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.     

Fluorescence properties of acrylodan-labeled tropomyosin and tropomyosin-actin: evidence for myosin subfragment 1 induced changes in geometry between tropomyosin and actin

The Cys groups of rabbit skeletal tropomyosin (Tm) and rabbit skeletal alpha alpha Tm were specifically labeled with acrylodan (AC). The probe on Tm is quite immobile yet exposed to solvent as indicated by its limiting polarization (P0 = 0.38) and fluorescence emission spectrum (lambda max = 520 nm) and its accessibility to solute quenching. Changes in the shape of the excitation spectrum with temperature correlated with the helix thermal pretransition and main transition without much spectral change of the emission spectrum. The probe environment of ACTm did not significantly change on binding to F-actin, but fluorescence energy transfer between tryptophan in actin and AC on Tm was indicated by a 15-20% increase in AC fluorescence and a few percent decrease in tryptophan fluorescence. This energy transfer increased when myosin subfragment 1 (S1) was bound to the ACTm-actin filament, in quantitative agreement with the postulated shift in state of Tm associated with the cooperative binding of S1 to actin (Hill et al., 1980). The increase in energy transfer shows that there is a change in the spatial relationship between Tm and actin associated with the S1-induced change in state of Tm.     

Labeling of a thiol residue in sarcoplasmic reticulum ATPase by pyrene maleimide. Solvent accessibility studied by fluorescence quenching

Sarcoplasmic reticulum ATPase was specifically labeled by the fluorescent probe N-(1-pyrene)maleimide which modified 1 mol of a highly reactive thiol residue per mol of ATPase under appropriate conditions, when the probe concentration was varied in the range 0.1-1.5 microM. Addition of inorganic phosphate to the labeling medium increased both the rate of labeling and the number of modified thiol residues. Addition of ATP gave a marked kinetic protection from labeling, suggesting that the label was attached to a protein domain which is sensitive to changes at the catalytic site. Quenching of pyrene fluorescence emission of labeled ATPase by acrylamide and cesium chloride gave linear Stern-Volmer plots. The Stern-Volmer quenching constants of pyrene-ATPase fluorescence were 10 times lower than the constant obtained for acrylamide quenching of the fluorescent adduct of pyrene-maleimide-cystein used as a control, indicating that the pyrene moiety of the probe was considerably shielded from the medium solvent when covalently attached to the ATPase. The efficiency of quenching of pyrene-ATPase fluorescence increased by a significant amount upon addition of 100 microM Ca2+, when compared to the quenching in the presence of a Ca2+ chelator. It suggests that occupancy of the high affinity Ca2+ sites of the ATPase increases the accessibility of medium solvent into hydrophobic domains of the enzyme. The fluorescence lifetime of the solubilized pyrene-ATPase emission was 144-149 ns. The fluorescence polarization of pyrene-ATPase solubilized by nonionic detergent C12E8 was rho = 0.10 and it increased with an increase in the viscosity of the medium yielding a linear Perrin plot. The rotational correlation time for the soluble ATPase was 532 ns, corresponding to the overall rotation of a detergent-pyrene-ATPase particle with radius of 87A.     

Fluorescence studies of the carboxyl-terminal domain of smooth muscle calponin effects of F-actin and salts.

The fluorescence parameters of the environment-sensitive acrylodan, selectively attached to Cys273 in the C-terminal domain of smooth muscle calponin, were studied in the presence of F-actin and using varying salt concentrations. The formation of the F-actin acrylodan labeled calponin complex at 75 mm NaCl resulted in a 21-nm blue shift of the maximum emission wavelength from 496 nm to 474 nm and a twofold increase of the fluorescent quantum yield at 460 nm. These spectral changes were observed at the low ionic strengths (< 110 mm) where the calponin : F-actin stoichiometry is 1 : 1 as well as at the high ionic strengths (> 110 mm) where the binding stoichiometry is a 1 : 2 ratio of calponin : actin monomers. On the basis of previous three-dimensional reconstruction and chemical crosslinking of the F-actin-calponin complex, the actin effect is shown to derive from the low ionic strength interaction of calponin with the bottom of subdomain-1 of an upper actin monomer in F-actin and not from its further association with the subdomain-1 of the adjacent lower monomer which occurs at the high ionic strength. Remarkably, the F-actin-dependent fluorescence change of acrylodan is qualitatively but not quantitatively similar to that earlier reported for the complexes of calponin and Ca2+-calmodulin or Ca2+-caltropin. As the three calponin ligands bind to the same segment of the protein, encompassing residues 145-182, the acrylodan can be considered as a sensitive probe of the functioning of this critical region. A distance of 29 A was measured by fluorescence resonance energy transfer between Cys273 of calponin and Cys374 of actin in the 1 : 1 F-actin-calponin complex suggesting that the F-actin effect was allosteric reflecting a global conformational change in the C-terminal domain of calponin.     

