Interactions of HIV-1 DNA Heterocyclic Bases with Viral Integrase

Integrase (IN) is responsible for one of the key stages in the replication cycle of human immunodeficiency virus type 1, namely, integration of a DNA copy of the viral RNA into the infected cell genome. IN recognizes the nucleotide sequences located at the ends of the U3 and U5 regions of long terminal repeats (LTRs) of the viral DNA and sequentially catalyzes the 3-end processing and strand transfer reactions. Analogs of U5 regions containing non-nucleoside insertions have been used to study the interaction between IN and viral DNA. Substrate modification has been demonstrated to have almost no effect on the rate of DNA binding by IN. However, the removal of heterocyclic bases from positions 5 and 6 of the substrate molecule and from position 3 of the processed strand almost completely inhibits IN enzymatic activity, which indicates the importance of these bases for the formation of an active enzyme–substrate complex. By contrast, modification of the third base of the nonprocessed strand stimulates 3-processing. Since the base removal disturbs the complementary and stacking interactions in DNA, these results indicate that double-helix destabilization near the cleaved bond promotes 3-end processing.     

Polarised pulse fluorimetry study on the conformational properties of wheat germ hexokinase LI

The conformational properties of wheat germ hexokinase LI, a monomeric enzyme showing non-Michaelian kinetics, have been studied by polarised pulse fluorimetry using synchrotron radiation as an excitation light source.The fluorescence decays and the fluorescence anisotropy decays of tryptophyl residues were measured with excitation at 300 nm. At pH 8.5, we found that the mnemonical temperature-dependent transition did not induce any detectable structural change in the protein. This rules out modifications of the aggregation state of hexokinase during the transition as well as important conformational changes in the tertiary structure. At pH 6.1, a temperature-dependent transition of the enzyme-glucose binary complex is observed: rapid, large amplitude, internal motions appear in the structure when the temperature is raised from-1°C to 30°C. Full standard activity is retained during this dynamic change.In the experiments described here we obtained an active fluorescent derivative by reacting hexokinase with N-(iodoacetylaminoethyl)-5-naphtylamine-1-sulfonic acid (1,5-IAEDANS), in the presence of glucose. Polarised fluorescence decay measurements indicate that the label is exposed to the solvent and very mobile, which makes it ineffective as a probe for the conformational properties of hexokinase.Abbreviations 1,5-IAEDANS N-(iodoacetylaminoethyl)-5-napthylamine-1-sulfonic acid - DTNB 5,5-dithiobis(2-nitrobenzoic acid)     

Micellization and interactions with phospholipid vesicles of the lipopeptide iturin a,as monitored by time-resolved fluorescence of a D-tyrosyl residue

The micellization and the interactions with lipid vesicles of the antifungal cyclic lipopeptide iturin A have been investigated by nanosecond pulse fluorometry of a D-tyrosyl residue. We show that this lipopeptide has three conformers in solution whose proportions are modified during the micellization process. Below the critical micellar concentration (CMC) iturin A does not self-associate inside the bilayer. Above the CMC all the molecules of iturin A interact with the vesicles and self-associate inside the membrane.     

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.     

Time-resolved fluorescence of the single tryptophan residue in rat alpha-fetoprotein and rat serum albumin: analysis by the maximum-entropy method.

The time-resolved fluorescence emissions of the lone tryptophan residues in rat alpha-fetoprotein (RFP) and rat serum albumin (RSA) were studied. The total fluorescence intensity decays in both proteins were multiexponential. Analysis of the data by nonlinear least squares as a sum of discrete exponentials showed that four exponentials were needed for a satisfactory fit for both proteins. Analysis by the maximum entropy method using 150 logarithmically equally spaced exponentials yielded four well-resolved excited-state lifetime classes with barycenters and relative amplitudes values (ci) that corresponded to those obtained from the nonlinear least-squares method. Changing the temperature affected the relative amplitudes of the lifetime classes but had little effect on the lifetime values themselves. This suggests that the four classes reflect local conformational substates that exchange slowly with respect to the time window of observation defined by the longest lifetime. The internal rotational dynamics of the tryptophan in each protein was monitored by fluorescence anisotropy decay measurements. The mobility of the tryptophan appeared to be larger and faster in RFP than in RSA. The nonlinear least-squares analysis suggests the existence of three rotational correlation times of 0.1, 3, and 55 ns for this protein. As a function of temperature, the long correlation time did not follow the Perrin's law expected for a rigid rotating body. This suggests that this correlation time may reflect not only the Brownian rotation of the whole protein but also the flexibilities of domains in the protein. For RSA a two-component model with correlation times of 0.4 and 31 ns was sufficient to describe the data.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mesure des déclins de l'intensité et de l'anisotropie de fluorescence du pérylène en présence de membranes d'érythrocyte de pigeon: Comparaison avec un milieu isotrope visqueux

