Convergent synthesis and properties of photoactivable NADPH mimics targeting nitric oxide synthases.

A new series of photoactivable NADPH mimics bearing one or two O-carboxymethyl groups on the adenosine moiety have been readily synthesized using click chemistry. These compounds display interesting one- or two-photon absorption properties. Their fluorescence emission wavelength and quantum yields (Φ) are dependent on the solvent polarity, with a red-shift in a more polar environment (λmax,em = 460-467 nm, Φ > 0.53 in DMSO, and λmax,em = 475-491 nm, Φ < 0.17 in Tris). These compounds show good binding affinity towards the constitutive nNOS and eNOS, confirming for the first time that the carboxymethyl group can be used as a surrogate of phosphate. Two-photon fluorescence imaging of nanotriggers in living cells showed that the presence of one carboxymethyl group (especially on the 3' position of the ribose) strongly favors the addressing of nanotriggers to eNOS in the cell context.

Structural investigations of basic amphipathic model peptides in the presence of lipid vesicles studied by circular dichroism, fluorescence, monolayer and modeling.

A cationic amphiphilic peptide made of 10 leucine and 10 lysine residues, and four of its fluorescent derivatives in which leucines were substituted by Trp residues at different locations on the primary sequence have been synthesized. The interactions of these five peptides with neutral anionic or cationic vesicles were investigated using circular dichroism, steady state and time-resolved fluorescence with a combination of Trp quenching by brominated lipid probes, monolayers, modeling with minimization and simulated annealing procedures. We show that all the five peptides interact with neutral and anionic DMPC, DMPG, DOPC or egg yolk PC vesicles. The binding takes place whatever the peptide conformation in solution is. In the case of DMPC bilayers the binding free energy DeltaG is estimated at -8 kcal mole-1 and the number of phospholipid molecules involved is about 20-25 per peptide molecule. Peptides are bound as single-stranded alpha helices orientated parallel to the bilayer surface. In the anchoring of phospholipid head groups around the peptides, the lipid molecules are not smeared out in a plane parallel to the membrane surface but are organized around the hydrophilic face of the alpha helices like 'wheat grains around an ear' and protrude outside the bilayer towards the solvent. We suggest that such a lipid arrangement generates transient structural defects responsible for the membrane permeability enhancement. When an electrical potential is applied, the axis of the peptide helices remains parallel to the membrane surface and does not reorient to give rise to a bundle of helix monomers that forms transmembrane channels via a 'barrel stave' mechanism. The penetration depth of alpha helices in relation to the position of phosphorus atoms in the unperturbed lipid leaflet is estimated at 3.2 A.

X-ray scattering titration of the quaternary structure transition of aspartate transcarbamylase with a bisubstrate analogue: influence of nucleotide effectors.

The regulation of aspartate transcarbamylase (ATCase) involves various conformational changes, including a large quaternary structure rearrangement. This is directly related to a major change in its solution X-ray scattering curve upon binding the bisubstrate analogue N-(phosphonacetyl)-L-aspartate (PALA), allowing us to monitor directly the amount of the different quaternary structures present in solution. Data were analysed by singular vector decomposition without any prior assumption as to the number of quaternary structure states. Scattering curves in the presence of variable concentrations of PALA, alone or with saturating CTP or ATP, can be accounted for with only two states. Consequently the method gives the fraction of molecules in either state. Whereas CTP slightly decreases the proportion of molecules in the R state, ATP has no detectable effect, whatever the amount of PALA ligated to ATCase. The requirement for only two quaternary structures, suggesting a concerted transition, promoted us to test the ability of the classical model, proposed by Monod, Wyman and Changeux, to account for our data. By and large, it is satisfactory as regards the homotropic effect of PALA and the observed effect of CTP, although it remains incompatible with some other observations, which support the involvement of more indirect mechanisms in the inhibitory properties of CTP. But ATP does not directly influence the T to R transition and consequently must act by a totally different mechanism.

Structural consequences of the replacement of Glu239 by Gln in the catalytic chain of Escherichia coli aspartate transcarbamylase.

