Fluorescence Quenching of Two Coumarin-3-carboxylic Acids by Trivalent Lanthanide Ions.

The effects of various trivalent lanthanide ions (acetates of Ce3+, Er3+, Eu3+, Nd3+) on the electronic absorption and fluorescence spectra of un-substituted coumarin-3-carboxylic acid (CCA) and 7-N,N-diethylamino-coumarin-3-carboxylic acid (DECCA) have been investigated in dimethylsulfoxide (DMSO) at room temperature. Depending on the lanthanide ion nature and concentration, significant spectral changes of absorption bands occurred for both coumarin derivatives. These spectral changes were attributed to the formation of ground-state complexes between the coumarin carboxylate derivatives and lanthanide ions. The fluorescence quenching of CCA and DECCA upon increasing the lanthanide ion concentration was studied. Different quantitative treatments, including the Stern-Volmer equation, the Perrin equation and a polynomial equation, were applied and compared in order to determine the nature of the quenching mechanisms for both coumarin derivatives. The results suggested the contribution of both dynamic and static quenching. Significant differences of CCA and DECCA fluorescence quenching efficiency were also observed, depending on the lanthanide ion. DECCA fluorescence lifetime measurements, performed in the absence and in the presence of Ln3+, confirmed a contribution of static quenching.

Synthesis and spectral properties of new fluorescent alkoxy-substituted thieno[3,2-b]indole derivatives.

The synthesis and optical properties of three new fluorescent alkoxy-substituted thieno[3,2-b]indole (TI) derivatives, including 7-methoxy thieno[3,2-b]indole (7-MeOTI), 6,7- methylenedioxythieno[3,2-b]indole (6,7-MDTI) and 6,7-dihexyloxythieno[3,2-b]indole, (6,7-DHTI), were investigated. Electronic absorption spectra, fluorescence excitation and emission spectra, fluorescence quantum yields (ΦF), lifetimes (τF), and other photophysical parameters of the three TI derivatives were measured in DMSO solutions at room temperature. Theoretical electronic absorption and fluorescence spectra were also calculated by means of a molecular orbital (MO) method. For all three alkoxy-TI derivatives, the fluorescence emission maximum wavelength was significantly red shifted relative to un-substituted TI, which was attributed to delocalization of the fused hetero-aromatic ring π electronic system by the electron-donating alkoxy group(s). ΦF values varied from 0.12 to 0.19, according to the compound. τF were short, in the range 0.56-1.13 ns.

Comparative study of the fatty acid binding process of a new FABP from Cherax quadricarinatus by fluorescence intensity, lifetime and anisotropy.

Fatty acid-binding proteins (FABPs) are small cytosolic proteins, largely distributed in invertebrates and vertebrates, which accomplish uptake and intracellular transport of hydrophobic ligands such as fatty acids. Although long chain fatty acids play multiple crucial roles in cellular functions (structural, energy metabolism, regulation of gene expression), the precise functions of FABPs, especially those of invertebrate species, remain elusive. Here, we have identified and characterized a novel FABP family member, Cq-FABP, from the hepatopancreas of red claw crayfish Cherax quadricarinatus. We report the characterization of fatty acid-binding affinity of Cq-FABP by four different competitive fluorescence-based assays. In the two first approaches, the fluorescent probe 8-Anilino-1-naphthalenesulfonate (ANS), a binder of internal cavities of protein, was used either by directly monitoring its fluorescence emission or by monitoring the fluorescence resonance energy transfer occurring between the single tryptophan residue of Cq-FABP and ANS. The third and the fourth approaches were based on the measurement of the fluorescence emission intensity of the naturally fluorescent cis-parinaric acid probe or the steady-state fluorescence anisotropy measurements of a fluorescently labeled fatty acid (BODIPY-C16), respectively. The four methodologies displayed consistent equilibrium constants for a given fatty acid but were not equivalent in terms of analysis. Indeed, the two first methods were complicated by the existence of non specific binding modes of ANS while BODIPY-C16 and cis-parinaric acid specifically targeted the fatty acid binding site. We found a relationship between the affinity and the length of the carbon chain, with the highest affinity obtained for the shortest fatty acid, suggesting that steric effects primarily influence the interaction of fatty acids in the binding cavity of Cq-FABP. Moreover, our results show that the binding affinities of several fatty acids closely parallel their prevalences in the hepatopancreas of C. quadricarinatus as measured under specific diet conditions.

