Excimer fluorescence of (+)-anti-benzo (a)pyrene diol epoxide covalently bound to poly(dG-dC): structural implications

Fluorescence of (+)-anti-benzo(a)pyrene diol epoxide [(+)-anti-BPDE] covalently bound to poly(dG-dC) has been studied with steady-state and time-resolved techniques. Extensive formation of excimers is found, even at small (0.008) BPDE/nucleotide ratios. This indicates favored covalent binding to bases close to already modified guanines. Both fluorescence excitation spectra and lifetime measurements reveal two populations of (+)-anti-BPDE adducts: one that can form excimers and one that cannot. Three excimer lifetimes (4.5, 29, and 83 ns) are observed. Differently shifted monomer and excimer excitation spectra are discussed in terms of pyrene-pyrene exciton interactions, consistent with a distance shorter than 7 A between the excimer-forming BPDE chromophores.     

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.     

Pyrene excimer fluorescence in rabbit skeletal alphaalphatropomyosin labeled with N-(1-pyrene)maleimide. A probe of sulfhydryl proximity and local chain separation

Rabbit skeletal alphaalphatropomyosin was specificially labeled at cysteine 190 with the fluorescent reagent, N-(1-pyrene)maleimide. Spectroscopically different products were obtained by labeling at pH 6.0 (PyrI-alphaalphaTm) or pH 7.5 (PyrII-alphaalphaTm). PyrII-alphaalphaTm results from a secondary reaction between the N-(1-pyrene)succinimido moiety at cysteine 190 of PyrI-alphaalphaTm and a lysine group on the same chain, probably lysine 189. Pyrene excimer fluorescence was present in the native state but absent in the unfolded state of both products, thus verifying the proximity of the--SH groups and the chain register model for the structure of tropomyosin. Studies of the guanidinium chloride-dependent unfolding of PyrII-alphaalphaTm showed that loss of excimer fluorescence precedes unfolding, providing evidence for a region of preferential instability in the molecule near cysteine 190. This work suggests that N-(1-pyrene)maleimide could be used to probe both--SH proximity and local conformation in any protein if the presence of two or more proximal--SH groups is suspected.     

Fluorescence studies of the conformation of pyrene-labeled tropomyosin: effects of F-actin and myosin subfragment 1

The fluorescence of pyrene-TM [rabbit skeletal tropomyosin (TM) labeled at Cys with N-(1-pyrenyl)maleimide] consists of monomer and excimer bands [Betcher-Lange, S., & Lehrer, S.S. (1978) J. Biol. Chem. 253, 3757-3760]; an increase in excimer fluorescence with temperature is due to a shift in equilibrium from a chain-closed state (N) to a chain-open state (X) associated with a helix pretransition [Graceffa, P., & Lehrer, S.S. (1980) J. Biol. Chem. 255, 11296-11300]. In this study, we show that the presence of appreciable excimer fluorescence at temperatures below the N----X pretransition (initial excimer) is due to perturbation of the TM chain-chain interaction by the pyrenes at Cys-190. Fluorescence and ATPase titrations indicated that the label caused a decrease in TM binding to F-actin primarily due to reduced end to end TM interactions on the actin filament. Under conditions where pyrene-TM was bound to F-actin, however, the excimer fluorescence did not increase with temperature, indicating that F-actin stabilizes tropomyosin by inhibiting the N----X transition. The binding of myosin subfragment 1 (S1) to pyrene-TM-F-actin at low ratios to actin caused time-dependent changes in fluorescence. After equilibrium was reached, the initial excimer fluorescence was markedly reduced and remained constant over the pretransition temperature range. Further stabilization of tropomyosin conformation on F-actin is therefore associated with S1 binding. Effects of the binding of S1 to the F-actin-tropomyosin thin filament on the state of tropomyosin were studied by monitoring the monomer fluorescence of pyrene-TM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monomer and excimer fluorescence of horse plasma gelsolin labelled with N-(1-pyrenyl)iodoacetamide.

