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J Sopkova

Publications and source records attributed to J Sopkova.

9 recordsLinked to original sources
PubMed2001

Inhibition of neuronal nitric oxide synthase by 7-methoxyindazole and related substituted indazoles.

The synthesis and pharmacological evaluation of methoxyindazoles as new inhibitors of neuronal nitric oxide synthase are presented. The 7-methoxyindazole, although less potent than 7-NI, is the most active compound of the series in an in vitro enzymatic assay of neuronal nitric oxide synthase activity. This result shows that the nitro-substitution is not indispensable to the biological activity of the indazole ring. 7-Methoxyindazole possesses in vivo NOS inhibitory as well and related antinociceptive properties.

Analgesics↗
PubMed2000

Modulation by Ca(2+) and by membrane binding of the dynamics of domain III of annexin 2 (p36) and the annexin 2-p11 complex (p90): implications for their biochemical properties.

The modulation of the local structure and dynamics of domain III of annexin 2 (Anx2), in both the monomeric (p36) and heterotetrameric forms (p90), by calcium and by membrane binding was studied by time-resolved fluorescence intensity and anisotropy measurements of the single tryptophan residue (W212). The results yield the same dominant excited-state lifetime (1.4 ns) in both p36 and p90, suggesting that the conformation and environment of W212 are very similar. The fluorescence anisotropy decay data were analyzed by associative (two-dimensional) as well as nonassociative (one-dimensional) models. Although no statistical criterion is decisive for one model versus the other, only the associative model allows recovery of a physically relevant value of the Brownian rotational correlation of the protein. Using the associative model, a nanosecond flexibility is detectable in p90 but not in p36. When Ca(2+) binds in the millimolar concentration range to both forms of Anx2, a conformational change takes place leading to an increase of the major excited-state lifetime (2.6 ns) and to a suppression of the W212 local flexibility of p90. Binding to membranes of either p36 or p90 in the presence of Ca(2+) does not induce any conformational change other than that provoked by Ca(2+) binding alone. The W212 local flexibility in both proteins increases significantly, however, in their membrane-bound forms. In the presence of membranes, the conformation change of domain III in p90 displays a sensitivity to Ca(2+) 2 orders of magnitude higher than that of p36, reaching intracellular sub-micromolar concentration ranges. This higher Ca(2+) sensitivity correlates with the Ca(2+)-dependent membrane aggregation but not with their Ca(2+)-dependent binding to membranes. The significance of these structural and dynamical changes for the function of the protein is discussed.

Animals↗
PubMed1999

Conformational flexibility of domain III of annexin V at membrane/water interfaces.

The conformational dynamics of domain III in annexin V bound to negatively charged phospholipid vesicles of 1-palmitoyl-2-oleoyl-sn-glycerophosphocholine and 1-palmitoyl-2-oleoyl-sn-glycerophosphoserine or incorporated into reverse micelles of water/sodium bis(2-ethylhexyl) sulfosuccinate in isooctane, used to mimic the phospholipid/water interface, was studied by steady-state and time-resolved fluorescence of its single tryptophan residue (W187). Upon interaction with sonicated phospholipid vesicles in the presence of calcium, or upon incorporation into reverse micelles without calcium, a progressive 12-14 nm red shift of the fluorescence emission spectrum of W187 is observed. The indole environment becomes therefore more polar than in the unbound protein. Three major lifetime populations describe the fluorescence intensity decays of W187 in both systems. A long-lived excited-state population characterizes the membrane-bound state of the protein. The existence of local conformers with different subnanosecond mobility is suggested by specific association between lifetimes and correlation times both for the protein in buffer and in interaction with the membrane surface. The interaction of the protein with the membrane surface preserves the existence of a rapid unhindered rotational motion, which is coupled with all three lifetimes. The longest lifetime is coupled to restricted motions in subnanosecond and nanosecond time scales. The overall amplitude of rotation of the indole ring is increased in the membrane-bound conformation of the protein. In reverse micelles, the local dynamics reported by W187 is also considerably increased whereas the overall folding of the protein remains unaffected. The same conformational change of domain III can therefore be provoked by different conditions: calcium binding at high concentration, mild acidic pH [Sopkova, J., Vincent, M., Takahashi, M., Lewit-Bentley, A. , and Gallay, J. (1998) Biochemistry 37, 11962-11970] and the interaction of the protein with the membrane surface. The high flexibility of domain III in the membrane-bound protein suggests that this domain may not be crucial for the interaction of the protein with the membrane, in contrast with previous models. Our data are compatible with atomic force microscopy results which suggest that domain III of annexin V does not interact strongly with the membrane surface [Reviakine, I., Bergma-Schutter, W., and Brisson, A. (1998) J. Struct. Biol. 121, 356-361].