Rate and mechanism of the assembly of tropomyosin with actin filaments

The rate of assembly of tropomyosin with actin filaments was measured by stopped-flow experiments. Binding of tropomyosin to actin filaments was followed by the change of the fluorescence intensity of a (dimethylamino)naphthalene label covalently linked to tropomyosin and by synchrotron radiation X-ray solution scattering. Under the experimental conditions (2 mM MgCl2, 100 mM KCl, pH 7.5, 25 degrees C) and at the protein concentrations used (2.5-24 microM actin, 0.2-3.4 microM tropomyosin) the half-life time of assembly of tropomyosin with actin filaments was found to be less than 1 s. The results were analyzed quantitatively by a model in which tropomyosin initially binds to isolated sites. Further tropomyosin molecules bind contiguously to bound tropomyosin along the actin filaments. Good agreement between the experimental and theoretical time course of assembly was obtained by assuming a fast preequilibrium between free and isolatedly bound tropomyosin.     

Interaction between caldesmon and tropomyosin in the presence and absence of smooth muscle actin

Cysteine residues of caldesmon were labeled with the fluorescent reagent N-(1-pyrenyl)maleimide. The number of sulfhydryl (SH) groups in caldesmon was around 3.5 on the basis of reactivity to 5,5'-dithiobis(2-nitrobenzoate); 80% of the SH groups were labeled with pyrene. The fluorescence spectrum from pyrene-caldesmon showed the presence of excited monomer and dimer (excimer). As the ionic strength increased, excimer fluorescence decreased, disappearing at salt concentrations higher than around 50 mM. The labeling of caldesmon with pyrene did not affect its ability to inhibit actin activation of heavy meromyosin Mg-ATPase and the release of this inhibition in the presence of Ca2+-calmodulin. Tropomyosin induced a change in the fluorescence spectrum of pyrene-caldesmon, indicating a conformational change associated with the interaction between caldesmon and tropomyosin. The affinity of caldesmon to tropomyosin was dependent on ionic strength. The binding constant was 5 x 10(6) M-1 in low salt, and the affinity was 20-fold less at ionic strengths close to physiological conditions. In the presence of actin, the affinity of caldesmon to tropomyosin was increased 5-fold. The addition of tropomyosin also changed the fluorescence spectrum of pyrene-caldesmon bound to actin filaments. The change in the conformation of tropomyosin, caused by the interaction between caldesmon and tropomyosin, was studied with pyrene-labeled tropomyosin. Fluorescence change was evident when unlabeled caldesmon was added to pyrene-tropomyosin bound to actin. These data suggest that the interaction between caldesmon and tropomyosin on the actin filament is associated with conformational changes on these thin filament associated proteins. These conformational changes may modulate the ability of thin filament to interact with myosin heads.     

Differential interaction of cardiac, skeletal muscle, and yeast tropomyosins with fluorescent (pyrene235) yeast actin

To monitor binding of tropomyosin to yeast actin, we mutated S235 to C and labeled the actin with pyrene maleimide at both C235 and the normally reactive C374. Saturating cardiac tropomyosin (cTM) caused about a 20% increase in pyrene fluorescence of the doubly labeled F-actin but no change in WT actin C374 probe fluorescence. Skeletal muscle tropomyosin caused only a 7% fluorescence increase, suggesting differential binding modes for the two tropomyosins. The increased cTM-induced fluorescence was proportional to the extent of tropomyosin binding. Yeast tropomyosin (TPM1) produced less increase in fluorescence than did cTM, whereas that caused by yeast TPM2 was greater than either TPM1 or cTM. Cardiac troponin largely reversed the cTM-induced fluorescence increase, and subsequent addition of calcium resulted in a small fluorescence recovery. An A230Y mutation, which causes a Ca(+2)-dependent hypercontractile response of regulated thin filaments, did not change probe235 fluorescence of actin alone or with tropomyosin +/- troponin. However, addition of calcium resulted in twice the fluorescence recovery observed with WT actin. Our results demonstrate isoform-specific binding of different tropomyosins to actin and suggest allosteric regulation of the tropomyosin/actin interaction across the actin interdomain cleft.     

