[Conformational changes of actin induced by strong or weak myosin subfragment-1 binding]. / Konformatsionnye izmeneniia aktina, vyzvannye sil'nym i slabym sviazyvaniem subfragmenta-1 miozina.

Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin (TRITC-falloidin, FITC-falloidin) and actin-bound nucleotide (e-ADP); 2) an increase in the orientation of dye oscillators located in the "front' surface of the small domain (where actin is viewed in the standard orientation with subdomains 1/2 and 3/4 oriented to the right and to the left, respectively); 3) a decrease in the angles of dye oscillators located on the "back" surface of subdomain-1. In contrast, a weak binding of S1 to actin induces the opposite effects in orientation of these probes. These data suggest that during the ATP hydrolysis cycle myosin heads induce a change in actin monomer (a tilt and twisting of its small domain). Presumably, these alterations in F-actin conformation play an important role in muscle contraction.

Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin.

Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation of F-actin. Strong binding by myosin heads produces changes in probe orientation that are opposite to those observed during weak binding.

Solution and interface aggregation states of Crotalus atrox venom phospholipase A2 by two-photon excitation fluorescence correlation spectroscopy.

The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate at 10 mM C12-PN in 10 mM Ca2+ and at 5 mM C12-PN in 1 mM EDTA. The PLA2 tryptophan fluorescence displayed spectral shifts and intensity changes upon interacting with C12-PN. On the basis of the FCS and tryptophan fluorescence results, we postulate an intermediate state where the two monomers are in loose interaction within a protein-lipid comicelle. As the concentration of C12-PN was increased, complete dissociation of the dimer was observed, inferred from the doubling of the particle number, and the average diffusion constant decreased to approximately 60 microm2/s, consistent with PLA2 associated with a C12-PN micelle. The presence of Ca2+ makes the comicelle intermediate more stable, retarding the separation of the monomers in the micellar suspension. Our data clearly indicate that PLA2, though a strong dimer in the absence of lipids, is dissociated by micellar C12-PN and supports the monomer hypothesis for PLA2 action.

Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase.

Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater conformational stability than the apoenzyme form. The data are discussed in light of the fact that the role of the coenzyme in structural stabilization varies among the pyridoxal 5'-phosphate-dependent enzymes and does not seem to be linked to the particular enzyme fold type.

Optical spectroscopy in studies of antibody-hapten interactions.

This article describes the use of optical spectroscopy in studying antibody-hapten interactions and in determining the equilibrium binding constants. Along with equilibrium binding data, spectroscopic tools often deliver structural information on binding-induced conformational changes of antibodies (or haptens). Structural implications of results from example antibody-hapten systems are included. Fluorescence spectroscopy has been particularly useful in the area of ligand binding, and thus steady-state fluorescence quenching and fluorescence polarization are the primary techniques under discussion. A brief description of fluorescence correlation spectroscopy is also provided. Absorption techniques, including circular dichroism, are mentioned to a lesser extent. A basic description of the mathematical models involved in the analysis of binding equilibria is provided along with references to more complete works. Simulated and experimental data are used to illustrate the various experimental protocols and the appropriate analytical methods. Typical sources of errors and experimental precautions are indicated throughout the general discussion.

Structural organization of the N-terminal domain of apolipoprotein A-I: studies of tryptophan mutants.

Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, the region surrounding Trp 108 appears to be more mobile than the rest of the protein in the lipid-free state. However, in rHDL particles, no significant domain motion was detected for any of the Trp residues. The results presented in this work are consistent with a model for monomeric lipid-free proapoA-I in which the N-terminal half of the molecule is organized into a bundle of helices.

Aggregation states of mitochondrial malate dehydrogenase.

The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.

Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.

Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of fructose-2,6-bisphosphate also significantly increased the anisotropy of W15. In the case of fructose-6-phosphate binding, the increased anisotropy was shown to be due to a restriction of the tryptophan residue's local mobility in the presence of bound ligand, which suggests that the N-terminus is located near the kinase active site. These increases in anisotropies were used to estimate the dissociation constants of fructose-6-phosphate and fructose-2,6-bisphosphate, which were 29 +/- 3 and 2.1 +/- 0.3 microM, respectively. These observations are considered in light of the recently published crystal structure for this bifunctional enzyme.