Low-angle X-ray scattering in solution has been used to probe the quaternary structure of a mutant version of Escherichia coli aspartate transcarbamylase in which Glu239 of the catalytic chain was replaced by glutamine by site-directed mutagenesis. X-ray crystallographic studies of the wild-type enzyme have shown that one set of intersubunit interactions involving Glu239 are lost, and are replaced by another set of intrachain interactions when the enzyme undergoes the allosteric transition from the T to the R state. Functional analysis of the mutant enzyme with glutamine in place of Glu239 indicates that homotropic co-operativity is lost without altering the maximal specific activity. The radius of gyration of the unligated mutant enzyme is larger than the unligated wild-type, indicating an alteration in quaternary structure of the mutant. However, the radius of gyration of the mutant enzyme in the presence of N-(phosphonoacetyl)-L-aspartate (PALA) is identical with the value for the wild-type enzyme in the presence of PALA. X-ray scattering at larger angles indicates that the mutant enzyme is in a new structural state different from the wild-type T and R structures. The scattering pattern in the presence of saturating concentrations of PALA is identical with that of the wild-type R structure. Saturating concentrations of carbamyl phosphate alone are sufficient to convert most of the mutant enzyme to the R structure, in the absence of aspartate. CTP shifts the scattering pattern of the mutant enzyme in the presence of saturating carbamyl phosphate towards the scattering curve of the unligated enzyme, but CTP has no effect on the scattering curve in the absence of carbamyl phosphate or in the presence of subsaturating PALA. However, in the presence of subsaturating PALA, ATP causes a strong shift towards the R structure. Neither ATP nor CTP has any effect on the activity of the mutant enzyme. These data suggest that the replacement of Glu239 by glutamine results in a new quaternary structure. These data also explain, on a structural basis, why co-operativity is lost in this mutant enzyme.

Quaternary structure changes in aspartate transcarbamylase studied by X-ray solution scattering. Signal transmission following effector binding.

The result of binding the effectors ATP and CTP to aspartate transcarbamylase was studied by X-ray solution scattering. Binding of substrate analogues produces a substantial change in the solution scattering curve, allowing us to monitor the proportion of the different quaternary structure states present in solution. In the initial solution this ratio was made roughly unity by adding either carbamyl phosphate and succinate, or N-(phosphonacetyl)-L-aspartate (PALA). ATP or CTP were then added, and their effect on the proportion of the different quaternary structure states was followed. When using carbamyl phosphate and succinate (weakly bound), ATP or CTP had a clear effect, as observed previously by monitoring the sedimentation rate (Changeux et al., 1968). However, when PALA (strongly bound) was used, the effect of CTP was very much smaller, and that of ATP was undetectable. This result supports the explanation by Tauc et al. (1982), that nucleotides act mostly through changing the affinity of the active sites for substrate, and only to a small extent by directly modifying the quaternary structure equilibrium in the case of CTP.

Kinetics of structure and activity changes during the allosteric transition of aspartate transcarbamylase.

For the first time, the structural change associated with an allosteric transition has been monitored by X-ray solution scattering. The kinetics of the quaternary structure change of aspartate transcarbamylase were first slowed by using acetyl phosphate instead of carbamyl phosphate, and by the presence of 10% or 30% ethylene glycol. At 6.5 degrees C, the quaternary structure change was found to have a time constant of about 11 seconds. This appears to be larger than that obtained for the switching of homotropic co-operativity, measured by chemical quench under the same conditions.

Differential behaviour of cationic triphenylamine derivatives in fixed and living cells: triggering and imaging cell death.

Triphenylamines are on/off fluorescent DNA minor groove binders, allowing nuclear staining of fixed cells. By contrast, they accumulate in the cytoplasm of living cells and efficiently trigger cell apoptosis upon prolonged visible light irradiation. This process occurs concomitantly with their subcellular re-localization to the nucleus, enabling fluorescence imaging of apoptosis.

Glu-50 in the catalytic chain of Escherichia coli aspartate transcarbamoylase plays a crucial role in the stability of the R quaternary structure.

Glu-50 of aspartate transcarbamoylase from Escherichia coli forms a set of interdomain bridging interactions between the 2 domains of the catalytic chain; these interactions are critical for stabilization of the high-activity high-affinity form of the enzyme. The mutant enzyme with an alanine substituted for Glu-50 (Glu-50-->Ala) exhibits significantly reduced activity, little cooperativity, and altered regulatory behavior (Newton CJ, Kantrowitz ER, 1990, Biochemistry 29:1444-1451). A study of the structural consequences of replacing Glu-50 by alanine using solution X-ray scattering is reported here. Correspondingly, in the absence of substrates, the mutant enzyme is in the same, so-called T quaternary conformation as is the wild-type enzyme. In the presence of a saturating concentration of the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA), the mutant enzyme is in the same, so-called R quaternary conformation as the wild-type enzyme. However, the Glu-50-->Ala enzyme differs from the wild-type enzyme, in that its scattering pattern is hardly altered by a combination of carbamoyl phosphate and succinate. Addition of ATP under these conditions does result in a slight shift toward the R structure. Steady-state kinetic studies indicate that, in contrast to the wild-type enzyme, the Glu-50-->Ala enzyme is activated by PALA at saturating concentrations of carbamoyl phosphate and aspartate, and that PALA increases the affinity of the mutant enzyme for aspartate. These data suggest that the enzyme does not undergo the normal T to R transition upon binding of the physiological substrates and verifies the previous suggestion that the interdomain bridging interactions involving Glu-50 are critical for the creation of the high-activity, high-affinity R state of the enzyme.

