Analysis of time-resolved fluorescence anisotropy decays.

We discuss the analysis of time-correlated single photon counting measurements of fluorescence anisotropy. Particular attention was paid to the statistical properties of the data. The methods used previously to analyze these experiments were examined and a new method was proposed in which parallel- and perpendicular-polarized fluorescence curves were fit simultaneously. The new method takes full advantage of the statistical properties of the measured curves; and, in some cases, it is shown to be more sensitive than other methods to systematic errors present in the data. Examples were presented using experimental and simulated data. The influence of fitting range on extracted parameters and statistical criteria for evaluating the quality of fits are also discussed.     

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

The subnanosecond fluorescence and motional dynamics of the tryptophan residue in the bacteriophage M13 coat protein incorporated within pure dioleoylphosphatidylcholine (DOPC) as well as dioleoylphosphatidylcholine/dioleoylphosphatidylglycerol (DOPC/DOPG) and dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol (DMPC/DMPG) bilayers (80/20 w/w) with various L/P ratio have been investigated. The fluorescence decay is decomposed into four components with lifetimes of about 0.5, 2.0, 4.5 and 10.0 ns, respectively. In pure DOPC and DOPC/DOPG lipid bilayers, above the phase transition temperature, the rotational diffusion of the protein molecules contributes to the depolarization and the anisotropy of tryptophan is fitted to a dual exponential function. The longer correlation time, describing the rotational diffusion of the whole protein, shortens with increasing temperature and decreasing protein aggregation number. In DMPC/DMPG lipid bilayers, below the phase transition, the rotational diffusion of the protein is slowed down such that the subnanosecond anisotropy decay of tryptophan in this system reflects only the segmental motion of the tryptophan residue. Because of a heterogeneous microenvironment, the anisotropy decay must be described by three exponentials with a constant term, containing a negative coefficient and a negative decay time constant. From such a decay, the tryptophan residue within the aggregate undergoes a more restricted motion than the one exposed to the lipids. At 20 degrees C, the order parameter of the transition moment of the isolated tryptophan is about 0.9 and that for the exposed one is about 0.5.     

DNA-induced polymerization of HIV-1 integrase analyzed with fluorescence fluctuation spectroscopy

Human immunodeficiency virus type 1 (HIV-1) integrase is essential for viral replication. Integrase inserts the viral DNA into the host DNA. We studied the association of integrase to fluorescently labeled oligonucleotides using fluorescence correlation spectroscopy. The binding of integrase to the fluorescent oligonucleotides resulted in the appearance of bright spikes during fluorescence correlation spectroscopy measurements. These spikes arise from the formation of high molecular mass protein-DNA complexes. The fluorescence of the free DNA was separated from the spikes with a statistical method. From the decrease of the concentration of free oligonucleotides, a site association constant was determined. The DNA-protein complexes were formed rapidly in a salt-dependent manner with site association constants ranging between 5 and 40 microm(-1) under different conditions. We also analyzed the kinetics of the DNA-protein complex assembly and the effect of different buffer components. The formation of the fluorescent protein-DNA complex was inhibited by guanosine quartets, and the inhibition constant was determined at 1.8 +/- 0.6 x 10(8) m(-1). Displacement of bound DNA with G-quartets allowed the determination of the dissociation rate constant and proves the reversibility of the association process.     

Oligomeric state of lipocalin-1 (LCN1) by multiangle laser light scattering and fluorescence anisotropy decay

Multiangle laser light scattering and fluorescence anisotropy decay measurements clarified the oligomeric states of native and recombinant tear lipocalin (lipocalin-1, TL). Native TL is monomeric. Recombinant TL (5-68 microM) with or without the histidine tag shows less than 7% dimer formation that is not in equilibrium with the monomeric form. Fluorescence anisotropy decay showed a correlation time of 9-10 ns for TL (10 microM-1 mM). Hydrodynamic calculations based on the crystallographic structure of a monomeric TL mutant closely concur with the observed correlation time. The solution properties calculated with HYDROPRO and SOLPRO programs from the available crystallographic structure of a monomeric TL mutant concur closely with the observed fluorescence anisotropy decay. The resulting model shows that protein topology is the major determinant of rotational correlation time and accounts for deviation from the Stokes-Einstein relation. The data challenge previous gel filtration studies to show that native TL exists predominantly as a monomer in solution rather than as a dimer. Delipidation of TL results in a formation of a complex oligomeric state (up to 25%). These findings are important as the dynamic processes in the tear film are limited by diffusional, translational as well as rotational, properties of the protein.     