Quantitative analysis of the effects of intrathylakoid pH and xanthophyll cycle pigments on chlorophyll a fluorescence lifetime distributions and intensity in thylakoids.

The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time-resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the approximately 0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous effects of the chloroplast trans-thylakoid membrane pH gradient and xanthophyll cycle pigments on the PSII Chl a fluorescence tau distributions and intensity. The model assumes that (1) there exists a specific binding site for Z (or antheraxanthin, A) among or in an inner antenna complex (primarily CP29), (2) this binding site is activated by a low intrathylakoid pH (pK approximately 4.5) that increases the affinity for Z (or A), (3) about one Z or A molecule binds to the activated site, and (4) this binding effectively "switches" the fluorescence tau distribution of the PSII unit to a state with a decreased fluorescence tau and emission intensity (a "dimmer switch" concept). This binding is suggested to cause the formation of an exciton trap with a rapid intrinsic rate constant of heat dissipation. Statistical analysis of the data yields an equilibrium association constant, Ka, that ranges from 0.7 to 3.4 per PSII for the protonated/activated binding site for Z (or A). The model explains (1) the relative fraction of the approximately 0.5 ns fluorescence component as a function of both Z and A concentration and intrathylakoid pH, (2) the dependence of the ratio of F'm/Fm on the fraction of the 0.5 ns fluorescence tau component (where F'm and Fm are maximal fluorescence intensities in the presence and the absence of a pH gradient), and (3) the dependence of the ratio of F'm/Fm on the concentration of Z and A and the intrathylakoid pH.

Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.

The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the cleft containing the Pi binding site. Cleft closure has a rate constant of 317 s-1 (pH 7.0, 5 degrees C), and opening has a rate constant of 4.5 s-1. The fluorescence increase is likely to arise from a change in the hydrophobic environment during this closure as the steady state fluorescence emission (lambdamax and intensity) on Pi binding is mimicked by the addition of ethanol to aqueous solutions of an MDCC-thiol adduct. Fluorescence lifetimes in the absence and presence of Pi were 0.3 and 2.4 ns, respectively, consistent with the change in quantum yield. The rotational correlation time of the coumarin increases only 2-fold from 15 to 26 ns on binding Pi as measured by time-resolved polarization, consistent with the main rotation being determined by the protein even in the open conformation, but with greater local motion. Circular dichroism of the coumarin induced by the protein is weak in the absence of Pi and increases strongly upon saturation by Pi. These data are also consistent with an open to closed conformational model.

Comparative time-resolved photosystem II chlorophyll a fluorescence analyses reveal distinctive differences between photoinhibitory reaction center damage and xanthophyll cycle-dependent energy dissipation.