Annexin A5↗
PubMed1999

Tryptophan octyl ester in detergent micelles of dodecylmaltoside: fluorescence properties and quenching by brominated detergent analogs.

The fluorescence properties of tryptophan octyl ester (TOE), a hydrophobic model of Trp in proteins, were investigated in various mixed micelles of dodecylmaltoside (DM) and 7,8-dibromododecyl beta-maltoside (BrDM) or 10,11-dibromoundecanoyl beta-maltoside (BrUM). This study focuses on the mechanism via which these brominated detergents quench the fluorescence of TOE in a micellar system. The experiments were performed at a pH at which TOE is uncharged and almost completely bound to detergent micelles. TOE binding was monitored by its enhanced fluorescence in pure DM micelles or its quenched fluorescence in pure BrUM or BrDM micelles. In DM/BrUM and DM/BrDM mixed micelles, the fluorescence intensity of TOE decreased, as a nonlinear function of the molar fraction of brominated detergent, to almost zero in pure brominated detergent. The indole moiety of TOE is therefore highly accessible to the bromine atoms located on the detergent alkyl chain because quenching by bromines occurs by direct contact with the fluorophore. TOE is simultaneously poorly accessible to iodide (I(-)), a water-soluble collisional quencher. TOE time-resolved fluorescence intensity decay is heterogeneous in pure DM micelles, with four lifetimes (from 0.2 to 4.4 ns) at the maximum emission wavelength. Such heterogeneity may arise from dipolar relaxation processes in a motionally restricted medium, as suggested by the time-dependent (nanoseconds) red shift (11 nm) of the TOE emission spectrum, and from the existence of various TOE conformations. Time-resolved quenching experiments for TOE in mixed micelles showed that the excited-state lifetime values decreased only slightly with increases in the proportion of BrDM or BrUM. In contrast, the relative amplitude of the component with the longest lifetime decreased significantly relative to that of the short-lived species. This is consistent with a mainly static mechanism for the quenching of TOE by brominated detergents. Molecular modeling of TOE (in vacuum and in water) suggested that the indole ring was stabilized by folding back upon the octyl chain, forming a hairpin conformation. Within micelles, the presence of such folded conformations, making it possible for the entire molecule to be located in the hydrophobic part of the micelle, is consistent with the results of fluorescence quenching experiments. TOE rotational correlation time values, in the nanosecond range, were consistent with a hindered rotation of the indole moiety and a rotation of the complete TOE molecule in the pure DM or mixed detergent micelles. These results, obtained with a simple micellar model system, provide a basis for the interpretation of fluorescence quenching by brominated detergents in more complex systems such as protein- or peptide-detergent complexes.

Biophysical Phenomena↗
PubMed1999

The crystal structure of a complex of p11 with the annexin II N-terminal peptide.

The aggregation and membrane fusion properties of annexin II are modulated by the association with a regulatory light chain called p11.p11 is a member of the S100 EF-hand protein family, which is unique in having lost its calcium-binding properties. We report the first structure of a complex between p11 and its cognate peptide, the N-terminus of annexin II, as well as that of p11 alone. The basic unit for p11 is a tight, non-covalent dimer. In the complex, each annexin II peptide forms hydrophobic interactions with both p11 monomers, thus providing a structural basis for high affinity interactions between an S100 protein and its target sequence. Finally, p11 forms a disulfide-linked tetramer in both types of crystals thus suggesting a model for an oxidized form of other S100 proteins that have been found in the extracellular milieu.

Animals↗
PubMed1998

Conformational flexibility of domain III of annexin V studied by fluorescence of tryptophan 187 and circular dichroism: the effect of pH.

