Ca2+-induced conformational changes in cardiac troponin C as measured by N-(1-pyrene)maleimide fluorescence

Bovine cardiac troponin C was modified by N-(1-pyrene)maleimide at Cys-35 and Cys-84; the Ca2+-induced conformational changes were followed by measuring pyrene fluorescence. In isolated troponin C, the saturation of Ca2+, Mg2+-sites leads to a simultaneous increase in the pyrene monomer as well as to a decrease in the pyrene excimer fluorescence, whereas the saturation of Ca2+-specific sites results in a slight decrease in the fluorescence of pyrene monomer. Troponin T does not influence the dependence of pyrene-troponin C fluorescence on Ca2+ concentration. Within the equimolar complex of troponin C and troponin I, the saturation of Ca2+, Mg2+-sites has no effect on pyrene fluorescence, whereas the saturation of Ca2+-specific sites leads to a simultaneous decrease of both pyrene monomer and pyrene excimer fluorescence. It is supposed that troponin I diminishes the conformational changes in troponin C that are induced by the saturation of Ca2+, Mg2+-sites and enhances the conformational changes induced by the saturation of Ca2+-specific sites of troponin C.     

Thermal denaturation and fluorescence study of nucleosomes containing non-histone chromosomal protein HMG2

Interaction of calf thymus non-histone chromosomal protein HMG2 with H1,H5-depleted nucleosomes from chicken erythrocytes was studied by means of thermal denaturation and an N-(3-pyrene)maleimide fluorescence probe. Under low ionic conditions (2 mM Tris buffer plus EDTA) addition of 1-2 molecules of HMG2 per nucleosome markedly stabilized the segment of the linker DNA against thermal denaturation. Under approximately physiological ionic conditions (0.1 M NaCl) addition of two HMG2 molecules per nucleosome, labeled by N-(3-pyrene)maleimide at the sulfhydryl groups of Cys-110 of histones H3, resulted in a decrease of the pyrene excimer fluorescence corresponding to the slight movement of the sulfhydryl groups of the two histone H3 molecules apart.     

Influence of hydrogen bonding,main chain–side chain interactions,and protecting groups on the excimer formation of bis(pyrenylalanine) peptides

Dipeptides of the aromatic fluorescent amino acid, pyrenylalanine, are studied using both stationary and transient fluorescence techniques. Since the conformational transitions of the peptide chain are slow compared to the decay of the pyrene excited state, both ground state conformations, adopted by the peptide, i.e., C₅ and C₇, can be monitored separately. Kinetic models are proposed to describe the molecular dynamics of the peptide chain as probed by the intramolecular excimer formation between both pyrene chromophores. These kinetic schemes explain the influence of solvent, chain chirality, main chain–side chain interactions, and nature of the protecting groups on the emission spectrum and the fluorescence decay profile of these model peptides. These schemes also provide a tool to calculate rate constants of conformational transitions and excimer formation. By comparing the kinetic and thermodynamic parameters of the various compounds, the influence of a structural modification on the molecular dynamics of the peptide chain is determined.     

Intermolecular histone H4 interactions in core nucleosomes

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.     

Incorporation of exogenous pyrene-labeled histone into Physarum chromatin: a system for studying changes in nucleosomes assembled in vivo

Treatment of Physarum histone with iodoacetoxypyrene selectively derivatives a single H3 cysteine with acetoxypyrene. Microplasmodia can incorporate this AP-H3 into nucleosomes. The distinction between blue monomeric pyrene fluorescene and green excimer pyrene fluorescence allows detection of changes in distance between the closely positioned H3 cysteines in nucleosomes. Fluorescence of nucleosomes labeled in vivo with AP-H3 is almost exclusively of the eximer form, indicating that H3 cysteines are within a few angstroms of each other in the nucleosome core. In histones recovered from these nucleosomes all detectable pyrene is covalently bound to H3. When Physarum is exposed sequentially to labeled followed by unlabeled histone, there is a rapid appearance of green excimer emission in nucleosomes after addition of labeled histone and no apparent switch from excimer to monomer fluorescence after several replications of the genome in the presence of unlabeled histone. These experiments provide evidence in favor of a model for conservative distribution of nucleosomal histones during chromatin replication.     