The photosystem II (PSII) reaction center in higher plants is susceptible to photoinhibitory molecular damage of its component pigments and proteins upon prolonged exposure to excess light in air. Higher plants have a limited capacity to avoid such damage through dissipation, as heat, of excess absorbed light energy in the PSII light-harvesting antenna. The most important photoprotective heat dissipation mechanism, induced under excess light conditions, includes a concerted effect of the trans-thylakoid pH gradient (delta pH) and the carotenoid pigment interconversions of the xanthophyll cycle. Coincidentally, both the photoprotective mechanism and photoinhibitory PSII damage decrease the PSII chlorophyll a (Chl a) fluorescence yield. In this paper we present a comparative fluorescence lifetime analysis of the xanthophyll cycle- and photoinhibition-dependent changes in PSII Chl a fluorescence. We analyze multifrequency phase and modulation data using both multicomponent exponential and bimodal Lorentzian fluorescence lifetime distribution models; further, the lifetime data were obtained in parallel with the steady-state fluorescence intensity. The photoinhibition was characterized by a progressive decrease in the center of the main fluorescence lifetime distribution from approximately 2 ns to approximately 0.5 ns after 90 min of high light exposure. The damaging effects were consistent with an increased nonradiative decay path for the charge-separated state of the PSII reaction center. In contrast, the delta pH and xanthophyll cycle had concerted minor and major effects, respectively, on the PSII fluorescence lifetimes and intensity (Gilmore et al., 1996, Photosynth. Res., in press). The minor change decreased both the width and lifetime center of the longest lifetime distribution; we suggest that this change is associated with the delta pH-induced activation step, needed for binding of the deepoxidized xanthophyll cycle pigments. The major change increased the fractional intensity of a short lifetime distribution at the expense of a longer lifetime distribution; we suggest that this change is related to the concentration-dependent binding of the deepoxidized xanthophylls in the PSII inner antenna. Further, both the photoinhibition and xanthophyll cycle mechanisms had different effects on the relationship between the fluorescence lifetimes and intensity. The observed differences between the xanthophyll cycle and photoinhibition mechanisms confirm and extend our current basic model of PSII exciton dynamics, structure and function.

Photosystem II chlorophyll a fluorescence lifetimes and intensity are independent of the antenna size differences between barley wild-type and chlorina mutants: Photochemical quenching and xanthophyll cycle-dependent nonphotochemical quenching of fluorescence.

Photosystem II (PS II) chlorophyll (Chl) a fluorescence lifetimes were measured in thylakoids and leaves of barley wild-type and chlorina f104 and f2 mutants to determine the effects of the PS II Chl a+b antenna size on the deexcitation of absorbed light energy. These barley chlorina mutants have drastically reduced levels of PS II light-harvesting Chls and pigment-proteins when compared to wild-type plants. However, the mutant and wild-type PS II Chl a fluorescence lifetimes and intensity parameters were remarkably similar and thus independent of the PS II light-harvesting antenna size for both maximal (at minimum Chl fluorescence level, Fo) and minimal rates of PS II photochemistry (at maximum Chl fluorescence level, Fm). Further, the fluorescence lifetimes and intensity parameters, as affected by the trans-thylakoid membrane pH gradient (ΔpH) and the carotenoid pigments of the xanthophyll cycle, were also similar and independent of the antenna size differences. In the presence of a ΔpH, the xanthophyll cycle-dependent processes increased the fractional intensity of a Chl a fluorescence lifetime distribution centered around 0.4-0.5 ns, at the expense of a 1.6 ns lifetime distribution (see Gilmore et al. (1995) Proc Natl Acad Sci USA 92: 2273-2277). When the zeaxanthin and antheraxanthin concentrations were measured relative to the number of PS II reaction center units, the ratios of fluorescence quenching to [xanthophyll] were similar between the wild-type and chlorina f104. However, the chlorina f104, compared to the wild-type, required around 2.5 times higher concentrations of these xanthophylls relative to Chl a+b to obtain the same levels of xanthophyll cycle-dependent fluorescence quenching. We thus suggest that, at a constant ΔpH, the fraction of the short lifetime distribution is determined by the concentration and thus binding frequency of the xanthophylls in the PS II inner antenna. The ΔpH also affected both the widths and centers of the lifetime distributions independent of the xanthophyll cycle. We suggest that the combined effects of the xanthophyll cycle and ΔpH cause major conformational changes in the pigment-protein complexes of the PS II inner or core antennae that switch a normal PS II unit to an increased rate constant of heat dissipation. We discuss a model of the PS II photochemical apparatus where PS II photochemistry and xanthophyll cycle-dependent energy dissipation are independent of the Peripheral antenna size.

Macromolecular arrangement in the aminoacyl-tRNA.elongation factor Tu.GTP ternary complex. A fluorescence energy transfer study.