Horse plasma gelsolin was labelled with the sulfhydryl-specific fluorescent reagent N-(1-pyrenyl)iodoacetamide. The level of incorporation of probe was 1.6 +/- 0.3 mol pyrene/mol gelsolin. The circular dichroism spectrum of pyrenyl-gelsolin and its ability to interact with muscle actin were not different from that found for unmodified gelsolin. The emission from pyrenyl-gelsolin was dominated by a broad emission band centred near 483 nm, characteristic of the presence of pyrene excimers. Analysis of excitation spectra for the monomer and excimer-type fluorescence suggested that ground-state interactions may occur between adjacent pyrenes in the gelsolin structure. In the case either of excimer formation or of ground-state pyrene-pyrene interactions in doubly labelled gelsolin molecules, the modified Cys residues must be in close proximity in the folded protein structure. Thermal denaturation of gelsolin could be monitored by observing the decrease in excimer emission that accompanied heating and unfolding of the tertiary structure. While heat treatment alone did not eliminate excimer fluorescence, digestion of gelsolin with chymotrypsin completely abolished such emission. Also, pyrenyl-gelsolin prepared and studied in 6 M guanidine-HCl exhibited fluorescence characteristic of pyrene monomers exclusively.     

Excimer fluorescence of pyrenyliodoacetamide-labeled tropomyosin: a probe of the state of tropomyosin in reconstituted muscle thin filaments

Rabbit skeletal tropomyosin (Tm) specifically labeled at cysteine groups with N-(1-pyrenyl)-iodoacetamide (PIA) exhibits excimer fluorescence. The excimer fluorescence was sensitive to the local conformation of Tm, to actin binding, and, in reconstituted thin filaments, to the Tm state change induced by binding of myosin subfragment 1 (S1). The properties of PIATm were similar to previously studied pyrenylmaleimide-labeled Tm (PMTm) [Ishii, Y., & Lehrer, S.S. (1985) Biochemistry 24, 6631] except that S1 binding to actin-Tm increased the excimer fluorescence in contrast to the time-dependent decrease seen for PMTm. The fluorescence properties of PIATm are sensitive to the Tm chain-chain interaction via equilibria among pyrene configurations and nonfluorescent dimer as well as the monomer and excimer-forming configurations. The effect of bound troponin (Tn) on the excimer fluorescence of PIATm in the reconstituted systems was dependent on ionic strength with a slight Ca2+ dependence. S1 titrations in the absence and presence of Tn and Ca2+ indicated that the excimer fluorescence probes the state change of Tm from the weak S1 binding state to the strong S1 binding state which is facilitated by Ca2+ [Hill et al. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 3186]. Binding of MgADP-S1 and MgAMPPNP-S1 produced the same total excimer fluorescence change as for nucleotide-free S1, showing that the strong S1 binding state of Tm-actin is independent of nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization of excimer-forming two-probe nucleic acid hybridization method with pyrene as a fluorophore.

A previously presented homogeneous assay method, named the excimer-forming two-probe nucleic acid hybridization (ETPH) method, is based on specific excimer formation between two pyrenes attached at the neighboring terminals of two sequential probe oligonucleotides complementary to a single target. In this study, we investigated assay conditions and optimal molecular design of probes for intense excimer emission using a pyrenemethyliodoacetamide-introduced 16mer probe, a pyrene butanoic acid-introduced 16merprobe and a target 32mer. The length of the linker between the pyrene residue and the terminal sugar moiety remarkably influenced the quantum efficiency of excimer emission; the pair of linker arms of these two probes was optimal. The quantum efficiency was also dependent upon the concentrations of dimethylformamide and NaCl added to the assay solution. Spectroscopic measurements and T m analysis showed that an optimal configuration of the two pyrene residues for intense excimer emission might be affected by pyrene-pyrene interaction, pyrene-duplex interaction (intercalation/stacking) and solvent conditions as a whole. We then demonstrated the practicality of the ETPH method with the optimal hybridization conditions thus attained by determining that the concentration of 16S rRNA in extracts from Vibrio mimicus ATCC 33655 cells in exponential growth phase is 18 500 16S rRNA molecules/cell on average.     

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.     

Quenching of fluorescence of pyrene-substituted lecithin by tetracyanoquinodimethane in liposomes.