Synthesis, electrochemical, and optical properties of new fluorescent, substituted thieno[3,2-b][1]benzothiophenes.

The synthesis, electrochemical and optical properties of three fluorescent substituted thieno[3,2-b][1]benzothiophenes (TBT) derivatives, including 3-methoxythieno[3,2-b][1]benzothiophene (3-MeO-TBT), 2,3-dimethylthieno[3,2-b][1]benzothiophene (2,3-diMe-TBT), and 6-methoxythieno[3,2-b][1]benzothiophene-2-carboxylate (6-MeO-TBT-2-COOMe), were investigated. The oxidation potential values varied between 1.40 and 1.20 V/SCE according to the electronic substituent effect, and electropolymerization attempts, performed in 0.1 M LiClO(4) acetonitrile solution, led to the formation of very thin films of poly(3-MeO-TBT) and poly(2,3-di-Me-TBT). Electronic absorption spectra, fluorescence excitation and emission spectra, fluorescence quantum yields (Φ(F)) , lifetimes (τ(F)), and other photophysical parameters of the three new TBT derivatives were measured in DMSO solutions at room temperature. For the methyl-and methoxy-substituted TBT derivatives, the fluorescence emission peak were slightly red shifted relative to that of unsubstituted TBT (Δλ(em) = 1-12 nm) whereas, in the case of 6-MeO-TBT-2-COOMe, a rather strong red-shift (Δλ(em) = 73 nm) was attributed to the existence of a "push-pull" electronic interaction of the MeO and COOMe groups. All Φ(F) values were rather high, varying between 0.11 and 0.35, according to the substituent effect. Fluorescence decays were mono-exponential and τ(F) values were very short, ranging between 0.11 and 0.30 ns for the substituted TBT derivatives until study.

Multiple Escherichia coli RecQ helicase monomers cooperate to unwind long DNA substrates: a fluorescence cross-correlation spectroscopy study.

The RecQ family helicases catalyze the DNA unwinding reaction in an ATP hydrolysis-dependent manner. We investigated the mechanism of DNA unwinding by the Escherichia coli RecQ helicase using a new sensitive helicase assay based on fluorescence cross-correlation spectroscopy (FCCS) with two-photon excitation. The FCCS-based assay can be used to measure the unwinding activity under both single and multiple turnover conditions with no limitation related to the size of the DNA strands constituting the DNA substrate. We found that the monomeric helicase was sufficient to perform the unwinding of short DNA substrates. However, a significant increase in the activity was observed using longer DNA substrates, under single turnover conditions, originating from the simultaneous binding of multiple helicase monomers to the same DNA molecule. This functional cooperativity was strongly dependent on several factors, including DNA substrate length, the number and size of single-stranded 3'-tails, and the temperature. Regarding the latter parameter, a strong cooperativity was observed at 37 degrees C, whereas only modest or no cooperativity was observed at 25 degrees C regardless of the nature of the DNA substrate. Consistently, the functional cooperativity was found to be tightly associated with a cooperative DNA binding mode. We also showed that the cooperative binding of helicase to the DNA substrate indirectly accounts for the sigmoidal dependence of unwinding activity on ATP concentration, which also occurs only at 37 degrees C but not at 25 degrees C. Finally, we further examined the influences of spontaneous DNA rehybridization (after helicase translocation) and the single-stranded DNA binding property of helicase on the unwinding activity as detected in the FCCS assay.

Wrapping nanocrystals with an amphiphilic polymer preloaded with fixed amounts of fluorophore generates FRET-based nanoprobes with a controlled donor/acceptor ratio.