Stereospecificity of reactions catalyzed by HIV-1 integrase

The retroviral integrase catalyzes two successive chemical reactions essential for integration of the retroviral genome into a host chromosome: 3' end processing, in which a dinucleotide is cleaved from each 3' end of the viral DNA; and the integration reaction itself, in which the resulting recessed 3' ends of the viral DNA are joined to the host DNA. We have examined the stereospecificity of human immunodeficiency virus type 1 integrase for phosphorothioate substrates in these reactions and in a third reaction, disintegration, which is macroscopically the reverse of integration. Integrase preferentially catalyzed end processing and integration of a substrate with the (R(p))-phosphorothioate stereoisomer at the reaction center and disintegration of a substrate with an (S(p))-phosphorothiate at the reaction center. These results suggest a model for the architecture of the active site of integrase, and its interactions with key features of the viral and target DNA.     

Probing DNA polymerase fidelity mechanisms using time-resolved fluorescence anisotropy

Prior to undergoing postsynthetic 3'-5' editing (proofreading), a defective DNA primer terminus must be transferred from the 5'-3' polymerase active site to a remote 3'-5' exonuclease site. To elucidate the mechanisms by which this occurs, we have used time-resolved fluorescence spectroscopy to study the interaction of dansyl-labeled DNA primer/templates with the Klenow fragment of Escherichia coli DNA polymerase I. The dansyl probe is positioned such that when the DNA substrate occupies the polymerase active site, the probe is solvent-exposed and possesses a short average fluorescence lifetime (4.7 ns) and extensive angular diffusion (42.5 degrees). Conversely, when the DNA substrate occupies the exonuclease active site, the probe becomes buried within the protein, resulting in an increase in the average lifetime (14.1 ns) and a decrease in the degree of angular diffusion (14.4 degrees ). If both polymerase and exonuclease binding modes are populated (lower limit approximately 5%), their markedly different fluorescence properties cause the anisotropy to decay with a characteristic "dip and rise" shape. Nonlinear least-squares analysis of these data recovers the ground-state mole fractions of exposed (x(e)) and buried (x(b)) probes, which are equivalent to the equilibrium proportions of the DNA substrate bound at the polymerase and exonuclease sites, respectively. The distribution between the polymerase and exonuclease binding modes is given by the equilibrium partitioning constant K(pe) (equal to x(b)/x(e)). The important determinants of the proofreading process can therefore be identified by changes made to either the protein or DNA that perturb the partitioning equilibrium and hence alter the magnitude of K(pe).     

Triple helix DNA oligomer melting measured by fluorescence polarization anisotropy

A synthetic DNA triple helix sequence was formed by annealing a pyrimidinic 21 mer single strand sequence onto the complementary purinic sequence centred on a 27 mer duplex DNA. Melting of the third strand was monitored by UV spectrophotometry in the temperature range 10-90 degrees C. The T(m) of the triplex, 37 degrees C, was well separated from the onset of duplex melting. When the same triple helix was formed on the duplex bearing one nick in the center of the pyrimidinic sequence the T(m) of the triplex was shifted to approximately 32 degrees C and overlapped the melting of the duplex. We have used fluorescence polarization anisotropy (FPA) measurements of ethidium bromide (EB) intercalated in duplex and triplex samples to determine the hydrodynamic parameters in the temperature range 10-40 degrees C. The fluorescence lifetime of EB in the samples of double and triple stranded DNA is the same (21.3 +/- 0.5 ns) at 20 degrees C, indicating that the geometries of the intercalation sites are similar. The values for the hydration radii of the duplex, normal triplex, and nicked triplex samples were 10.7 +/- 0.2, 12.2 +/- 0.2, and 12.0 +/- 0.2 A. FPA measurements on normal triplex DNA as a function of temperature gave a melting profile very similar to that derived by UV absorption spectroscopy. For the triplex carrying a nick, the melting curve obtained using FPA showed a clear shift compared with that obtained for the normal triplex sample. The torsional rigidity of the triplex forms was found to be higher than that of the duplex form.     