Excimer fluorescence of equine platelet tropomyosin labeled with N-(1-pyrenyl)iodoacetamide

Tropomyosin from equine platelets was reacted with N-(1-pyrenyl)iodoacetamide, a sulfhydryl-specific fluorescent reagent, to give an average extent of incorporation of 1.12 pyrene (Py) groups per platelet tropomyosin (P-TM) chain. The predominant site of reaction on P-TM was the penultimate COOH-terminal residue, Cys-246. The high proportion of the total emission that is due to pyrene ecximers and the pretransition observed in thermal denaturation of Py-P-TM point to a rather loose structure for the COOH-terminal amino acid residues of P-TM. The label on Cys-246 also reports on end-to-end overlap interactions that occur between two different tropomyosin molecules. Additions to a Py-P-TM solution at low ionic strength of unlabeled P-TM, rabbit cardiac tropomyosin (C-TM), or a carboxypeptidase A treated, nonpolymerizable derivative of C-TM all reduce the extent of excimer fluorescence from the sample. Addition of salt greatly reduces the effects of the unlabeled TM species on the Py-P-TM emission spectrum. Circular dichroism measurements indicate Py-P-TM still to be greater than 95% helical. However, analysis of excimer fluorescence levels in samples that contained a constant protein concentration but different mole ratios of labeled to unlabeled P-TM suggests that the bulky pyrene group may diminish the tendency of Py-P-TM to polymerize in an end-to-end manner.     

Evaluation of the binding of Acanthamoeba profilin to pyrene-labeled actin by fluorescence enhancement

We have used a fluorescence assay to measure the binding of Acanthamoeba profilin to monomeric Acanthamoeba and rabbit skeletal muscle actin labeled on cysteine-374 with pyrene iodoacetamide. The wavelengths of the pyrene excitation and emission maxima are constant at 346 and 386 nm, but the fluorescence is enhanced up to 50% by profilin. The higher fluorescence is largely due to higher absorbance in the presence of profilin. The fluorescence enhancement has a hyperbolic dependence on the concentration of profilin, suggesting a single class of binding sites. Linear Scatchard plots yield an estimate of the dissociation constant, Kd, of the complex of profilin with pyrenyl-actin. In low-ionic-strength buffers with 2 to 6 mM imidazole (pH 7.0) and 0.1 mM CaCl2 the Kd is 9 microM for both muscle and Acanthamoeba actin. In 50 mM KCl the Kd for the complex with Acanthamoeba actin is 16 microM, while the Kd for the complex with muscle actin is greater than 50 microM.     

Linear dichroism of acrylodan-labeled tropomyosin and myosin subfragment 1 bound to actin in myofibrils.

Muscle contraction can be activated by the binding of myosin heads to the thin filament, which appears to result in thin filament structural changes. In vitro studies of reconstituted muscle thin filaments have shown changes in tropomyosin-actin geometry associated with the binding of myosin subfragment 1 to actin. Further information about these structural changes was obtained with fluorescence-detected linear dichroism of tropomyosin, which was labeled at Cys 190 with acrylodan and incorporated into oriented ghost myofibrils. The fluorescence from three sarcomeres of the fibril was collected with the high numerical aperture objective of a microscope and the dichroic ratio, R (0/90 degrees), for excitation parallel/perpendicular to the fibril, was obtained, which gave the average probe dipole polar angle, Theta. For both acrylodan-labeled tropomyosin bound to actin in fibrils and in Mg2+ paracrystals, Theta congruent to 52 degrees +/- 1.0 degrees, allowing for a small degree of orientational disorder. Binding of myosin subfragment 1 to actin in fibrils did not change Theta; i.e., the orientation of the rigidly bound probe on tropomyosin did not change relative to the actin axis. These data indicate that myosin subfragment 1 binding to actin does not appreciably perturb the structure of tropomyosin near the probe and suggest that the geometry changes are such as to maintain the parallel orientation of the tropomyosin and actin axes, a finding consistent with models of muscle regulation. Data are also presented for effects of MgADP on the orientation of labeled myosin subfragment 1 bound to actin in myofibrils.     