Replacement of Asp-162 by Ala prevents the cooperative transition by the substrates while enhancing the effect of the allosteric activator ATP on E. coli aspartate transcarbamoylase.

The available crystal structures of Escherichia coli aspartate transcarbamoylase (ATCase) show that the conserved residue Asp-162 from the catalytic chain interacts with essentially the same residues in both the T- and R-states. To study the role of Asp-162 in the regulatory properties of the enzyme, this residue has been replaced by alanine. The mutant D162A shows a 7700-fold reduction in the maximal observed specific activity, a twofold decrease in the affinity for aspartate, a loss of homotropic cooperativity, and decreased activation by the nucleotide effector adenosine triphosphate (ATP) compared with the wild-type enzyme. Small-angle X-ray scattering (SAXS) measurements reveal that the unliganded mutant enzyme adopts the T-quaternary structure of the wild-type enzyme. Most strikingly, the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) is unable to induce the T to R quaternary structural transition, causing only a small alteration of the scattering pattern. In contrast, addition of the activator ATP in the presence of PALA causes a significant increase in the scattering amplitude, indicating a large quaternary structural change, although the mutant does not entirely convert to the wild-type R structure. Attempts at modeling this new conformation using rigid body movements of the catalytic trimers and regulatory dimers did not yield a satisfactory solution. This indicates that intra- and/or interchain rearrangements resulting from the mutation bring about domain movements not accounted for in the simple model. Therefore, Asp-162 appears to play a crucial role in the cooperative structural transition and the heterotropic regulatory properties of ATCase.

Structural consequences of a one atom mutation on aspartate transcarbamylase from E. coli.

Tyr-240 of the catalytic chain of aspartate transcarbamylase from E. coli has been substituted by Phe using site-directed mutagenesis. The regulatory mechanisms of the mutant enzyme have been shown to be slightly less effective than the wild-type enzyme. A study of the structural consequences of the mutation using solution X-ray scattering and computer simulations is reported here. No significant change from the wild-type enzyme is detectable in the quaternary structure. Simulations suggest that the only effect of the mutation is an increased mobility of the mutated side chain.

Structural kinetics of the allosteric transition of aspartate transcarbamylase produced by physiological substrates.

We have studied the kinetics of the quaternary structure change associated with the allosteric transition of aspartate transcarbamylase (ATCase) (E. coli), inducing this change by exposure to the natural substrates (carbamyl phosphate and L-aspartate). The presence of 30% ethylene glycol slowed the quaternary structure change sufficiently for it to be followed by stopped-flow X-ray scattering at -5 degrees C. After adding substrates to the enzyme, the change occurred, with a half-life of a few seconds, yielding a mixture of the two standard quaternary structures (or, conceivably, a state intermediate between them). This mixture persisted until the enzyme reduced the substrate concentration below a threshold value.

New triarylamine-based far-red DNA stainers with high two-photon absorption properties.

A series of red emitting vinyl-triphenyamines (TP) have been synthesized and evaluated for their two photon absorption (2PA) properties. These compounds are virtually non fluorescent in the free state but exhibit a bright red fluorescence upon binding to double-stranded DNA, both in one- and two-photon absorption. This feature allows one- and two-photon confocal imaging in cells of nuclear DNA with an excellent contrast. Derivatizable analogues for covalent bioconjugation to oligonucleotides are described and variation on the structure is discussed.

L-alanosine: a noncooperative substrate for Escherichia coli aspartate transcarbamylase.