In this work we have applied a kinetic scheme derived from fluorescence kinetics of pyrene-labeled phosphatidylcholine in phosphatidylcholine membrane to explain the fluorescence quenching of 1-palmitoyl-2-(10-[pyrenl-yl]-sn-glycerol-3-phosphatidylchol ine (PPDPC) liposomes by tetracyanoquinodimethane (TCNQ). The scheme was also found to be applicable to neat PPDPC and the effect of the quencher could be attributed to certain steps of the proposed mechanism. The TCNQ molecules influence the fluorescence of pyrene moieties in PPDPC liposome in two ways. Firstly, an interaction between the quencher molecule and the pyrene monomer in the excited state quenches monomer fluorescence and effectively prevents the diffusional formation of the excimer. Secondly, an interaction between the quencher molecule and the excited dimer quenches the excimer fluorescence. The TCNQ molecule does not prevent the formation of the excimer in pyrene moieties aggregated in such a way that they require only a small rotational motion to attain excimer configuration. The diffusional quenching rate constant is calculated to be 1.0 x 10(8) M-1 s-1 for the pyrene monomer quenching and 1.3 x 10(7) M-1 s-1 for the pyrene excimer quenching. The diffusion constant of TCNQ is 1.5 x 10(-7) cm2 s-1 for the interaction radii of 0.8-0.9 nm. The TCNQ molecules are practically totally partitioned in the membrane phase.     

The reaction of N-(1-pyrene)maleimide with sarcoplasmic reticulum.

The excimer fluorescence of the adduct of N-(1-pyrene)maleimide (PMI) with the Ca2+-ATPase was proposed as a probe of ATPase-ATPase interactions in sarcoplasmic reticulum (Lüdi and Hasselbach, Eur. J. Biochem., 1983, 130:5-8). We tested this proposition by analyzing the spectral properties and stoichiometry of the adducts of pyrenemaleimide with sarcoplasmic reticulum and with dithiothreitol and by comparing the effects of various detergents on the excimer fluorescence of the two adducts, with their influence on the sedimentation characteristics, ATPase activity, and light scattering of the pyrenemaleimide-labeled sarcoplasmic reticulum. These studies indicate that pyrenemaleimide reacts nearly randomly with several SH groups on the Ca2+-ATPase, and suggest that the observed excimer fluorescence of pyrenemaleimide-labeled sarcoplasmic reticulum may reflect intramolecular phenomena rather than ATPase-ATPase interactions. Further work is required to establish the relative contribution of intra- and intermolecular mechanisms to the excimer fluorescence.     

A comparison study of the interaction between β-lactoglobulin and retinol at two different conditions: spectroscopic and molecular modeling approaches

The interaction between bovin β-Lactoglobulin (β-LG) and retinol at two different pH values was investigated by multispectroscopic, zeta potential, molecular modeling, and conductometry measurements. The steady state and polarization fluorescence spectroscopy revealed that complex formation at two different pH values could occur through a remarkable static quenching. According to fluorescence quenching, one set of binding site at pH 2 and two sets of binding sites at pH 7 were introduced for binding of retinol to β-LG that show the enhancement of saturation score of β-LG to retinol in dimmer condition. The polarization fluorescence analysis represented that there is more affinity between β-LG and retinol at pH 7 rather than at pH 2. The effect of retinol on β-LG was studied by UV-visible, circular dichroism (CD), and synchronous fluorescence, which indicated that retinol induced more structural changes on β-LG at pH 7. β-LG–retinol complex formation at two different pH values was recorded via applying resonance light scattering (RLS) and zeta potential. Conductometry and RLS showed two different behaviors of interaction between β-LG and retinol at two different pH values; therefore, dimmer formation played important roles in different behaviors of interaction between β-LG and retinol. The zeta potential was the implied combination of electrostatic and hydrophobic forces which are involved in β-LG–retinol complex at two different pH values, and the hydrophobic interactions play a dominant role in complex formation. Molecular modeling was approved by all experimental results. The acquired results suggested that monomer and dimmer states of β-LG can be induced by retinol with different behaviors.     