The distance between the corner of the L-shaped transfer RNA and the GTP bound to elongation factor Tu (EF-Tu) in the aminoacyl-tRNA.EF-Tu.GTP ternary complex was measured using fluorescence energy transfer. The donor dye, fluorescein (Fl), was attached covalently to the 4-thiouridine base at position 8 of tRNAPhe, and aminoacylation yielded Phe-tRNAPhe-Fl8. The ribose of GTP was covalently modified at the 2'(3') position with the acceptor dye rhodamine (Rh) to form GTP-Rh. Formation of the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex was verified both by EF-Tu protection of the aminoacyl bond from chemical hydrolysis and by an EF-Tu.GTP-dependent increase in fluorescein intensity. Spectral analyses revealed that both the emission intensity and lifetime of fluorescein were greater in the Phe-tRNAPhe-Fl8.EF-Tu.GTP ternary complex than in the Phe-tRNAPhe-Fl8.EF-Tu.GTP-Rh ternary complex. These spectral differences disappeared when excess GTP was added to replace GTP-Rh in the latter ternary complex, thereby showing that excited-state energy was transferred from fluorescein to rhodamine in the ternary complex. The efficiency of singlet-singlet energy transfer was low (10-12%), corresponding to a distance between the donor and acceptor dyes in the ternary complex of 70 +/- 7 A, where the indicated uncertainty reflects the uncertainty in dye orientation. After correction for the lengths of the probe attachment tethers, the 2'(3')-oxygen of the GTP ribose and the sulfur in the s4U are separated by a minimum of 49 A. This large distance limits the possible arrangements of the EF-Tu and the tRNA in the ternary complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Xanthophyll cycle-dependent quenching of photosystem II chlorophyll a fluorescence: formation of a quenching complex with a short fluorescence lifetime.

Excess light triggers protective nonradiative dissipation of excitation energy in photosystem II through the formation of a trans-thylakoid pH gradient that in turn stimulates formation of zeaxanthin and antheraxanthin. These xanthophylls when combined with protonation of antenna pigment-protein complexes may increase nonradiative dissipation and, thus, quench chlorophyll a fluorescence. Here we measured, in parallel, the chlorophyll a fluorescence lifetime and intensity to understand the mechanism of this process. Increasing the xanthophyll concentration in the presence of a pH gradient (quenched conditions) decreases the fractional intensity of a fluorescence lifetime component centered at approximately 2 ns and increases a component at approximately 0.4 ns. Uncoupling the pH gradient (unquenched conditions) eliminates the 0.4-ns component. Changes in the xanthophyll concentration do not significantly affect the fluorescence lifetimes in either the quenched or unquenched sample conditions. However, there are differences in fluorescence lifetimes between the quenched and unquenched states that are due to pH-related, but nonxanthophyll-related, processes. Quenching of the maximal fluorescence intensity correlates with both the xanthophyll concentration and the fractional intensity of the 0.4-ns component. The unchanged fluorescence lifetimes and the proportional quenching of the maximal and dark-level fluorescence intensities indicate that the xanthophylls act on antenna, not reaction center processes. Further, the fluorescence quenching is interpreted as the combined effect of the pH gradient and xanthophyll concentration, resulting in the formation of a quenching complex with a short (approximately 0.4 ns) fluorescence lifetime.

Solution dynamics of p21ras proteins bound with fluorescent nucleotides: a time-resolved fluorescence study.

