P. V. Vrzheshch

Denaturation and partial renaturation of a tightly tetramerized DsRed protein under mildly acidic conditions

The red fluorescent protein, DsRed, recently cloned from coral Discosoma sp. has one of the longest fluorescence waves and one of the most complex absorbance spectra among the family of fluorescent proteins. In this work we found that with time DsRed fluorescence decreases under mildly acidic conditions (pH 4.0–4.8) in a pH‐dependent manner, and this fluorescence inactivation could be partially recovered by subsequent re‐alkalization. The DsRed absorbance and circular dichroism spectra under these conditions revealed that the fluorescence changes were caused by denaturation followed by partial renaturation of the protein. Further, analytical ultracentrifugation determined that native DsRed formed a tight tetramer under various native conditions. Quantitative analysis of the data showed that several distinct states of protein exist during the fluorescence inactivation and recovery, and the inactivation of fluorescence can be caused by protonation of a single ionogenic group in each monomer of DsRed tetramer.

Kinetic analysis of maturation and denaturation of DsRed, a coral-derived red fluorescent protein.

The red fluorescent protein DsRed recently cloned from Discosoma coral, with its significantly red-shifted excitation and emission maxima (558 and 583 nm, respectively), has attracted great interest because of its spectral complementation to other fluorescent proteins, including the green fluorescent protein and its enhanced mutant EGFP. We demonstrated that the much slower DsRed fluorescence development could be described by a three-step kinetic model, in contrast to the fast EGFP maturation, which was fitted by a one-step model. At pH below 5.0 DsRed fluorescence gradually decreased, and the rate and degree of this fluorescence inactivation depended on the pH value. The kinetics of fluorescence inactivation under acidic conditions was fitted by a two-exponential function where the initial inactivation rate was proportional to the fourth power of proton concentration. Subsequent DsRed alkalization resulted in partial fluorescence recovery, and the rate and degree of such recovery depended on the incubation time in the acid. Recovery kinetics had a lag-time and was fitted minimally by three exponential functions. The DsRed absorbance and circular dichroism spectra revealed that the fluorescence loss was accompanied by protein denaturation. We developed a kinetic mechanism for DsRed denaturation that includes consecutive conversion of the initial state of the protein, protonated by four hydrogen ions, to the denatured one through three intermediates. The first intermediate still emits fluorescence, and the last one is subjected to irreversible inactivation. Because of tight DsRed tetramerization we have suggested that obligatory protonation of each monomer results in the fluorescence inactivation of the whole tetramer.

Supercooperativity in platelet aggregation: Substituted pyridyl isoxazoles, a new class of supercooperative platelet aggregation inhibitors

The phenomenon of supercooperativity in platelet aggregation is manifested by the occurence of clear‐cut thresholds in dose—response relationships; in such cases the Hill coefficient has unusually high values. Approximation, by the Hill equation, of the relationship of the rate of arachidonate‐induced platelet aggregation to the concentrations of either the inducer or inhibitors such as substituted pyridyl isoxazoles (synthesized by us), indomethacin, and pinane thromboxane A2, demonstrated that the Hill coefficients ranged from 30 to 100. 3‐(3‐Pyridyl)‐5‐phenylisoxazole, which exhibited maximal anti‐aggregatory activity among the synthesized compounds, inhibited neither cyclooxygenase nor thromboxane synthase. The compounds affected the signal transduction pathway at/or posterior to the stage of thromboxane A2 reception.

Molecular oxygen (a substrate of the cyclooxygenase reaction) in the kinetic mechanism of the bifunctional enzyme prostaglandin-H-synthase.

Prostaglandin-H-synthase is a bifunctional enzyme catalyzing conversion of arachidonic acid into prostaglandin H2 as a result of cyclooxygenase and peroxidase reactions. The dependence of the rate of the cyclooxygenase reaction on oxygen concentration in the absence and in the presence of electron donor was determined. A two-dimensional kinetic scheme accounting for independent proceeding and mutual influence of the cyclooxygenase and peroxidase reactions and also for hierarchy of the rates of these reactions was used as a model. In the context of this model, it was shown that there are irreversible stages in the mechanism of the cyclooxygenase reaction between points of substrate donation (between donation of arachidonic acid and the first oxygen molecule and also between donation of two oxygen molecules).

Cyclooxygenase and peroxidase inactivation of prostaglandin-H-synthase during catalysis.

Prostaglandin-H-synthase (PGHS) is a bifunctional enzyme catalyzing cyclooxygenase and peroxidase reactions and undergoing irreversible inactivation during catalysis. A new method for kinetic studies of both PGHS activities in the course of cyclooxygenase as well as peroxidase reactions and also preincubation with hydroperoxides is suggested. It is shown that peroxidase activity is retained after complete cyclooxygenase inactivation and cyclooxygenase activity is retained after complete peroxidase inactivation. Two-stage cyclooxygenase inactivation occurs on preincubation of PGHS with hydrogen peroxide. Studies on inactivation under various conditions indicate that chemical mechanisms of cyclooxygenase and per-oxidase inactivation are different. The data allow development of kinetic models.