Colloidal nanocrystal (NC) donors wrapped with a polymer coating including multiple organic acceptor molecules are promising scaffolds for fluorescence resonance energy transfer (FRET)-based nanobiosensors. Over other self-assembling donor-acceptor configurations, our preloaded polymers have the virtue of producing compact assemblies with a fixed donor/acceptor distance. This property, together with the possibility of stoichiometric polymer loading, allowed us to directly address how the FRET efficiency depended on the donor/acceptor. At the population level, nanoprobes based on commercial as well as custom CdSe/ZnS donors displayed the expected dose-dependent rise in transfer efficiency, saturating from about five ATTO dyes/NC. However, for a given acceptor concentration, both the intensity and lifetime of single-pair FRET data revealed a large dispersion of transfer efficiencies, highlighting an important heterogeneity among nominally identical FRET-based nanoprobes. Rigorous quality check during synthesis and shell assembly as well as postsynthesis sorting and purification are required to make hybrid semiconductor-organic nanoprobes a robust and viable alternative to organic or genetically encoded nanobiosensors.

NO formation by neuronal NO-synthase can be controlled by ultrafast electron injection from a nanotrigger.

Nitric oxide synthases (NOSs) are unique flavohemoproteins with various roles in mammalian physiology. Constitutive NOS catalysis is initiated by fast hydride transfer from NADPH, followed by slower structural rearrangements. We used a photoactive nanotrigger (NT) to study the initial electron transfer to FAD in native neuronal NOS (nNOS) catalysis. Molecular modeling and fluorescence spectroscopy showed that selective NT binding to NADPH sites close to FAD is able to override Phe1395 regulation. Ultrafast injection of electrons into the protein electron pathway by NT photoactivation through the use of a femtosecond laser pulse is thus possible. We show that calmodulin, required for NO synthesis by constitutive NOS, strongly promotes intramolecular electron flow (6.2-fold stimulation) by a mechanism involving proton transfer to the reduced FAD(-) site. Site-directed mutagenesis using the S1176A and S1176T mutants of nNOS supports this hypothesis. The NT synchronized the initiation of flavoenzyme catalysis, leading to the formation of NO, as detected by EPR. This NT is thus promising for time-resolved X-ray and other cellular applications.

Insight into the integrase-DNA recognition mechanism. A specific DNA-binding mode revealed by an enzymatically labeled integrase.

Integration catalyzed by integrase (IN) is a key process in the retrovirus life cycle. Many biochemical or structural human immunodeficiency virus, type 1 (HIV-1) IN studies have been severely impeded by its propensity to aggregate. We characterized a retroviral IN (primate foamy virus (PFV-1)) that displays a solubility profile different from that of HIV-1 IN. Using various techniques, including fluorescence correlation spectroscopy, time-resolved fluorescence anisotropy, and size exclusion chromatography, we identified a monomer-dimer equilibrium for the protein alone, with a half-transition concentration of 20-30 mum. We performed specific enzymatic labeling of PFV-1 IN and measured the fluorescence resonance energy transfer between carboxytetramethylrhodamine-labeled IN and fluorescein-labeled DNA substrates. FRET and fluorescence anisotropy highlight the preferential binding of PFV-1 IN to the 3'-end processing site. Sequence-specific DNA binding was not observed with HIV-1 IN, suggesting that the intrinsic ability of retroviral INs to bind preferentially to the processing site is highly underestimated in the presence of aggregates. IN is in a dimeric state for 3'-processing on short DNA substrates, whereas IN polymerization, mediated by nonspecific contacts at internal DNA positions, occurs on longer DNAs. Additionally, aggregation, mediated by nonspecific IN-IN interactions, occurs preferentially with short DNAs at high IN/DNA ratios. The presence of either higher order complex is detrimental for specific activity. Ionic strength favors catalytically competent over higher order complexes by selectively disrupting nonspecific IN-IN interactions. This counteracting effect was not observed with polymerization. The synergic effect on the selection of specific/competent complexes, obtained by using short DNA substrates under high salt conditions, may have important implications for further structural studies in IN.DNA complexes.

Effects of water-borne copper on the gills and hepatopancreas of Macrobrachium rosenbergii.