Using synchrotron radiation as the excitation light, we studied the fluorescence parameters of perylene incubated with pigeon erythrocyte membranes and with an isotropic viscous medium, the Primol 342 oil.From 4 to 37°C, we observed a single lifetime of 4.5 ns in the oil and two with the membrane (τ₁ = 1−1.4 ns and τ₂ = 5.4−6.1 ns). The dependence upon temperature of the rotation correlation time of perylene () in the oil was characteristic of an isotropic medium, whereas the limiting value of anitropy (r ∞) was zero. With the membrane, γ ∞ decreased from 0.14 to 0.06 and from 2.9 to 0.5 ns, indicating a greater amplitude and frequency of molecular motions.The addition of chlorpromazine, indomethacine, tetracaine, n-octylamine, octanol or octanoic acid to the membrane decreased the τ₁ and τ₂ values. This would stem from the desorganization of the membrane induced by the drugs.     

Determinants of Mg2+-dependent activities of recombinant human immunodeficiency virus type 1 integrase

The relationship between Mg(2+)-dependent activity and the self-assembly state of HIV-1 integrase was investigated using different protein preparations. The first preparations, IN(CHAPS) and IN(dial), were purified in the presence of detergent, but in the case of IN(dial), the detergent was removed during a final dialysis. The third preparation, IN(zn), was purified without any detergent. The three preparations displayed comparable Mn(2+)-dependent activities. In contrast, the Mg(2+)-dependent activity that reflects a more realistic view of the physiological activity strongly depended on the preparation. IN(CHAPS) was not capable of using Mg(2+) as a cofactor, whereas IN(zn) was highly active under the same conditions. In the accompanying paper [Deprez, E., et al. (2000) Biochemistry 39, 9275-9284], we used time-resolved fluorescence anisotropy to demonstrate that IN(CHAPS) was monomeric at the concentration of enzymatic assays. Here, we show that IN(zn) was homogeneously tetrameric under similar conditions. Moreover, IN(dial) that exhibited an intermediary Mg(2+)-dependent activity existed in a monomer-multimer equilibrium. The level of Mg(2+)- but not Mn(2+)-dependent activity of IN(dial) was altered by addition of detergent which plays a detrimental role in the maintenance of the oligomeric organization. Our results indicate that the ability of integrase to use Mg(2+) as a cofactor is related to its self-assembly state in solution, whereas Mn(2+)-dependent activity is not. Finally, the oligomeric IN(zn) was capable of binding efficiently to DNA regardless of the cationic cofactor, whereas the monomeric IN(CHAPS) strictly required Mn(2+). Thus, we propose that a specific conformation of integrase is a prerequisite for its binding to DNA in the presence of Mg(2+).     

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.     

HIV-1 integrase catalytic core: molecular dynamics and simulated fluorescence decays.

Two molecular dynamics simulations have been carried out on the HIV-1 integrase catalytic core starting from fully determined crystal structures. During the first one, performed in the absence of divalent cation (6-ns long), the catalytic core took on two main conformations. The conformational transition occurs at approximately 3.4 ns. In contrast, during the second one, in the presence of Mg(2+) (4-ns long), there were no such changes. The molecular dynamics simulations were used to compute the fluorescence intensity decays emitted by the four tryptophan residues considered as the only chromophores. The decay was computed by following, frame by frame, the amount of chromophores that remained excited at a certain time after light absorption. The simulation took into account the quenching through electron transfer to the peptide bond and the fluorescence resonance energy transfer between the chromophores. The fit to the experimental intensity decays obtained at 5 degrees C and at 30 degrees C is very good. The fluorescence anisotropy decays were also simulated. Interestingly, the fit to the experimental anisotropy decay was excellent at 5 degrees C and rather poor at 30 degrees C. Various hypotheses such as dimerization and abnormal increase of uncorrelated internal motions are discussed.     

Analyzing the Distribution of Decay Constants in Pulse-Fluorimetry Using the Maximum Entropy Method

The maximum entropy method (MEM) is used to analyze time-resolved pulse-fluorescence spectrometry. The central problem in such analyses is the recovery of the distribution of exponentials describing the decay of the fluorescence (i.e., inverting the Laplace transform) which is, in turn, convolved by the shape of the excitation flash. MEM is shown to give high quality results from both computer-generated “noisy” data and experimental data from chemical and biological molecules.

The use of the Shannon-Jaynes entropy function is justified and both the theoretical and practical advantages of MEM are presented. The MEM results are easy to interpret and can help to overcome some experimental limitations. In particular MEM could be a powerful tool to analyze the heterogeneity of fluorescent emission of biological macromolecules which can be correlated with their conformational dynamics in solution.