Quantitative fluorescence analysis of the adsorption of lysozyme to phospholipid vesicles

Experiments directed to measure the interaction of lysozyme with liposomes consisting of phosphatidylcholine (PC) and phosphatidylserine (PS) have been conducted by monitoring both protein and lipid fluorescence and fluorescence anisotropy of the protein. The binding of lysozyme to the unilamellar vesicles was quantified using a novel method of analysis in which the fractional contribution at moderate binding conditions is determined from either total fluorescence decay or anisotropy decay curves of tryptophan at limiting binding conditions. In the energy transfer experiments PC and PS lipids labelled with two pyrene acyl chains served as energy acceptors of the excited tryptophan residues in lysozyme. The binding was strongly dependent on the molar fraction of negatively charged PS in neutral PC membranes and on the ionic strength. Changes in the tryptophan fluorescence decay characteristics were found to be connected with long correlation times, indicating conformational rearrangements induced by binding of the protein to these lipid membranes. The dynamics of membrane bound protein appeared to be dependent on the physical state of the membrane. Independent of protein fluorescence studies, formation of a protein-membrane complex can also be observed from the lipid properties of the system. The interaction of lysozyme with di-pyrenyl-labelled phosphatidylserine in anionic PS/PC membranes resulted in a substantial decrease of the intramolecular excimer formation, while the excimer formation of dipyrenyl-labelled phosphatidylcholine in neutral PC membranes barely changed in the presence of lysozyme.Abbreviations dipyr₄ sn-1,2-(pyrenylbutyl) - dipyr₁₀ sn-1,2-(pyrenyldecanoyl). - DMPC dimyristoyl-phosphatidylcholine - DOPC dioleoyl-phosphatidylcholine - DPPC dipalmitoyl-phosphatidylcholine - DPPC dipalmitoylphosphatidylcholine - PC phosphatidylcholine - PS phosphatidylserine Correspondence to: A. J. W. G. Visser     

Dynamics of benzo[a]pyrene diol epoxide adducts in poly(dG-dC).(dG-dC) studied by synchrotron excited fluorescence polarization anisotropy decay.

Time-resolved fluorescence studies have been performed on (+)-anti-7,8-dihydrodiol-9,10-epoxybenzo[a]pyrene adducts in double-stranded poly(dG-dC).(dG-dC). Part of the adduct population gives rise to excimer fluorescence. The heterogeneous fluorescence emission decay curves at 22 degrees C could be resolved into three components with lifetimes: 0.4 ns, 3 ns and 24 ns for the total fluorescence (monomer and excimer emission), and 0.5 ns, 5 ns and 24 ns, respectively, for excimer emission alone. The relative amplitudes for the longer lifetimes were larger for the pure excimer population than for the mixed population. The fluorescence polarization anisotropy decay curves were resolved into two components of rotational correlation times: 0.4 ns and 25 ns for the total fluorescence and 0.3 ns and 33 ns for the excimer fluorescence. We interpret the two rotational correlation times to correspond to local motion of the adduct and segmental motion of the polynucleotide, respectively.     

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.     

Coupled responses of the regions near cysteine-190 and the carboxy terminus of rabbit cardiac tropomyosin: fluorescence and circular dichroism studies

Rabbit cardiac tropomyosin was labeled at Cys-190 with either N-(1-pyrenyl)iodoacetamide (Py) or 6-acryloyl-2-(dimethylamino)naphthalene (AD, acrylodan). Half of the labeled sample then was treated with carboxypeptidase A to produce an identically labeled nonpolymerizable form of tropomyosin, NPTM. Investigation of temperature-dependent changes in pyrene excimer emission, acrylodan fluorescence polarization, and tyrosyl circular dichroism in different samples of tropomyosin and NPTM reveals that absence of the COOH-terminal portion of tropomyosin modifies the response of the Cys-190 region to heat. Removal of the COOH terminus releases certain conformational constraints from the coiled-coil back to and including the Cys-190 region without causing a severe drop in the net alpha-helical content of the protein. Observation of changes in pyrene excimer fluorescence and in fluorescence polarization of acrylodan with time after addition of carboxypeptidase A to samples of labeled tropomyosin directly demonstrates this relaxation process. Thermally induced reduction in tyrosyl circular dichroism, together with consideration of the distribution of tyrosyl residues on tropomyosin, also supports the proposal.     