A time-resolved fluorescence assay to identify small-molecule inhibitors of HIV-1 fusion

Fusion of host cell and human immunodeficiency virus type 1 (HIV-1) membranes is mediated by the 2 "heptad-repeat" regions of the viral gp41 protein. The collapse of the C-terminal heptad-repeat regions into the hydrophobic grooves of a coiled-coil formed by the corresponding homotrimeric N-terminal heptad-repeat regions generates a stable 6-helix bundle. This brings viral and cell membranes together for membrane fusion, facilitating viral entry. The authors developed an assay based on soluble peptides derived from the gp41 N-terminal heptad-repeat region (IQN36) as well as from the C-terminal region (C34). Both peptides were labeled with fluorophores, IQN36 with allophycocyanin (APC) and C34 with the lanthanide europium (Eu3+). Formation of the 6-helix bundle brings both fluorophores in close proximity needed for F?rster resonance energy transfer (FRET). Compounds that interfere with binding of C34-Eu with IQN36-APC suppress the FRET signal. The assay was validated with various peptides and small molecules, and quenching issues were addressed. Evaluation of a diversified compound collection in a high-throughput screening campaign enabled identification of small molecules with different chemical scaffolds that inhibit this crucial intermediate in the HIV-1 entry process. This study's observations substantiate the expediency of time-resolved FRET-based assays to identify small-molecule inhibitors of protein-protein interactions.     

A time-resolved fluorescence anisotropy study of bilayer membranes containing alpha-tocopherol

Rotational mobility in fluid phase dipalmitoylphosphatidylcholine unilamellar vesicles containing alpha-tocopherol has been studied by time-resolved anisotropy measurements of fluorescence from a diphenylhexatriene-phosphatidylcholine conjugate. The results are analysed using a simple wobbling-in-cone model. The diphenylhexatriene probe shows an increasing order parameter and more restricted wobbling with increasing alpha-tocopherol content of the membrane. The diffusional rate for wobbling was found not to change significantly.     

Microsecond rotational motions of eosin-labeled myosin measured by time-resolved anisotropy of absorption and phosphorescence

We have studied submicrosecond and microsecond rotational motions within the contractile protein myosin by observing the time-resolved anisotropy of both absorption and emission from the long-lived triplet state of eosin-5-iodoacetamide covalently bound to a specific site on the myosin head. These results, reporting anisotropy data up to 50 microseconds after excitation, extend by two orders of magnitude the time range of data on time-resolved site-specific probe motion in myosin. Optical and enzymatic analyses of the labeled myosin and its chymotryptic digests show that more than 95% of the probe is specifically attached to sulfhydryl-1 (SH1) on the myosin head. In a solution of labeled subfragment-1 (S-1) at 4 degrees C, absorption anisotropy at 0.1 microseconds after a laser pulse is about 0.27. This anisotropy decays exponentially with a rotational correlation time of 210 ns, in good agreement with the theoretical prediction for end-over-end tumbling of S-1, and with times determined previously by fluorescence and electron paramagnetic resonance. In aqueous glycerol solutions, this correlation time is proportional to viscosity/temperature in the microsecond time range. Furthermore, binding to actin greatly restricts probe motion. Thus the bound eosin is a reliable probe of myosin-head rotational motion in the submicrosecond and microsecond time ranges. Our submicrosecond data for myosin monomers (correlation time 400 ns) also agree with previous results using other techniques, but we also detect a previously unresolvable slower decay component (correlation time 2.6 microseconds), indicating that the faster motions are restricted in amplitude. This restriction is not consistent with the commonly accepted free-swivel model of S-1 attachment in myosin. In synthetic thick filaments of myosin, both fast (700 ns) and slow (5 microseconds) components of anisotropy decay are observed. In contrast to the data for monomers, the anisotropy of filaments has a substantial residual component (26% of the initial anisotropy) that does not decay to zero even at times as long as 50 microseconds, implying significant restriction in overall rotational amplitude. This result is consistent with motion restricted to a cone half-angle of about 50 degrees. The combined results are consistent with a model in which myosin has two principal sites of segmental flexibility, one giving rise to submicrosecond motions (possibly corresponding to the junction between S-1 and S-2) and the other giving rise to microsecond motions (possibly corresponding to the junction between S-2 and light meromyosin).(ABSTRACT TRUNCATED AT 400 WORDS)     