We focused on elucidating the toxic effects of water-borne copper on the giant freshwater prawn Macrobrachium rosenbergii. After seven days of exposure to copper (Cu(2+) ) at concentrations ranging from 0.01 mg L(-1) to 0.5 mg L(-1) , three isozymes, malate dehydrogenase, alkaline phosphatase and esterase, were analyzed and compared using polyacrylamide electrophoresis (PAGE) and biochemical staining. The results indicated that the electrophoretic patterns of the isozymes showed a copper-concentration-related difference. Low doses of copper stimulated strong expression of the three isozymes. Electrophoretic patterns of malate dehydrogenase and alkaline phosphatase gradually became weaker or even lost as the level of copper increased. In contrast, esterase patterns exhibited an increased molecular heterogeneity at higher copper concentrations. A transmission electron microscope was used to study ultrastructure differences in the gills and hepatopancreas of M. rosenbergii, and the results showed significant structural damage at increased levels of copper compared with the control group. The basement membranes and mitochondira in the gills were seriously damaged, the cuticle electron density distribution was not homogeneous, and an infolded basement membrane, circularized nucleus, disintegrated nuclear membrane, and decreased mitochondria number and size were observed in the gills. Similarly, flowing out of karyoplasms, partly falling microvilli, decreased mitochondrion, partly disappeared mitochondrial cristae, and a thinned matrix were observed in the hepatopancreas. These findings indicate that exposure to elevated copper levels might damage the ultrastructure of the gills and hepatopancreas of M. rosenbergii and might further weaken their normal physical activities. Isozymes were quite sensitive to environmental stress and changes in isozyme elctrophoretic patterns might be effective biomarkers of environmental contamination.

Intrinsic fluorescence of B and Z forms of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA: spectra and lifetimes by the maximum entropy method.

A study has been made of the fluorescence of poly d(G-m5C).poly d(G-m5C), a synthetic double-stranded DNA, in buffered neutral aqueous solution at room temperature, excited by synchrotron radiation at 280 nm and 250 nm and by a frequency-doubled pulse dye laser at 290 nm. Exciting at 280 nm, the B form shows a uni-modal UV spectrum with lambdaf(max) approximately 340 nm. The Z form has in addition a visible emission lambdaf(max) at 450 nm. The spectral positions remain unchanged on exciting at 250 nm but the relative intensities change considerably. Decay profiles have been obtained at 360 nm and 450 nm for both the B and Z forms and have been analyzed by fitting to a pseudo-continuous distribution of 100 (and occasionally 200) exponentials, ranging from 10 ps to 20 ns, by optimizing the 'entropy' of the signal (the method of maximum entropy). We find the mean lifetimes for both wavelengths of emission and for both structural forms fall into three well-separated regions in the ranges indicated tau1 approximately 0.04-0.21 ns, tau2 approximately 0.9-1.26 ns, and tau3 approximately 5.1-6.5 ns. The UV emission, from its spectral position and half-width, correlates with monomeric emission from m5C (and from C for poly d(G-C)). However the lifetime tau1 is approximately 2 orders of magnitude longer than the monomers and points to an involvement of protonated guanosine (GH+, tauf approximately 200 ps) in the overall absorption/emission sequence. In the UV the tau3 emission is predominant, with fractional time-integrated emission approximately 86% for B DNA and approximately 64% for Z. We suggest it results from exciton (stacked) absorption followed by dissociative emission. For Z DNA the visible (450 nm) emission is dominated by a tau3 species (approximately 91%) with a lifetime of 6.5 ns and we suggest it represents a hetero-excimer emission consequent upon absorption by the strongly overlapped base-stacking, which differs from that in B DNA. The weak emission corresponding to tau2 is made more apparent by scanned gated detection of the emission from laser excitation (290 nm) of single-crystal d(m5C-G)3. A central role is attributed to the tight stacking of the bases in the Z form which correlates with enhanced hypochromism at 250 nm vs. 280 nm and with the reversal of the fluorescence intensity ratios UV-visible between these wavelengths.

Synthesis and characterization of polymer-coated quantum dots with integrated acceptor dyes as FRET-based nanoprobes.