The solution dynamics of normal and transforming p21ras proteins in both the GTP- and GDP-bound forms were examined with time-resolved fluorescence spectroscopy. The fluorescent 2'(3')-O-(N-methylanthraniloyl) derivatives (mant derivatives) of GTP, dGTP, and GDP and the aminocoumarin and fluorescein derivatives of GTP and GDP were synthesized and used as reporter groups. The fluorescence lifetimes at 5 degrees C of the mant nucleotide derivatives increased from approximately 4 ns in solution to approximately 9 ns when bound to p21ras. At 30 degrees C, there was a 7.8% difference in lifetime between normal p21ras.mantGTP and p21ras.mantGDP, but no difference between similar complexes of the [Asp-12]p21ras protein. These data are consistent with steady-state fluorescence intensity differences among p21ras.mantGTP, p21ras.mantGDP, and the free nucleotides. Rotational correlation times for the mantGTP- and mantGDP-bound p21 proteins, N-ras, K-ras, and H-ras, were similar at 26 ns (5 degrees C), which is significantly longer than the 15-ns rotational correlation time predicted for a globular 21,000-Da protein. The p21-bound fluorescein and aminocoumarin nucleotide derivatives reported correlation times of 19 and 29 ns, respectively. Global analysis of the three fluorophore.p21 complexes with linked protein rotational correlation functions were best fit with a common rotational correlation time of 28 ns. Gel permeation chromatography of the GDP and mantGDP complexes of normal p21N-ras also showed greater apparent molecular weights than were expected in both cases, demonstrating that the high rotational correlation times obtained from time-resolved fluorescence measurements were not a result of the introduction of the fluorophore.(ABSTRACT TRUNCATED AT 250 WORDS)

Elucidation of anti-ssDNA autoantibody BV 04-01 binding interactions with homooligonucleotides.

Binding interactions of various synthetic oligohomonucleotides with anti-ssDNA autoantibody BV 04-01 (IgG2b, kappa) and the corresponding single-chain antibody (SCA) 04-01/212 were studied. Oligonucleotide binding to IgG or SCA resulted in quenching of the protein's tryptophan fluorescence permitting direct assessment of ligand binding under equilibrium conditions. The effect of oligothymidylate length, (dT)n, on tryptophan quenching was evaluated. The equilibrium dissociation constants (Kd) for the binding of (dT)6 and (dT)8 were the same [(1.3 +/- 0.02) x 10(-7) M], while decreasing the length of the oligothymidylate to (dT)3 increased the Kd an order of magnitude. To assess base specificity, the comparative binding of other hexahomonucleotides was examined. Neither (dA)6 nor (dC)6 showed measurable binding, while the dissociation constant for (dG)6 was (7.1 +/- 0.3) x 10(-7) M. Fluorescence lifetime quenching data correlated with the steady-state binding results and indicated that the quenching process contains both dynamic and static components. The ability of BV 04-01 to bind (dT)6 and (dG)6 nucleotides was further supported by fluorescence anisotrophy studies with fluorescein-labeled hexadeoxynucleotides. Various levels of tryptophan fluorescence quenching upon titration with oligothymidylates of different length, as well as the similar affinities for (dT)6 and (dG)6, supported the concept that the groove-type binding pocket in BV 04-01 consists of binding subsites that cooperatively adapt for efficient binding of oligonucleotides.

Examination of elongation factor Tu for aluminum fluoride binding sites using fluorescence and 19F-NMR methodologies.

This article reports on a comparison of the interaction of Al3+ and F- with two GTP-binding proteins, elongation factor Tu (EF-Tu) and the hormone sensitive regulatory protein (G protein) G0 alpha. The methodologies chosen to elucidate possible interactions between protein and aluminum fluoride were fluorescence spectroscopy and nuclear magnetic resonance (19F-NMR). Both proteins have tryptophan residues near their nucleotide binding sites, the purported site of aluminum fluoride interaction. It has been assumed for G proteins (including G0 alpha) that aluminum fluoride, in the presence of Mg2+ mimics the magnesium coordinated gamma-phosphate group for the GDP-form of the protein and shifts the protein's conformation toward the active GTP-form. Indeed, changes in intrinsic fluorescence of G0 alpha effected by aluminum fluoride are observed. The presence of aluminum fluoride did not affect the intrinsic fluorescence, spectra or lifetimes, of EF-Tu.GDP 19F-NMR was then used to directly test for bound F-. Fluoride alone or in the presence of either protein gave a single 19F-NMR peak at -10 ppm, characteristic of free F-. With the addition of aluminum to the protein and F- samples a second peak, shifted upfield from the first to -29 ppm, was observed for G0 alpha.GDP. This second peak, which has been assigned to protein-bound F-, was not observed for EF-Tu.GDP. These observations show that the interaction of Al3+ and F-, in the presence of Mg2+, may be quite different between the hormone-sensitive G proteins, which bind aluminum fluoride, and the GTP-binding proteins as a whole, which include EF-Tu. Care must therefore be exercised when structural data on the elongation factor, specifically on the nucleotide site, are used to interpret data or compose models intended to describe the hormone-sensitive regulatory G proteins.