Fluorescence-coupled CD conformational monitoring of filament formation of tau microtubule-binding repeat domain

To clarify the contribution of the three- or four-repeated peptide moiety in tau microtubule-binding domain (MBD) to paired helical filament (PHF) formation, conformational transition accompanied by heparin-induced filament formation was investigated stepwise for four repeat peptides (R1-R4), one three-repeated R1-R3-R4 peptide (3RMBD), and one four-repeated R1-R2-R3-R4 peptide (4RMBD) using a combination of thioflavin S fluorescence and circular dichroism (CD) measurements in a neutral buffer (pH 7.6). The comparison of the fluorescence profile of each repeat peptide with those of 3RMBD and 4RMBD showed the synergistic contribution of R1-R4 to PHF formation of MBD. The CD spectrum measured as a function of filament formation time indicates that: (i) two conformational transitions occur for the filament formations of R3 (from the random structure to the beta-sheet structure) and 3RMBD (from the random structure to the alpha-helix structure), (ii) the filament formations of R2 and 4RMBD proceed via the synchronized conformational transitions of the alpha-helix and random structures, and (iii) the filament formation of 4RMBD is dependent on the aggregation behavior of R2. These data are useful for elucidating the MBD conformational transition in tau PHF formation.     

Evidence that pyrene excimer formation in membranes is not diffusion-controlled

Kinetic and steady-state measurements of pyrene fluorescence in a variety of model membranes are evaluated in terms of the theory of collisional excimer formation. In the region of 10(-3)-0.1 M pyrene, molecular fluorescence decay in membranes is biphasic and the two component lifetimes do not depend on the pyrene concentration. The lifetime data are consistent with the rate constant for collisional excimer formation being of the order 10(6) M-1 X s-1 or less. The concentration dependence of the component amplitudes is inconsistent with the theory of collisional excimer formation and suggests that pyrene exists in two forms in membranes: a slowly diffusing monomeric form and an aggregated form. The component of molecular fluorescence decay associated with aggregated pyrene is highly correlated with steady-state excimer fluorescence, suggesting that excimer fluorescence in membranes arises from aggregated pyrene in which excimers are formed by a static rather than a collisional mechanism. It is suggested that the concentration dependence of excimer to molecular fluorescence intensity ratios in membranes is related to the equilibrium constant for exchange between monomeric and aggregated pyrene forms rather than to the collisional excimer formation rate constant.     

Effects of the state of the succinimido-ring on the fluorescence and structural properties of pyrene maleimide-labeled alpha alpha-tropomyosin.

Rabbit skeletal alpha alpha-tropomyosin was labeled at Cys-190 with pyrene maleimide to form (S-[N-(1-pyrene)succinimido])2-tropomyosin (pyreneI-Tm). The product with cleaved succinimido-rings, pyreneII-Tm was also prepared by incubation of pyreneI-Tm at pH greater than 7.5. The pH dependence of the rate of cleavage indicated that hydrolysis rather than aminolysis was the more likely reaction. PyreneI-Tm exhibited a loss in helix content and end-to-end polymerization compared with unlabeled Tm, which increased upon formation of pyreneII-Tm. The cleavage resulted in increased interchain excited state excimer fluorescence originating from pyrene-pyrene interaction between the chains. Thus, increased pyrene-pyrene interaction at Cys 190 leads to an increase in unfolding, the effects of which appear to be transmitted to the ends of tropomyosin. The fluorescence properties of the two types of pyrene-succinimide adducts of dithiothreitol were very similar to the corresponding adducts of pyrene-Tm indicating excimer formation through ground state pyrene-pyrene interaction.     

Stopped flow kinetics of pyrene transfer between human high density lipoproteins.