A fluorescence resonance energy transfer pair consisting of a colloidal quantum dot donor and multiple organic fluorophores as acceptors is reported and the photophysics of the system is characterized. Most nanoparticle-based biosensors reported so far use the detection of specific changes of the donor/acceptor distance under the influence of analyte binding. Our nanoparticle design on the other hand leads to sensors that detect spectral changes of the acceptor (under the influence of analyte binding) at fixed donor/acceptor distance by the introduction of the acceptor into the polymer coating. This approach allows for short acceptor-donor separation and thus for high-energy transfer efficiencies. Advantageously, the binding properties of the hydrophilic polymer coating further allows for addition of poly(ethylene glycol) shells for improved colloidal stability.

Molecular interaction and energy transfer between human serum albumin and polyoxometalates.

As a step toward the elucidation of the mechanistic pathways governing the known bioactivity of polyoxometalates (POMs), two representative molecules of this class of chemicals, the wheel-shaped [NaP(5)W(30)O(110)]14- (P(5)W(30)) and the Keggin-type anion [H(2)W(12)O(40)]6- (H(2)W(12)), are shown, by two independent techniques, to interact with the fatty-acid-free human serum albumin (HSA). The excited-state lifetime of the single tryptophan molecule of this protein is dramatically decreased by the binding. The quenching mechanism is found to constitute the first example of energy transfer between HSA and POMs. Such molecular recognition is believed to be a key step for subsequent evolution of the systems. Circular dichroism (CD) was used to assess the structural effects of POM binding on HSA and to confirm the interaction revealed by fluorescence studies. CD experiments showed that the two POMs have different effects on the secondary structure of the protein. Binding P(5)W(30) partially unfolds the protein whereas H(2)W(12) has no remarkable effect on the structure of the protein.

Relationship between the oligomeric status of HIV-1 integrase on DNA and enzymatic activity.

The 3'-processing of the extremities of viral DNA is the first of two reactions catalyzed by HIV-1 integrase (IN). High order IN multimers (tetramers) are required for complete integration, but it remains unclear which oligomer is responsible for the 3'-processing reaction. Moreover, IN tends to aggregate, and it is unknown whether the polymerization or aggregation of this enzyme on DNA is detrimental or beneficial for activity. We have developed a fluorescence assay based on anisotropy for monitoring release of the terminal dinucleotide product in real-time. Because the initial anisotropy value obtained after DNA binding and before catalysis depends on the fractional saturation of DNA sites and the size of IN.DNA complexes, this approach can be used to study the relationship between activity and binding/multimerization parameters in the same assay. By increasing the IN:DNA ratio, we found that the anisotropy increased but the 3'-processing activity displayed a characteristic bell-shaped behavior. The anisotropy values obtained in the first phase were predictive of subsequent activity and accounted for the number of complexes. Interestingly, activity peaked and then decreased in the second phase, whereas anisotropy continued to increase. Time-resolved fluorescence anisotropy studies showed that the most competent form for catalysis corresponds to a dimer bound to one viral DNA end, whereas higher order complexes such as aggregates predominate during the second phase when activity drops off. We conclude that a single IN dimer at each extremity of viral DNA molecules is required for 3'-processing, with a dimer of dimers responsible for the subsequent full integration.

Kinetic study of the HIV-1 DNA 3'-end processing.

The 3'-processing of viral DNA extremities is the first step in the integration process catalysed by human immunodeficiency virus (HIV)-1 integrase (IN). This reaction is relatively inefficient and processed DNAs are usually detected in vitro under conditions of excess enzyme. Despite such experimental conditions, steady-state Michaelis-Menten formalism is often applied to calculate characteristic equilibrium/kinetic constants of IN. We found that the amount of processed product was not significantly affected under conditions of excess DNA substrate, indicating that IN has a limited turnover for DNA cleavage. Therefore, IN works principally in a single-turnover mode and is intrinsically very slow (single-turnover rate constant = 0.004 min(-1)), suggesting that IN activity is mainly limited at the chemistry step or at a stage that precedes chemistry. Moreover, fluorescence experiments showed that IN-DNA product complexes were very stable over the time-course of the reaction. Binding isotherms of IN to DNA substrate and product also indicate tight binding of IN to the reaction product. Therefore, the slow cleavage rate and limited product release prevent or greatly reduce subsequent turnover. Nevertheless, the time-course of product formation approximates to a straight line for 90 min (apparent initial velocity), but we show that this linear phase is due to the slow single-turnover rate constant and does not indicate steady-state multiple turnover. Finally, our data ruled out the possibility that there were large amounts of inactive proteins or dead-end complexes in the assay. Most of complexes initially formed were active although dramatically slow.

Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles.

The multifunctional DNA repair enzymes apurinic/apyrimidinic (AP) endonucleases cleave DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases in the base excision repair pathway. Alternatively, in the nucleotide incision repair (NIR) pathway, the same AP endonucleases incise DNA 5' of a number of oxidatively damaged bases. At present, the physiological relevance of latter function remains unclear. Here, we report genetic dissection of AP endonuclease functions in base excision repair and NIR pathways. Three mutants of Escherichia coli endonuclease IV (Nfo), carrying amino acid substitutions H69A, H109A, and G149D have been isolated. All mutants were proficient in the AP endonuclease and 3'-repair diesterase activities but deficient in the NIR. Analysis of metal content reveals that all three mutant proteins have lost one of their intrinsic zinc atoms. Expression of the nfo mutants in a repair-deficient strain of E. coli complemented its hypersensitivity to alkylation but not to oxidative DNA damage. The differential drug sensitivity of the mutants suggests that the NIR pathway removes lethal DNA lesions generated by oxidizing agents. To address the physiological relevance of the NIR pathway in human cells, we used the fluorescence quenching mechanism of molecular beacons. We show that in living cells a major human AP endonuclease, Ape1, incises DNA containing alpha-anomeric 2'-deoxyadenosine, indicating that the intracellular environment supports NIR activity. Our data establish that NIR is a distinct and separable function of AP endonucleases essential for handling lethal oxidative DNA lesions.

HIV-1 integrase complexes with DNA dissociate in the presence of short oligonucleotides conjugated to acridine.

The human immunodeficiency virus type 1 (HIV-1) integrase is an essential enzyme in the life cycle of the virus and is therefore an attractive target for the development of new antiviral drugs. Among them, inhibitors which are capable of targeting the preassembled integrase/DNA complex are of particular interest, because they could suppress integrase activity in the context of the HIV-1 preintegration complex. Here, we study the mechanism of action of 11-mer oligonucleotides, which are efficient inhibitors of the catalytic activity of integrase, provided that they are conjugated to a hydrophobic compound, acridine. To understand the mechanism of the conjugate inhibitory action, we used a steady-state fluorescence anisotropy assay, which allowed us to study the stability of the integrase/DNA complex in various conditions. We found that oligonucleotide-acridine conjugates induced the efficient dissociation of preassembled integrase/DNA complexes. The simultaneous presence of both acridine and an oligonucleotidic moiety is required for the inhibitory activity of conjugates. However, the dissociation effect is not dependent on the oligonucleotide sequence. Finally, our results suggest that the conjugates bind directly to integrase within its complex with DNA at a site different from the viral DNA binding site.

Mechanism of HIV-1 integrase inhibition by styrylquinoline derivatives in vitro.