Proximity of sulfhydryl groups in lens proteins. Excimer fluorescence of pyrene-labeled crystallins

Lens proteins labeled with the -SH-specific reagents N-(1-pyrene)-maleimide (PM) and N-(1-pyrene)-iodo-acetamide (PIA) exhibited pyrene excimer fluorescence around 480 nm. Among the gamma-fractions, only gamma II showed excimer band at room temperature with both probes PM and PIA. As the temperature increased, PM-labeled gamma IIIA, gamma IIIB, and gamma IV also began to exhibit excimer around 55 degrees C, which did not disappear at a very high temperature (85 degrees C). With PIA, gamma IIIA and gamma IVA did not show excimer at any temperature. The beta-crystallins, on the other hand, revealed a very strong excimer/monomer intensity ratio at room temperature, which decreased with an increase in temperature. Life-time measurements indicated a difference in the micro-environments around the labeled -SH residues. The origin of the excimer band as well as temperature effects on this band have been explained on the basis of intra- and inter-molecular interaction among the Cys residues in the lens proteins. The temperature-dependent CD studies further indicated retention of thermodynamic stability of the crystallins after chemical modifications. Both PM and PIA could be used conveniently to probe -SH proximity, determine the ease and extent of disulfide formation, and monitor the dynamics of lens protein conformation, all of which are critically important with regard to cataract formation.     

Lipid-triggered conformational switch of apolipophorin III helix bundle to an extended helix organization

Apolipophorin III (ApoLp-III) from the Sphinx moth, Manduca sexta, is an 18kDa protein that binds reversibly to hydrophobic surfaces generated on metabolizing lipoprotein particles. It is comprised of amphipathic alpha-helices (H1-H5) organized in an up-and-down topology forming a helix bundle in the lipid-free state. Upon interaction with lipids, apoLp-III has been proposed to undergo a dramatic conformational change, involving helix bundle opening about putative hinge loops such that H1, H2 and H5 move away from H3 and H4. In the present study, we examine the relative spatial disposition of H1 and H5 on discoidal phospholipid complexes and spherical lipoproteins. Cysteine residues were engineered at position 8 in H1 and/or at position 138 in H5 in apoLp-III (which otherwise lacks Cys) yielding A8C-, A138C- and A8C/A138C-apoLp-III. Tethering of H1 and H5 by a disulfide bond between A8C and A138C abolished the ability of apoLp-III to transform phospholipid vesicles to discoidal particles, or to interact with lipoproteins, demonstrating that these helices are required to reposition during lipid interaction. Site-specific labeling of A8C/A138C-apoLp-III with N-(1-pyrene)maleimide in the lipid-free state resulted in intramolecular pyrene "excimer" fluorescence emission indicative of spatial proximity between these sites. Upon association with dimyristoylphosphatidylcholine (DMPC) discoidal complexes, the intramolecular excimer was replaced by intermolecular excimer fluorescence due to proximity between pyrene moieties on A8C and A138C in neighboring apoLp-III molecules on the discoidal particle. No excimer emission was observed in the case of pyrene-A8C-apoLp-III/DMPC or pyrene-A138C-apoLp-III/DMPC complexes. However, equimolar mixing of the two labeled single-cysteine mutants prior to disc formation resulted in excimer emission. In addition, intramolecular pyrene excimer formation was diminished upon binding of pyrene-A8C/A138C-apoLp-III to spherical lipoproteins. The data are consistent with repositioning of H1 away from H5 upon encountering a lipid surface, resulting in an extended conformation of apoLp-III that circumscribes the discoidal bilayer particle.     

Fluorescence decay of 1-methylpyrene in small unilamellar l-alpha-dimyristoylphosphatidylcholine vesicles. A temperature and concentration dependence study

The fluorescence decay kinetics of 1-methylpyrene in small unilamellar l-alpha-dimyristoylphosphatidylcholine vesicles above the phase transition temperature has been studied as a function of concentration and temperature. When the 1-methylpyrene/phospholipid ratio equals 1:2000 no excimer is observed and the fluorescence decay is monoexponential. When this ratio is equal to or higher than 1 200, excimer is observed and the monomer and excimer decays can be adequately described by two exponential terms. The deviation of the monomer decays from monoexponentiality cannot be described by a model where the diffusion-controlled excimer formation is time dependent. The observed decays are compatible with the excimer formation scheme which is valid in an isotropic medium. The activation energy of excimer formation is found to be 29-9 +/-1.4 kJ mol . The (apparent) excimer formation constant and the excimer lifetime at different temperatures have been determined. The diffusion coefficient associated with the excimer formation process varies between 2 x 10(-10) m(2)/s at 70 degrees C to 4 x 10(-11) m(2)/s at 25 degrees C.     