Time-resolved fluorescence studies on the ternary complex formed between bacterial elongation factor Tu, guanosine 5'-triphosphate, and phenylalanyl-tRNAPhe.

Time-resolved fluorescence spectroscopy was used to investigate the solution dynamics of Escherichia coli tRNAPhe, Phe-tRNAPhe, and Phe-tRNAPhe associated with GTP and elongation factor Tu (EF-Tu) in a ternary complex. Two fluorescence probes were employed: fluorescein, covalently bound to Phe-tRNAPhe at the s4U8 base (Phe-tRNAPhe-Fl8), and ethidium bromide, noncovalently associated with the tRNA (EB.Phe-tRNAPhe). The lifetimes observed for ethidium bromide were 1.89 ns, free in solution, and 26.3 ns, bound to its tight binding site on tRNA. Fluorescein-labeled tRNA had a lifetime of 4.3 ns, with no significant difference among the values for aminoacylated, unacylated, and EF-Tu-bound Phe-tRNAPhe-Fl8. Differential phase and modulation data for each fluorophore-tRNA system were fit with local and global Debye rotational relaxation times. Local motion of the labeled fluorescein in Phe-tRNAPhe-Fl8, tRNAPhe-Fl8, and Phe-tRNAPhe-Fl8.EF-Tu.GTP was characterized by rotational relaxation times of 2.7 +/- 0.5, 2.4 +/- 0.4, and 2.4 +/- 0.1 ns, respectively. These values are equal, within experimental error, and suggest that the rotational mobility of the s4U8-conjugated dye is unaffected by either tRNAPhe aminoacylation or ternary complex formation. Global rotational relaxation times for Phe-tRNAPhe-Fl8, 97 ns, and EB.Phe-tRNAPhe, 140 ns, were equivalent to those determined for the unacylated species, denoting little change in the overall size or shape of the tRNA molecule upon aminoacylation. These values for (Phe-)tRNA were larger than expected for a hydrated sphere of equivalent volume, 83 ns, and therefore confirm the asymmetric nature of the tRNA structure in solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipid-induced aggregation of phospholipase A2: sucrose density gradient ultracentrifugation and crosslinking studies.

The aggregation behavior of cobra venom (Naja naja naja) phospholipase A2 in the presence of lipids and Ca2+ was examined using ultracentrifugation and crosslinking techniques. Velocity sedimentation experiments were performed in sucrose gradients. The sedimentation coefficients of the cobra phospholipase A2 and various controls, including bovine serum albumin (BSA), malate dehydrogenase, carbonic anhydrase and pancreatic phospholipase A2, were calculated both in the presence and absence of ligands. The monomeric phospholipid, diheptanoylphosphatidylcholine, and the phospholipid analogue, dodecylphosphocholine (DPC), increased the sedimentation coefficient of the cobra phospholipase A2 from 2.2 S to 2.9 S, a value that is consistent with the formation of an enzyme dimer. The control proteins were unaffected by the presence of phospholipid, except for BSA, which apparently binds large amounts of DPC. Crosslinking experiments with glutaraldehyde showed that in the presence of diheptanoylphosphatidylcholine or DPC, the amount of crosslinked enzyme increased. Ca2+ had no effect on the aggregation state of the enzyme as measured by either technique. Both the ultracentrifugation data and crosslinking data are consistent with the hypothesis that the cobra venom phospholipase A2 exists as a dimer or higher-order aggregate in the presence of lipid substrate, although it is yet to be determined whether the functional subunit is a monomer, dimer or higher-order oligomer.