Degradation of HIV-1 integrase by the N-end rule pathway

Human immunodeficiency virus type-1 (HIV-1) integrase catalyzes the irreversible insertion of the viral genome into host chromosomal DNA. We have developed a mammalian expression system for the synthesis of authentic HIV-1 integrase in the absence of other viral proteins. Integrase, which bears a N-terminal phenylalanine, was found to be a short-lived protein in human embryo kidney 293T cells. The degradation of integrase could be suppressed by proteasome inhibitors. N-terminal phenylalanine is recognized as a degradation signal by a ubiquitin-proteasome proteolytic system known as the N-end rule pathway. The replacement of N-terminal phenylalanine with methionine, valine, or glycine, which are stabilizing residues in the N-end rule, resulted in metabolically stabilized integrase proteins (half-life of N-terminal Met-integrase was at least 3 h). Conversely, the substitution of N-terminal phenylalanine with other destabilizing residues retained the metabolic instability of integrase. These findings indicate that the HIV-1 integrase is a physiological substrate of the N-end rule. We discuss a possible functional similarity to the better understood turnover of the bacteriophage Mu transposase and functions of integrase instability to the maintenance and integrity of the host cell genome.     

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.     

Cheek cell membrane fluidity measured by fluorescence recovery after photobleaching and steady-state fluorescence anisotropy

Membrane fluidity of human cheek cells was determined using fluorescence recovery after photobleaching (FRAP) and steady-state fluorescence anisotropy. The FRAP data showed that the lateral diffusion coefficient (D) and mobile fraction (%R) of lipid in the plasma membrane of control cells were 2.01×10^–9 cm²/ sec and 54.25%, respectively. Trypsin treatment increased D and %R to 6.4×10^–9 cm²/sec and 72.15%. In contrast, the anisotropy (r) for control cells was 0.270 which remained unchanged by trypsin treatment. The results show that diffusion of lipids in the plane of the membrane is restricted by trypsin-sensitive barriers.     

Erythrocyte membranes alteration in a shear stress measured by fluorescence anisotropy

An experimental setup has been designed to allow fluorescence anisotropy measurements on labeled cell membranes under shear stress. An important change is observed when increasing the shear stress and varying the experimental parameters indicates that a decrease in membrane cohesion leads to a subsequent increase in the membrane alteration under shear stress. A model has been developed that shows, in agreement with experiment, that the effect observed is mainly the result of the alteration of the membrane, elongation, and orientation with respect to the fluid flow, which can be estimated.     

Rational design of 2-pyrrolinones as inhibitors of HIV-1 integrase

HIV-1 integrase is an essential enzyme for viral replication and a validated target for the development of drugs against AIDS. With an aim to discover new potent inhibitors of HIV-1 integrase, we developed a pharmacophore model based on reported inhibitors embodying structural diversity. Eight compounds of 2-pyrrolinones fitting all the features of the pharmacophore query were found through the screening of an in-house database. These candidates were successfully synthesized, and three of them showed strand transfer inhibitory activity, in which, one compound showed antiviral activity. Further mapping analysis and docking studies affirmed these results.     

Motion of myosin cross-bridges in skeletal muscle fibers studied by time-resolved fluorescence anisotropy decay

The time-resolved fluorescence polarization anisotropy signal has been measured from fluorescent-labeled myosin cross-bridges in single glycerinated muscle fibers in the relaxed and rigor states. In one experimental configuration, the polarization of the excitation light and the fiber axis are aligned, and the anisotropy is sensitive to rotational motions of the probes about axes other than the fiber axis. The rotational correlation times are approximately 1000 ns for relaxed fibers and greater than 7000 ns for rigor fibers. In another experimental configuration, the excitation light polarization is perpendicular to the fiber axis, and its propagation vector has a component parallel to the fiber axis so that the anisotropy is sensitive to probe rotational motion about different axes, including the fiber axis. In this configuration, the rotational correlation times are approximately 300 ns for both relaxed and rigor fibers. The theory of rotational diffusion in a potential described in a related paper [Burghardt, T.P. (1985) Biophys. J. (in press)] is applied to the relaxed fiber data.     

Erythrocyte membranes alteration in a shear stress measured by fluorescence anisotropy.

An experimental setup has been designed to allow fluorescence anisotropy measurements on labeled cell membranes under shear stress. An important change is observed when increasing the shear stress and varying the experimental parameters indicates that a decrease in membrane cohesion leads to a subsequent increase in the membrane alteration under shear stress. A model has been developed that shows, in agreement with experiment, that the effect observed is mainly the result of the alteration of the membrane, elongation, and orientation with respect to the fluid flow, which can be estimated.