The effects of polar and/or ionizable residues in the core and flanking regions of hydrophobic helices on transmembrane conformation and oligomerization

To explore the influence of amino acid composition on the behavior of membrane-inserted alpha-helices, we examined the behavior of Lys-flanked polyleucyl (pLeu) helices containing a single polar/ionizable residue within their hydrophobic core. To evaluate the location of the helices within the membrane by fluorescence, each contained a Trp residue at the center of the sequence. When incorporated into dioleoylphosphatidylcholine (DOPC) model membrane vesicles, pLeu helices with or without a single Ser, Asn, Lys, or Asp residue in the hydrophobic core maintained a transmembrane state (named the N state) at neutral and acidic pH. In this state, the central Trp exhibited highly blue-shifted fluorescence, and fluorescence quenching by nitroxide-labeled lipids showed it located at the bilayer center. A state in which Trp fluorescence red-shifted by several nanometers (named the B state) was observed above pH 10-11. B state formation appears to result from deprotonation of the flanking Lys residues. Despite the red shift in Trp emission, fluorescence quenching showed that in the B state the Trp at most is only slightly shallower than in the N state, suggesting the B state also is a transmembrane or near-transmembrane structure. The B state is characterized by increased helix oligomerization, as shown by the dependence of Trp lambda(max) on the concentration of the peptide within the bilayer at high pH. The pLeu peptide with a Asp residue in the core underwent a pH-dependent transition at a lower pH than the other peptides (pH 8-9). At high pH, it exhibited both a more highly red-shifted fluorescence and shallower Trp location than the other peptides. This state (named the S state) did not exhibit a concentration-dependent Trp lambda(max). We attribute S state behavior to the formation of a charged Asp residue at high pH, and a consequent movement of the Asp toward the membrane surface, resulting in the formation of a nontransmembrane state. We conclude that a polar or ionizable residue can readily be tolerated in a single transmembrane helix, but that the charges on ionizable residues in the core and regions flanking the helix significantly modulate the stability of transmembrane insertion and/or helix-helix association.     

Role of caldesmon in the Ca2+ regulation of smooth muscle thin filaments: evidence for a cooperative switching mechanism

Smooth muscle thin filaments are made up of actin, tropomyosin, caldesmon, and a Ca(2+)-binding protein and their interaction with myosin is Ca(2+)-regulated. We suggested that Ca(2+) regulation by caldesmon and Ca(2+)-calmodulin is achieved by controlling the state of thin filament through a cooperative-allosteric mechanism homologous to troponin-tropomyosin in striated muscles. In the present work, we have tested this hypothesis. We monitored directly the thin filament transition between the ON and OFF state using the excimer fluorescence of pyrene iodoacetamide (PIA)-labeled smooth muscle alphaalpha-tropomyosin homodimers. In steady state fluorescence measurements, myosin subfragment 1 (S1) cooperatively switches the thin filaments to the ON state, and this is exhibited as an increase in the excimer fluorescence. In contrast, caldesmon decreases the excimer fluorescence, indicating a switch of the thin filament to the OFF state. Addition of Ca(2+)-calmodulin increases the excimer fluorescence, indicating a switch of the thin filament to the ON state. The excimer fluorescence was also used to monitor the kinetics of the ON-OFF transition in a stopped-flow apparatus. When ATP induces S1 dissociation from actin-PIA-tropomyosin, the transition to the OFF state is delayed until all S1 molecules are dissociated actin. In contrast, caldesmon switches the thin filament to the OFF state in a cooperative way, and no lag is displayed in the time course of the caldesmon-induced fluorescence decrease. We have also studied caldesmon and Ca(2+)-calmodulin-caldesmon binding to actin-tropomyosin in the ON and OFF states. The results are used to discuss both caldesmon inhibition and Ca(2+)-calmodulin-caldesmon activation of actin-tropomyosin.