Styrylquinoline derivatives (SQ) efficiently inhibit the 3'-processing activity of integrase (IN) with IC50 values of between 0.5 and 5 microM. We studied the mechanism of action of these compounds in vitro. First, we used steady-state fluorescence anisotropy to assay the effects of the SQ derivatives on the formation of IN-viral DNA complexes independently of the catalytic process. The IC50 values obtained in activity and DNA-binding tests were similar, suggesting that the inhibition of 3'-processing can be fully explained by the prevention of IN-DNA recognition. SQ compounds act in a competitive manner, with Ki values of between 400 and 900 nM. In contrast, SQs did not inhibit 3'-processing when IN-DNA complexes were preassembled. Computational docking followed or not by molecular dynamics using the catalytic core of HIV-1 IN suggested a competitive inhibition mechanism, which is consistent with our previous data obtained with the corresponding Rous sarcoma virus domain. Second, we used preassembled IN-preprocessed DNA complexes to assay the potency of SQs against the strand transfer reaction, independently of 3'-processing. Inhibition occurred even if the efficiency was decreased by about 5- to 10-fold. Our results suggest that two inhibitor-binding modes exist: the first one prevents the binding of the viral DNA and then the two subsequent reactions (i.e., 3'-processing and strand transfer), whereas the second one prevents the binding of target DNA, thus inhibiting strand transfer. SQ derivatives have a higher affinity for the first site, in contrast to that observed for the diketo acids, which preferentially bind to the second one.

The Escherichia coli RecQ helicase functions as a monomer.

The RecQ helicases belong to an important family of highly conserved DNA helicases that play a key role in chromosomal maintenance, and their defects have been shown to lead to several disorders and cancer in humans. In this work, the conformational and functional properties of the Escherichia coli RecQ helicase have been determined using a wide array of biochemical and biophysical techniques. The results obtained clearly indicate that E. coli RecQ helicase is monomeric in solution up to a concentration of 20 microM and in a temperature range between 4 and 37 degrees C. Furthermore, these properties are not affected by the presence of ATP, which is strictly required for the unwinding and translocating activity of the protein, or by its nonhydrolyzable analogue 5'-adenylyl-beta,gamma-imidodiphosphate. Consistent with the structural properties, functional analysis shows that both DNA unwinding activity and single-stranded DNA-stimulated ATPase specific activity were independent of RecQ concentration. The monomeric state was further confirmed by the ATPase-deficient mutants of RecQ protein. The rate of unwinding was unchanged when the wild type RecQ helicase was mixed with the ATPase-deficient mutants, indicating that nonprotein-protein interactions were involved in the unwinding processes. Taken together, these results indicate that RecQ helicase functions as a monomer and provide new data on the structural and functional properties of RecQ helicase that may help elucidate its mechanism action.

Disulfide-linked integrase oligomers involving C280 residues are formed in vitro and in vivo but are not essential for human immunodeficiency virus replication.

The human immunodeficiency virus type 1 integrase (IN) forms an oligomer that integrates both ends of the viral DNA. The nature of the active oligomer is unclear. Recombinant IN obtained under reducing conditions is always in the form of noncovalent oligomers. However, disulfide-linked oligomers of IN were recently observed within viral particles. We show that IN produced from a baculovirus expression system can form disulfide-linked oligomers. We investigated which residues are responsible for the disulfide bridges and the relationship between the ability to form covalent dimers and IN activity. Only the mutation of residue C280 was sufficient to prevent the formation of intermolecular disulfide bridges in oligomers of recombinant IN. IN activity was studied under and versus nonreducing conditions: the formation of disulfide bridges was not required for the in vitro activities of the enzyme. Moreover, the covalent dimer does not dissociate into individual protomers on disulfide bridge reduction. Instead, IN undergoes a spontaneous multimerization process that yields a homogenous noncovalent tetramer. The C280S mutation also completely abolished the formation of disulfide bonds in the context of the viral particle. Finally, the replication of the mutant virus was investigated in replicating and arrested cells. The infectivity of the virus was not affected by the C280S IN mutation in either dividing or nondividing cells. The disulfide-linked form of the IN oligomers observed in the viral particles is thus not required for viral replication.

Investigation of the effect of high hydrostatic pressure on proteins and lipidic membranes by dynamic fluorescence spectroscopy.

Dynamic fluorescence spectroscopy brings new insight into the functional and structural changes of biological molecules under moderate and high hydrostatic pressure. The principles of time-resolved fluorescence methods are briefly described and the resulting type of information is summarized. A first set of selected applications of the use of dynamic fluorescence on pressure effects on proteins in terms of denaturation, ternary and quaternary structure, aggregation and also interaction with DNA are presented. A second set of applications is devoted to the effect of pressure and of cholesterol on lateral heterogeneity of lipidic membranes.