The transfer of pyrene between high density lipoproteins was studied as a model of lipid exchange. When high density lipoprotein containing pyrene was mixed with unlabeled lipoprotein, pyrene excimer fluorescence decreased with a half-time of approximately 3 ms. The rate of pyrene transfer was invariant over a 100-fold range of unlabeled lipoprotein concentrations. Since a decrease in excimer fluorescence indicates a decrease in the microscopic concentrations of pyrene, the observed fluorescence change relfects pyrene transfer to unlabeled lipoproteins, and, therefore, dilution of the pyrene molecules. When high density lipoprotein labeled with pyrene was rapidly diluted 1:14 into buffer, a small decrease in excimer fluorescence was observed. The half-time of this fluorescence change was also about 3 ms and represents the half-time for the dissociation of pyrene from high density lipoprotein into water. The latter observation, coupled with the invariant exchange rate with lipoprotein concentration suggests strongly that the limiting step in the transfer of pyrene between high density lipoproteins is the dissociation of pyrene into solvent. Finally, regardless of mechanism, the exchange of pyrene, and presumably other hydrophobic aromatic compounds, among serum high density lipoproteins is extremely fast. This result indicates that these types of compounds can be rapidly assimilated and transported through the body by plasma lipoproteins.     

Excimer fluorescence of pyrene-tropomyosin adducts

Studies of the fluorescence of N-(1-pyrene)maleimide and N-(1-pyrenyl)iodoacetamide with actin from rabbit skeletal muscle tropomyosin revealed the presence of excimer fluorescence characterized by a broad emission band at 480 nm with a shoulder at 505 nm. Monomer fluorescence decay exhibited different lifetimes, viz., about 3, 22 and 87 ns for the pyrenemaleimide adduct; about 2.5, 11 and 51 ns for the aminolyzed maleimide adduct: and about 2.5, 15 and 74 ns for the pyrenyliodoacetamide adduct. Almost identical excimer fluorescence lifetimes were found for all adducts; about 9, 35 and 65 ns. Excimer fluorescence was sensitive to changes in ionic strength and pH of the medium while monomer fluorescence did not change. The protein denaturants guanidine hydrochloride and urea caused dissociation of the two tropomyosin subunits and partial disappearance of excimer fluorescence, but not as effectively as the hydrophobic surfactant sodium dodecyl sulfate. The sensitivity of excimer fluorescence to changes in the micro-environment make these pyrene derivatives very useful probes for studying conformational changes and binding interaction of tropomyosin with other contractile proteins. The unique location of the excimer probe at tropomyosin Cys-190 and its characteristic long lifetimes could make it useful in time-resolved anisotropy studies and fluorescence energy-transfer experiments.     

Interaction of glucagon with dimyristoyl glycerophosphocholine

Glucagon can form amphipathic helices and can interact with dimyristoyl glycerophosphocholine at temperatures below the phase transition leading to a shift in the fluorescence emission maximum of tryptophan from 350 to 338 nm and a 3-fold enhancement of fluorescence intensity as well as a change in the polarization of fluorescence. The circular dichroism properties of the lipid-associated glucagon indicates that it has an increased content of alpha-helix. The phase transition temperature of the lipid as monitored by pyrene excimer fluorescence is not altered by interaction with glucagon although at higher glucagon/lipid ratios a decrease in excimer formation is noted at low temperature. Above the phase transition temperature, the addition of lipid has no effect on the fluorescence emission or circular dichroism of glucagon. Thus this hormone can interact with dimyristoyl glycerophosphocholine and this interaction is stronger below the phase transition temperature than above it.     

