Takayoshi Iio

Fluorescent rotors and their applications to the study of G-F transformation of actin

New fluorescent rotor molecules having hydrophilic functional groups, which are derivatives of p-(N,N-dialkylamino)benzylidenemalononitrile, were synthesized. Their properties as fluorescent rotors were confirmed by an observation of solvent viscosity-dependent fluorescence. Incorporation of hydrophilic groups into the molecules increased the solubility of fluorescent rotors in aqueous media; the application of the compounds to biochemical systems became feasible as a consequence. To demonstrate this applicability, we attempted to monitor the G-F transformation of rabbit skeletal muscle actin with these newly synthesized compounds. All the compounds carrying a malononitrile moiety showed greater fluorescence in F-actin. Among them, 1-(2-hydroxyethyl)-6-[(2,2-dicyano)vinyl]-2,3,4-trihydroquinoli ne gave the best result by the criteria of the difference in fluorescence quantum yield for G- and F-actin, solubility, and stability of the compound. The method has the major advantage of not requiring covalent modification of actin.

Reconstitution of bacteriorhodopsin from a mixture of a proteinase V8 fragment and two synthetic peptides.

The peptide and retinal mixture of bacteriorhodopsin, composed of two synthetic peptides corresponding to helices F (160-197) and G (202-237) and a proteinase V8-derived fragment V1 (1-166), generated the characteristic features of bacteriorhodopsin with absorbance maximum at 550 nm and fluorescence quenching as in two synthetic peptides corresponded to helix A (sequence 7-31) and B (41-65) and a chymotryptic fragment (72-248). The recovery of reconstitution estimated from the absorption and the fluorescence quenching of these mixture was 16-19% and 25-32% of the native purple membrane, respectively, whereas mixtures lacking any one of the peptides exhibited no absorption recovery Circular dichroism of each peptide fragment showed complete formation of alpha-helical structure in a membrane-mimetic medium of sodium dodecyl sulfate. These results indicate that the specific interactions or mutual recognitions between alpha-helices in lipid bilayers are essential for correct bundling of the seven helices and formation of the retinal binding pocket.

Calcium-bindings of wild type and mutant troponin Cs of Caenorhabditis elegans

Apparent Ca(2+)-binding constant (K(app)) of Caenorhabditis elegans troponin C (CeTnC) was determined by a fluorescence titration method. The K(app) of the N-domain Ca(2+)-binding site of CeTnC was 7.9+/-1.6 x 10(5) M(-1) and that of the C-domain site was 1.2+/-0.6 x 10(6) M(-1), respectively. Mg(2+)-dependence of the K(app) showed that both Ca(2+)-binding sites did not bind competitively Mg(2+). The Ca(2+) dissociation rate constant (k(off)) of CeTnC was determined by the fluorescence stopped-flow method. The k(off) of the N-domain Ca(2+)-binding site of CeTnC was 703+/-208 s(-1) and that of the C-domain site was 286+/-33 s(-1), respectively. From these values we could calculate the Ca(2+)-binding rate constant (k(on)) as to be 5.6+/-2.8 x 10(8) M(-1) s(-1) for the N-domain site and 3.4+/-2.1 x 10(8) M(-1) s(-1) for the C-domain site, respectively. These results mean that all Ca(2+)-binding sites of CeTnC are low affinity, fast dissociating and Ca(2+)-specific sites. Evolutional function of TnC between vertebrate and invertebrate and biological functions of wild type and mutant CeTnCs are discussed.

Fluorescence energy transfer measurements between the nucleotide binding site and CYS-373 in actin and their application to the kinetics of actin polymerization

Intramonomer fluorescence energy transfer between the donor epsilon-ATP bound to the nucleotide-binding site and the acceptor 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole bound to Cys-373 in G-actin was measured by steady-state fluorimetry. Assuming for the orientation factor its dynamic limit K2 = 2/3, the donor and acceptor distance in a G-actin molecule was calculated to be about 3 nm. The intermonomer energy transfer in F-actin occurring between the donor bound to an actin monomer and the acceptor bound to the nearest-neighbour actin monomer was also measured and the distance was calculated to be about 4 nm. The kinetics of the actin polymerization process was studied by following the decrease in fluorescence intensity upon addition of salts to G-actin solution. The initial velocity of the fluorescence intensity change was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to the square of the initial G-actin concentration. The temperature dependence of the velocity was proportional to exp(-10/RT). These results indicated that the initial fluorescence intensity change corresponds to monomer-dimer transformation and its activation enthalpy was 10 kcal/mol.

Kinetic analysis on nitric oxide binding of recombinant Prolixin-S, a nitric oxide transport protein from the bloodsucking bug, Rhodnius prolixus

Kinetics of the NO binding and removal reaction of recombinant Prolixin-S (rProlixin-S) were analyzed using stopped-flow spectrophotometry. The reaction was observed as a biphasic process. The rate constant of the fast phase increased linearly as NO concentration increased. The rate constant at the slow phase increased as NO concentrations increased at low NO concentration, then reached a plateau at high NO concentration. These NO dependencies of the reaction are characteristic of a bimolecular two-step consecutive reaction. The reaction consisted of the fast NO binding reaction of rProlixin-S and the following slow structural change of NO-protein complex. Kinetic studies revealed that the NO binding rate constant was independent of pH, but the rate constant of the NO removal reaction increased as pH increased. The apparent NO dissociation constant (Kd) of rProlixin-S was also calculated from the values of the kinetic parameters obtained in this work. The Kd value increased as pH and temperature increased. The Kd value of rProlixin-S and NO was 10-300 nM in regular physiological condition, which is 103 higher and 103 lower than those of the other ferric and ferrous hemoproteins and NO, respectively. These results indicate that Prolixin-S is one of NO transport proteins regulating blood pressure.

Kinetics of the conformational change of troponin-C induced by magnesium-binding or removal.

The kinetics of the conformation change of troponin-C (TN-C) induced by magnesium-binding or removal were studied in the absence of calcium ion by measuring the fluorescence intensity change of BIPM bound to TN-C by stopped-flow spectrofluorometry. The kinetic process of the conformational change was biphasic. The rate constants of the two phases were determined as a function of free magnesium ion concentration ([Mg]) of the solution. The [Mg]-dependence of the rate constants was explained by a simple molecular kinetic mechanism: (formula: see text) The dissociation constant of magnesium bound to TN-C was also determined to be 1 x 10(-3) M in the kinetic study.