The conformation and dynamics of domain III of annexin V was studied by steady-state and time-resolved fluorescence of its single tryptophan residue (Trp187) as a function of pH in the absence of calcium. At neutral pH, the maximum of emission occurs at 326 nm, in agreement with the hydrophobic location of the tryptophan residue seen in the three-dimensional structure. Upon decreasing the pH, a progressive red-shift by about 12 nm of the fluorescence emission spectrum is observed. The effect is complete between pH 6 and 4.5, and most likely involves at least one and maybe two carboxylic group(s). Circular dichroism mesurements give evidence for a preservation of the native folding of the protein in these mild acidic conditions. A fluorescence red-shift of smaller amplitude is also observed at high pH (approximately 11). The aggregation state of the protein is affected by pH: while at neutral pH, the protein is monomeric (rotational correlation time = 14 ns); it forms aggregates larger than a dimer (rotational correlation time > 40 ns) in acidic pH conditions. These results suggest that electrostatic interactions are probably important for the stabilization of the folding of domain III without calcium. The conformational change may be related to the aggregation state of the molecule. Examination of the protein crystal structures with and without calcium ion in domain III shows an interplay of salt bridges implying charged amino acid side chains at the molecule surface of domain III. These observations may provide a further clue to the mechanism of the conformational change of domain III of annexin V induced by high calcium concentrations and interaction at the membrane/water interface.

Acrylamide↗
PubMed1994

The dynamic behavior of annexin V as a function of calcium ion binding: a circular dichroism, UV absorption, and steady-state and time-resolved fluorescence study.

The binding of calcium ions to annexin V in the absence of phospholipids has been studied by UV-difference spectroscopy, circular dichroism, and steady-state and time-resolved fluorescence. In the absence of calcium, the unique tryptophan 187, located in domain III of annexin V, is surrounded by a strongly hydrophobic environment, as indicated by its "blue" fluorescence emission maximum (325 nm). This corresponds well with the description of the structure determined by X-ray crystallography of several crystal forms. The Trp187 time-resolved fluorescence decay shows the existence of a fast (picosecond) excited-state reaction which can involve the formation of an H-bond between the indole NH group and the proximate epsilon-OH and/or alpha-carbonyl groups of Thr224. Titration with calcium tends to stabilize the overall structure, as shown by circular dichroism, while leading to large modifications of the local structure around Trp187 making it accessible to the solvent as shown by UV-difference spectra, circular dichroism spectra, and the displacement of its fluorescence emission maximum at saturating concentrations of calcium (350 nm). A rapid (picosecond) formation of an excited-state complex, probably involving one or a few water molecules of the solvation shell, is observed. These observations correlate well with the conformational change observed in crystal structures obtained in high calcium concentrations, involving the removal of Trp187 from the buried position to the surface of the molecule [Sopkova, J., Renouard, M., & Lewit-Bentley, A. (1993) J. Mol. Biol. 234, 816-825; Concha, N. O., Head, J. F., Kaetzel, M. A., Dedman, J. R., & Seaton, B. A. (1993) Science 261, 1321-1324]. In the solvent-exposed conformation, the indole ring becomes mobile in the subnanosecond and nanosecond time range. This conformational change and the increase in local flexibility can be important for the accommodation of the protein on the surface of phospholipid membranes.

Annexin A5↗
PubMed1993

The crystal structure of a new high-calcium form of annexin V.

Annexin V was crystallized in the presence of a high concentration of calcium and the structure refined at 1.9 A resolution. The crystals are triclinic (P1) with three molecules per asymmetric unit and pseudo-R3 symmetry, reflecting a tendency of annexin to form trimers. The overall structure of the protein is similar to that seen in other crystal forms. There are, however, significant changes in domain III, where a new calcium site is formed. The whole region surrounding this site is reorganized in our structure, rendering annexin V more symmetrical and more alike annexin I. The formation of the new calcium site causes the displacement of Trp187 from a buried to an exposed conformation, a change that has recently been demonstrated by fluorescence measurements. The affinity of the different potential calcium sites is modulated: there is no calcium bound in domains II and IV, while up to two secondary calcium ions sites (in domains I and III) can substitute, depending on the calcium concentration present. We suggest that annexin can act as a calcium buffer, binding or releasing calcium depending on its local concentration. Our results also show that annexin displays inherent mobility which, together with its capacity to modulate the calcium affinity of its sites, can be of importance for its function on the membrane surface.

Amino Acid Sequence↗