L-Alanosine, an antibiotic produced by Streptomyces alanosinicus, can be used by Escherichia coli aspartate transcarbamylase as a substrate instead of L-aspartate. The Michaelis constant of the catalytic subunit for this analogue is about 10 times higher than that for the physiological substrate, and the catalytic constant is about 30 times lower. The saturation curve of the native enzyme for L-alanosine indicates the lack of homotropic cooperative interactions between the catalytic sites for the utilization of this compound. It appears therefore that L-alanosine is unable to promote the allosteric transition. However, N-(phosphonoacetyl)-L-aspartate, a "bisubstrate analogue" of the physiological substrates, stimulates the reaction. This phenomenon is very similar to that reported by Foote and Lipscomb [Foote, J., & Lipscomb, W. N. (1981) J. Biol. Chem. 256, 11428-11433] concerning the reverse reaction using carbamylaspartate. The reaction is normally sensitive to the physiological effectors ATP and CTP. The significance of these results for the mechanism of the allosteric regulation is discussed.

Apparent cooperativity for carbamoylphosphate in Escherichia coli aspartate transcarbamoylase only reflects cooperativity for aspartate.

The reaction catalyzed by Escherichia coli aspartate transcarbamoylase (ATCase) proceeds through an ordered mechanism, in which carbamoylphosphate binds first, followed by aspartate; upon binding of this second substrate, the enzyme undergoes a concerted transition from a low-affinity T state to a high-affinity R state. In various studies, conflicting results were obtained concerning the existence of positive cooperativity for the first substrate, carbamoylphosphate. It is shown here that cooperativity for this substrate is only apparent. Indeed, saturation curves for carbamoylphosphate display sigmoidicity only if the aspartate concentration used is high enough to shift ATCase into the R state. Furthermore, it is shown that succinate, an unreactive aspartate analogue which is able to promote the T-->R conformational transition, also induces the appearance of cooperativity for carbamoylphosphate. Similar results were obtained in the course of continuous-flow-dialysis experiments, which show that the binding of carbamoylphosphate is apparently cooperative only in the presence of a concentration of succinate high enough to shift the enzyme into the R state. Taken together, these data show that the apparent cooperativity for carbamoylphosphate is not an intrinsic property of ATCase, as it only reflects the cooperativity for the second substrate, aspartate, as a consequence of the process of ordered substrate binding.

Pressure effects on the physical properties of lipid bilayers detected by trans-parinaric acid fluorescence decay.

The effects of hydrostatic pressure on the physical properties of large unilamellar vesicles of single lipids dipalmitoyl phosphatidylcholine (DPPC) and dimyristoyl phosphatidylcholine (DMPC) and lipid mixtures of DMPC/DPPC have been studied from time-resolved fluorescence of trans-parinaric acid. Additional experiments were carried out using diphenylhexatriene to compare the results extracted from both probes. Fluorescence decays were analyzed by the maximum entropy method. Pressure does not influence the fluorescence lifetime distribution of trans-parinaric acid in isotropic solvents. However, in pressurized lipid bilayers an abrupt change was observed in the lifetime distribution which was associated with the isothermal pressure-induced phase transition. The pressure to temperature equivalence values, dT/dP, determined from the midpoint of the phase transitions, were 24 and 14.5 degrees C kbar-1 for DMPC and POPC, respectively. Relatively moderate pressures of about 500 bar shifted the DMPC/DPPC phase diagram 11.5 degrees C to higher temperatures. The effects of pressure on the structural properties of these lipid vesicles were investigated from the anisotropy decays of both probes. Order parameters for all systems increased with pressure. In the gel phase of POPC the order parameter was smaller than that obtained in the same phase of saturated phospholipids, suggesting that an efficient packing of the POPC hydrocarbon chains is hindered.

The pAR5 mutation and the allosteric mechanism of Escherichia coli aspartate carbamoyltransferase.

Mutation pAR5 replaces residues 145'-153' at the C terminus of the regulatory (r) chains of Escherichia coli ATCase by a new sequence of six residues. The mutated enzyme has been shown to lack substrate cooperativity and inhibition by CTP. Solution X-ray scattering curves demonstrate that, in the absence of ligands, its structure is intermediate between the T form and the R form. In the presence of N-phosphonacetyl-L-aspartate, the mutant is similar to the wild type. An examination of the crystal structure of unligated ATCase reveals that the mutated site is at an interface between r and catalytic (c) chains, which exists only in the T allosteric form. A computer simulation by energy minimization suggests that the pAR5 mutation destabilizes this interface and induces minor changes in the tertiary structure of r chains. The resulting lower stability of the T form explains the loss of substrate cooperativity. The lack of allosteric inhibition may be related to a new electrostatic interaction made in mutant r chains between the C-terminal carboxylate and a lysine residue of the allosteric domain.

