrease consistently resulted from its substitution by leucine and, to a greater extent, by lysine. None of the amino acid properties that dominate protein behavior, e.g. hydrophobicity, conformational preferences and electrostatic interactions, accounts completely for the experimental results, but each of them must be taken into account to explain the different binding energies. It is suggested that the invariance of Gln11 depends on the fact that its hydrophilic side-chain lacks cationic character in the free globule and anionic character in the enzymic process.

Near-ultraviolet difference absorption and circular dichroism studies on partially synthetic ribonucleases S'.

Near-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-,1epsilon,7epsilon-diguanidono-[Asn14]-, [Phe(F)8, Orn10]- and 1epsilon, 7epsilon-diguanidino-S-peptide, with S-protein. Environmental alterations of Phe-8 in the S-peptide and Tyr-25 in the S-protein, derived from the association process, lead to strong optical signals whose location and magnitude were clearly defined by means of a comparative analysis of the above spectra. Additionally, the spectroscopic effects resulting from insertion of a tyrosyl residue into an hydrophobic environment in the presence or absence of hydrogen-bonding partners were identified and compared with similar findings obtained from the model compound p-cresol.

Circular dichroism and fluorescence studies to probe the conformational properties of Rhus vernicifera laccase

Abstract The conformational properties of native, apo- and type-2 copper depleted laccase from Rhus vernicifera have been investigated by circular dichroism and fluorescence spectroscopies. Circular dichroism experiments reveal a high prevalence of random-coil structure in all laccase derivatives, the content of α-helix and β-sheet not exceeding 8% and 21%, respectively. Nevertheless, the microenvironment of the tryptophan residues is deeply shielded from the external medium and exhibits a marked stability against pH-denaturation. Fluorescence data suggest that a class of tryptophan residues close to type-2 site contributes 50% to the overall fluorescence emitted by apo- and type-2 copper depleted laccase. These residues are masked in the native enzyme, due to quenching effects by copper ions and adjacent amino acid side chains. The interaction of 1-anilino-8-naphthalene sulfonate (ANS) with laccase has been studied by following the fluorescence changes of the dye upon binding. Native laccase does not bind ANS. The type-2 copper depleted derivative and apo-laccase bind one and three moles of dye per mole of protein, respectively. These findings suggest that the three ANS binding sites correspond with the three different copper binding sites in laccase. These can be distinguished on the basis of their dissociation constants (K D ) for ANS. The type-2 copper site exhibits the highest affinity for ANS.

The Role of Lysine-7 in Ribonuclease-A

Bovine pancreatic ribonuclease A(RNase A) catalyzes the hydrolysis of RNA by a two-stage mechanism. The first step is a transphosphorylation to give an oligonucleotide terminating in a pyrimidine 2′-3′-cyclic phosphate. The second is the hydrolysis of the cyclic phosphate to give a terminal 3′-phosphate. At present it is not clear whether the invariant lysine-7 plays any part in the enzyme activity. Chemical modifications of the e-ammonium group give derivatives with 15–30% activity, but substrate binding has been observed to be unaffected. On the other hand, the X-ray crystallography data, while confirming that Lys-7 is located relatively close to and with free access to the active site, strongly suggest that this residue is still too far away from the site to interact with the substrate (1).