Pyrene excimer fluorescence: a spatially sensitive probe to monitor lipid-induced helical rearrangement of apolipophorin III

Manduca sexta apolipophorin III (apoLp-III), an 18-kDa, monomeric, insect hemolymph apolipoprotein, is comprised of five amphipathic alpha-helices arranged as a globular bundle in the lipid-free state. Upon lipid binding, it is postulated that the bundle opens, exposing a continuous hydrophobic surface which becomes available for lipid interaction. To investigate lipid binding-induced helical rearrangements, we exploited the unique fluorescence characteristics of N-(1-pyrene)maleimide. Pyrene is a spatially sensitive extrinsic fluorescent probe, which forms excited-state dimers (excimers) upon close encounter with another pyrene molecule. Cysteine residues were introduced into apoLp-III (which otherwise lacks cysteine) at Asn 40 (helix 2) and/or Leu 90 (helix 3), creating two single-cysteine mutants (N40C-apoLp-III and L90C-apoLp-III) and N40C/L90C-apoLp-III, a double-cysteine mutant, which were labeled with pyrene maleimide. Pyrene-labeled N40C/L90C-apoLp-III, but not the pyrene-labeled single-cysteine mutants, exhibited strong excimer fluorescence in the lipid-free, monomeric state. Guanidine hydrochloride titration and temperature studies revealed a loss in excimer fluorescence, accompanied by a loss in the molar ellipticity of the protein. When apoLp-III interacts with phospholipid vesicles to form disklike complexes, a significant loss in excimer fluorescence was noted, indicating that the helices bearing the pyrene moieties diverge from each other. Pyrene excimer fluorescence was further employed to examine the relative orientation of lipid-bound apoLp-III molecules. Pyrene-labeled N40C- or L90C-apoLp-III displayed no excimer fluorescence in the disk complexes, while complexes prepared with an equal mixture of both single-labeled mutants did emit excimer fluorescence, indicating apoLp-III adopts a preferred nonrandom orientation around the perimeter of the bilayer disk. These studies establish pyrene excimer fluorescence as a useful spectroscopic tool to address intra- and intermolecular interactions of exchangeable apolipoproteins upon binding to lipid.     

Pyrene-labeled cardiac troponin C. Effect of Ca2+ on monomer and excimer fluorescence in solution and in myofibrils.

The two cysteine residues (Cys-35 and Cys-84) of bovine cardiac troponin C (cTnC) were labeled with the pyrene-containing SH-reactive compounds, N-(1-pyrene) maleimide, and N-(1-pyrene)iodoacetamide in order to study conformational changes in the regulatory domain of cTnC associated with cation binding and cross-bridge attachment. The labeled cTnC exhibits the characteristic fluorescence spectrum of pyrene with two sharp monomer fluorescence peaks and one broad excimer fluorescence peak. The excimer fluorescence results from dimerization of adjacent pyrene groups. With metal binding (Mg2+ or Ca2+) to the high affinity sites of cTnC (sites III and IV), there is a small decrease in monomer fluorescence but no effect on excimer fluorescence. In contrast, Ca2+ binding to the low affinity regulatory (site II) site elicits an increase in monomer fluorescence and a reduction in excimer fluorescence. These results can be accounted for by assuming that the pyrene attached to Cys-84 is drawn into a hydrophobic pocket formed by the binding of Ca2+ to site II. When the labeled cTnC is incorporated into the troponin complex or substituted into cardiac myofibrils the monomer fluorescence is enhanced while the excimer fluorescence is reduced. This suggests that the association with other regulatory components in the thin filament might influence the proximity (or mobility) of the two pyrene groups in a way similar to that of Ca2+ binding. With the binding of Ca2+ to site II the excimer fluorescence is further reduced while the monomer fluorescence is not changed significantly. In myofibrils, cross-bridge detachment (5 mM MgATP, pCa 8.0) causes a reduction in monomer fluorescence but has no effect on excimer fluorescence. However, saturation of the cTnC with Ca2+ reduces excimer fluorescence but causes no further change in monomer fluorescence. Thus, the pyrene fluorescence spectra define the different conformations of cTnC associated with weak-binding, cycling, and rigor cross-bridges.     