Solvent effects on allosteric equilibria: stabilization of T and R conformations of Escherichia coli aspartate transcarbamylase by organic solvents.

The activity of Escherichia coli aspartate transcarbamylase (ATCase) is markedly influenced by the addition of organic solvents to the assay medium. The cosolvents tested, which include simple aliphatic alcohols, amides, and ureas, as well as acetone and dioxane, fall into two different classes: the most polar ones (formamide, acetamide, N-methylformamide, and urea) stimulate the enzyme activity for all concentrations tested. In contrast, solvents that are less polar than water inhibit the enzyme at low concentrations but stimulate it at higher concentrations. No comparable effects are observed in the case of the isolated catalytic subunits, a non-regulated form of ATCase. Extensive kinetic studies on ATCase and on two of its Michaelian derivatives, 2-thioU-ATCase and carbamylated ATCase, indicate that solvents modulate the same allosteric transition that is responsible for homotropic interactions between the catalytic sites. The stabilization of the R state of ATCase by comparatively high concentrations of cosolvents is reminiscent of similar findings made on hemoglobin and glycogen phosphorylase, suggesting a common underlying mechanism. Addition of organic cosolvents to water is known to reduce hydrophobic interactions, and we suggest that this effect may preferentially stabilize the more "relaxed" conformations of allosteric proteins, because they have a larger surface exposed to solvent [Chothia, C. (1974) Nature (London) 248, 338-339]. On the other hand, we suggest that the stabilization of the T state by low concentrations of all but the most polar cosolvents simply reflects stronger electrostatic interactions in this conformation.

Pressure effects on the lateral distribution of cholesterol in lipid bilayers: a time-resolved spectroscopy study.

The effects of hydrostatic pressure and temperature on the phase behavior and physical properties of the binary mixture palmitoyloleoylphosphatidylcholine/cholesterol, over the 0-40 molar % range of cholesterol compositions, were determined from the changes in the fluorescence lifetime distribution and anisotropy decay parameters of the natural lipid trans-parinaric acid (t-PnA). Pressurized samples were excited with a Ti-sapphire subpicosecond laser, and fluorescence decays were analyzed by the quantified maximum entropy method. Above the transition temperature (T(T) = -5 degrees C), at atmospheric pressure, two liquid-crystalline phases, alpha and beta, are formed in this system. At each temperature and cholesterol concentration below the transition pressure, the fluorescence lifetime distribution pattern of t-PnA was clearly modulated by the pressure changes. Pressure increased the fraction of the liquid-ordered beta-phase and its order parameter, but it decreased the amount of cholesterol in this phase. Palmitoyloleoylphosphatidylcholine/cholesterol phase diagrams were also determined as a function of temperature and hydrostatic pressure.

Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy.

Self-assembly properties of HIV-1 integrase were investigated by time-resolved fluorescence anisotropy using tryptophanyl residues as a probe. From simulation analyses, we show that suitable photon counting leads to an accurate determination of long rotational correlation times in the range of 20-80 ns, permitting the distinction of the monomer, dimer, and tetramer from higher oligomeric forms of integrase. The accuracy of correlation times higher than 100 ns is too low to distinguish the octamer from other larger species. The oligomeric states of the widely used detergent-solubilized integrase were then studied in solution under varying parameters known to influence the activity. In the micromolar range, integrase exists as high-order multimers such as an octamer and/or aggregates and a well-defined tetramer, at 25 and 35 degrees C, respectively. However, integrase is monomeric at catalytically active concentrations (in the sub-micromolar range). Detergents (NP-40 and CHAPS) and divalent cation cofactors (Mg(2+) and Mn(2+)) have a clear dissociative effect on the high multimeric forms of integrase. In addition, we observed that Mg(2+) and Mn(2+) have different effects on both the oligomeric state and the conformation of the monomer. This could explain in part why these two metal cations are not equivalent in terms of catalytic activity in vitro. In contrast, addition of Zn(2+) stimulates dimerization. Interestingly, this role of Zn(2+) in the multimerization process was evident only in the presence of Mg(2+) which by itself does not induce oligomerization. Finally, it is highly suggested that the presence of detergent during the purification procedure plays a negative role in the proper self-assembly of integrase. Accordingly, the accompanying paper [Leh, H., et al. (2000) Biochemistry 39, 9285-9294] shows that a detergent-free integrase preparation has self-assembly and catalytic properties different from those of the detergent-solubilized enzyme.