Steady-state kinetics of bifunctional enzymes. Taking into account kinetic hierarchy of fast and slow catalytic cycles in a generalized model.

A steady-state approximation of the generalized two-dimensional model of a bifunctional enzyme catalyzing independent proceeding of two one-pathway reactions is considered in a case of mutual influence of the active sites. Coexistence of fast and slow catalytic cycles in the reaction mechanism is analyzed. Conditions when the hierarchy of fast and slow catalytic cycles allows simplification of a two-dimensional model and its reduction to the one-dimensional cyclic schemes were determined. Kinetic equations describing these simplified schemes are presented.

Mutants of monomeric red fluorescent protein mRFP1 at residue 66: Structure modeling by molecular dynamics and search for correlations with spectral properties

To study the interrelation between the spectral and structural properties of fluorescent proteins, structures of mutants of monomeric red fluorescent protein mRFP1 with all possible point mutations of Glu66 (except replacement by Pro) were simulated by molecular dynamics. A global search for correlations between geometrical structure parameters and some spectral characteristics (absorption maximum wavelength, integral extinction coefficient at the absorption maximum, excitation maximum wavelength, emission maximum wavelength, and quantum yield) was performed for the chromophore and its 6 environment in mRFP1, Q66A, Q66L, Q66S, Q66C, Q66H, and Q66N. The correlation coefficients (0.81–0.87) were maximal for torsion angles in phenolic and imidazolidine rings as well as for torsion angles in the regions of connection between these rings and chromophore attachment to β-barrel. The data can be used to predict the spectral properties of fluorescent proteins based on their structures and to reveal promising positions for directed mutagenesis.

Kinetic mechanism of the bifunctional enzyme prostaglandin-H-synthase. Effect of electron donors on the cyclooxygenase reaction

Prostaglandin-H-synthase (PGHS, EC 1.14.99.1) catalyzes the first committed step in biosynthesis of all prostaglandins, thromboxanes, and prostacyclins by converting arachidonic acid to prostaglandin H2 (PGH2). PGHS exhibits two enzymatic activities: cyclooxygenase activity converting arachidonic acid to prostaglandin G2 (PGG2) and peroxidase activity reducing the hydroperoxide PGG2 to the corresponding alcohol, PGH2. Despite the many investigations of the kinetics of PGHS, many features such as the absence of competition and mutual activation between the cyclooxygenase and peroxidase activities cannot be explained in terms of existing schemes. In this work we have studied the influence of different electron donors (N,N,N′,N′-tetramethyl-p-phenylenediamine, L-epinephrine, 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonic acid), potassium ferrocyanide) on the PGHS activities. The proposed scheme describes independent but interconnected cyclooxygenase and peroxidase activities of PGHS. It also explains the experimental data obtained in the present work and known from the literature.

Kinetics of merthiolate-induced aggregation of human platelets

Incubation of human platelets (in the form of platelet rich plasma or washed platelet suspension) with sodium merthiolate (ethyl mercuric salicylate inhibiting the arachidonic acid incorporation into phospholipids) induces their irreversible aggregation, which is accompanied by TxB2 synthesis. The merthiolate-induced aggregation has a lag-period of 0.5-10 min, whose magnitude is inversely correlated with the merthiolate concentration. The concentration dependencies of the rate of the merthiolate-induced and arachidonate-induced aggregation are threshold ones; the Hill coefficients are more than 30. The merthiolate-induced aggregation occurs in two phases: a slow phase which is independent of the arachidonic acid cyclooxygenase metabolism and a fast phase which is fully blocked by indomethacin. This aggregation is inhibited by PGE1 and ajoene (an inhibitor of the fibrinogen interaction with the fibrinogen receptor, GPIIb/IIIa). Quantitative and qualitative analyses of the experimental data were performed, using a model which took account of: (a) increase in the concentration of free endogenous arachidonic acid resulting from the inhibition by merthiolate of the arachidonic acid re-incorporation into phospholipids, and (b) existence of a threshold intracellular arachidonic acid concentration needed for the irreversible aggregation of platelets.

Cell response kinetics: The phenomenon of supercooperativity in aggregation of human platelets

Concentration-response relationships of human platelet aggregation rates were analyzed for a variety of agonists and inhibitors. Their approximation by the Hill equation showed that the values of the Hill coefficient (h) were agonist-dependent and increased as follows: hADP = hL-EPINEPHRINE = hPAF = hPGH2 = hU46619 less than hPMA less than hA23187 less than hMERTHIOLATE = hARACHIDONATE. The results were interpreted in terms of a model assuming varying degrees of cooperativity for each step of signal transduction involved in platelet aggregation. Super-high values of h (greater than 30) obtained with arachidonate and merthiolate, as well as in the case of inhibition of an arachidonate-induced response by indomethacin and PTA2, suggested that at least one region of signal transduction pathway leading to aggregation exhibited supercooperative properties.