Role of histone pairs H2A,H2B and H3,H4 in the self-assembly of nucleosome core particles

The role of the histone pairs H2A,H2B and H3,H4 in the kinetics of core particle formation was investigated by using N-(1-pyrene)maleimide-labeled histone H3. The excimer emission intensity of a DNA-core histone complex prepared by direct mixing of DNA and histones in 0.2 m-NaCl is reduced by half when H2A,H2B is omitted. Fluorescence quenching studies and lifetime measurements indicate that the emission differences are probably due to static quenching. In a correctly folded nucleosome or a DNA-(H3,H4) complex, the two pyrene rings are buried and are held very close. DNA-(H3,H4) can interact with additional copies of H3,H4, but only when two dimers of H2A,H2B are correctly bound is there a specific twofold increase in excimer emission.The kinetics of the reaction of H3,H4 with DNA in 0.2 m-NaCl were followed by measuring the increase in 460 nm fluorescence. The apparent rate constant of the dominant kinetic component is ~ 2 × 10^?1 s^?1. If histones H2A,H2B are added immediately after the preparation of the DNA-(H3,H4) complex, an increase in excimer fluorescence is observed, with an apparent rate constant of ~ 6 × 10^?3 s^?1. However, if histones H2A,H2B are added one hour after DNA-(H3,H4) complex formation, there is no increase in excimer fluorescence. These results suggest that an intermediate involving the H3,H4 tetramer is formed first in nucleosome assembly. In the presence of H2A,H2B, this intermediate evolves to the final folded nucleosome, but in the absence of H2A,H2B it rearranges to an unmaturable dead-end complex. Additional experiments show that a very fast transfer of histone pairs (probably H2A,H2B) can take place between partially reconstituted nucleosomes.     

Pyrene phospholipid as a biological fluorescent probe for studying fusion of virus membrane with liposomes

We are using fluorescent endogenous phospholipids in virus membranes to study the factors that promote fusion on interaction with receptor membranes. To this end, vesicular stomatitis virus (VSV) grown in baby hamster kidney (BHK-21) cells was biologically labeled with fluorescent lipids, primarily phosphatidylcholine and phosphatidylethanolamine, derived from pyrene fatty acids. The pyrene lipids present in the virions showed a fluorescence spectrum typical of pyrene with an intense monomer and a broad excimer. Interaction of pyrene lipid labeled VSV with serum lipoproteins led to a spontaneous fast transfer of the small amount of pyrene fatty acids present in the envelope (t1/2 less than or equal to 7 min), followed by a considerably slower transfer of pyrene phospholipids from the membrane of the virions (t1/2 greater than or equal to 12 h). Incubation of pyrene phospholipid labeled VSV with phosphatidylserine small unilamellar vesicles resulted in fusion at low pH (pH 5.0) as measured by the change in the excimer/monomer fluorescence intensity ratio. Fusion kinetics was rapid, reaching a plateau after 4 min at pH 5.0 and 37 degrees C. Only negligible fusion was noted at neutral pH or at 4 degrees C. Fully infectious virions labeled biologically with fluorescent lipids provide a useful tool for studying mechanisms of cell-virus interactions and neutralization of viral infectivity by specific monoclonal antibodies reactive with viral membrane glycoprotein.     

Fluorescent-labeled crosslinks in collagen: Pyrenebutyrylhydrazine

The aldehydes present in acid-soluble type I collagen react with pyrenebutyrylhydrazine to form various types of complexes under different reaction conditions. These complexes exhibit one or more of three different pyrene fluorescence bands: monomer, excimer, and aggregate fluorescence. Collagen, whose aldehydes have been reduced with NaBH₄, does not react with this fluorescent hydrazine, confirming that the hydrazine reacts specifically with aldehyde groups to form hydrazones. The absence of a reaction with pepsin-treated collagen also shows that the fluorescent labels are primarily in the nonhelical terminal telopeptides. Upon dialysis, the pyrene label bound to a saturated aldehyde in an α-chain is lost; whereas that bound to an unsaturated aldehyde remains on the protein. The pyrene monomer fluorescence in the β-chain of old collagen is stronger than that of young collagen. The formation of the pyrene excimer fluorescence implies the proximity of two pyrene molecules, probably attached to two adjacent aldehydes. Upon changing from acidic to neutral pH, both excimer and aggregate fluorescence bands disappear within a few seconds, revealing a very rapid alteration at the telopeptides.     

Conformational biosensor for diagnosis of prion diseases

A fluorescence technology to monitor the proliferation of amyloidogenic neurological disorders is proposed. A crude brain homogenate (0.01%) from animals infected with a transmissible spongiform encephalopathy is employed as a catalytic medium initiating conformational changes in 520 nM polypeptide biosensors (Tris/trifluoroethanol 50% mixture at pH 7). The fluorescence methods utilize pyrene residues covalently attached to the peptide ends. The coil-to-β-strand transitions in biosensor molecules cause elevation of a distinct fluorescence band of the pyrene aggregates (i.e. excimers). This approach enables the detection of infectious prion proteins at fmol, does not require antibody binding or protease treatment. Technology might be adopted for diagnosing a large variety of conformational disorders as well as for generic high-throughput screening of the amyloidogenic potential in plasma.     

Rapid segmental and subdomain motions of DNA polymerase beta

DNA polymerase (pol) beta is a two-domain DNA repair enzyme that undergoes structural transitions upon binding substrates. Crystallographic structures indicate that these transitions include movement of the amino-terminal 8-kDa lyase domain relative to the 31-kDa polymerase domain. Additionally, a polymerase subdomain moves toward the nucleotide-binding pocket after nucleotide binding, resulting in critical contacts between alpha-helix N and the nascent base pair. Kinetic and structural characterization of pol beta has suggested that these conformational changes participate in stabilizing the ternary enzyme-substrate complex facilitating chemistry. To probe the microenvironment and dynamics of both the lyase domain and alpha-helix N in the polymerase domain, the single native tryptophan (Trp-325) of wild-type enzyme was replaced with alanine, and tryptophan was strategically substituted for residues in the lyase domain (F25W/W325A) or near the end of alpha-helix N (L287W/W325A). Influences of substrate on the fluorescence anisotropy decay of these single tryptophan forms of pol beta were determined. The results revealed that the segmental motion of alpha-helix N was rapid ( approximately 1 ns) and far more rapid than the step that limits chemistry. Binding of Mg(2+) and/or gapped DNA did not cause a noticeable change in the rotational correlation time or angular amplitude of tryptophan in alpha-helix N. More important, binding of a correct nucleotide significantly limited the angular range of the nanosecond motion within alpha-helix N. In contrast, the segmental motion of the 8-kDa domain was "frozen" upon DNA binding alone, and this restriction did not increase further upon nucleotide binding. The dynamics of alpha-helix N are discussed from the perspective of the "open" to "closed" conformational change of pol beta deduced from crystallography, and the results are more generally discussed in the context of reaction cycle-regulated flexibility for proteins acting as molecular motors.     

Comparison of properties on non-protected fluid room temperature phosphorescence of some tetra-ring aromatic hydrocarbons.

A comparative study of the photoluminescence properties of three kinds of tetra-ring aromatic hydrocarbon (1-sodium pyrenesulphonate, benz[alpha]anthracene and chrysene) solution in the absence of any protecting medium is described. It was found that a room temperature phosphorescence signal with different intensities can be induced for these solutions, using only TlNO3 or KI as a heavy atom perturber (HAP) and Na2SO3 as a deoxygenator. An appropriate amount of organic solvent added to the systems of pyrene, benz[alpha]anthracene and chrysene is necessary for increasing the solubility and phosphorescence intensity, and the preferable solvent is acetonitrile. For the pyrene, pyrenesulphonate and chrysene systems, a delayed excimer fluorescence accompanied with the room temperature phosphorescence (RTP) emission can be observed, but that for benz[alpha]anthracene cannot. The ratio of delayed excimer fluorescence and phosphorescence signals for pyrene, pyrenesulphonate and chrysene systems can be controlled by adjusting the concentration of luminophor, kinds and amount of both organic solvents and HAP. Under the optimal conditions, the RTP signals are proportional to the concentration of the four aromatic hydrocarbons, which means that the RTP properties of the four tetra-ring aromatic hydrocarbons can be used for quantitative analysis.