19F NMR study of the myosin and tropomyosin binding sites on actin

Actin was labeled with pentafluorophenyl isothiocyanate at Lys-61. The label was sufficiently small not to affect the rate or extent of actin polymerization unlike the much larger fluorescein 5-isothiocyanate which completely inhibits actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. Furthermore, the label resonances in the 376.3-MHz 19F NMR spectrum were unaffected by actin polymerization. However, the binding of the relaxing protein tropomyosin resulted in the fluorinated Lys-61 resonances broadening out beyond detection due to a substantial increase in the effective correlation time of the label. Similarly, the binding of myosin subfragment 1 to F-actin resulted in the dramatic broadening of the labeled Lys-61 resonances. Thus, Lys-61 on actin appears to be closely associated with the binding sites for both tropomyosin and myosin, suggesting that both these proteins can compete for the same site on actin. The other region of actin known to be involved in myosin binding, Cys-10, was found to be more remote from the actin-actin interfaces than Lys-61. Labels on Cys-10 exhibited substantially greater mobility than fluorescein 5-isothiocyanate attached to Lys-61 which appeared to be held down on the surface of the actin monomer. This may sterically hinder the actin-actin interaction about 1 nm from the tropomyosin/myosin binding site.     

Transformation of actin-encapsulating liposomes induced by cytochalasin D.

Liposomes encapsulating actin filaments were prepared by swelling at 0 degrees C lipid film consisting of a mixture of dimyristoyl phosphatidylcholine and cardiolipin (equal amounts by weight) in 100 microM rabbit skeletal muscle actin and 0.5 mM CaCl2 followed by polymerization of actin at 30 degrees C. Liposomes initially assumed either disk or dumbbell shape, but when cytochalasin D was added to the medium surrounding the liposomes, they were found to become spindle shaped. Liposomes containing bovine serum albumin that were given cytochalasin D and actin-containing liposomes that were given dimethylformamide, the solvent for cytochalasin D, did not transform. These results indicated actin-cytochalasin interaction is involved in the transformation process. Falling-ball viscometry and sedimentation analysis of actin solution indicated that cytochalasin cleaved actin filaments and caused depolymerization. The observation of polarized fluorescence of encapsulated actin labeled with acrylodan indicated that the actin filaments in the transformed liposomes aligned along the long axis of the liposomes. Because the actin filaments in the disk- or dumbbell-shaped liposomes formed bundles running along the liposome contour, the transformation was likely to be accompanied by the change in the actin filament arrangement in the liposomes, which was induced by actin-cytochalasin interaction.     

Distribution of fluorescently labeled actin and tropomyosin after microinjection in living tissue culture cells as observed with TV image intensification

Skeletal muscle F-actin and smooth muscle tropomyosin separately labeled with the fluorescent reporter group 5-iodoacetamidofluorescein (5-IAF) were further purified to yield G-actin fully competent to polymerize and tropomyosin able to bind specifically to F-actin. The two fluorescent proteins (dye content of 0.4–0.5 moles/mole of protein) were microinjected into tissue culture cells and their intracellular distribution was followed by TV image intensification. Fluorescent actin is found in the stress fibers and in the lamellopodia and ruffling edges of the cells. In addition a general cytoplasmic fluorescence is observed as well as fluorescent patches, which could be actin paracrystals. In contrast tropomyosin is not incorporated into ruffles although it is clearly seen along the stress fibers and gives rise to general cytoplasmic fluorescence. Control experiments using fluorescent serum albumin show no specific visualization of either stress fibers or ruffles. The specificity of the incorporation of the fluorescently labeled contractile proteins into the microfilament structures is further documented by the preparation of detergent resistant cytoskeletons which retain actin and tropomyosin in the appropriate structures but are devoid of fluorescent serum albumin. In addition the distribution of the contractile proteins in the living cells is affected by the microfilament specific drugs phalloidin and cytochalasin B (CB). The results obtained on live cells are in excellent agreement with conclusions drawn from immunofluorescence microscopical observations on fixed cells. In addition they directly prove the rather obvious point that contractile proteins are constantly rearranged in tissue culture cells.     

Segmental mobility in glycogen phosphorylase b

The dynamics and structuredness of the pyridoxal 5'-phosphate-binding region in glycogen phosphorylase b (EC 2.4.1.1) has been investigated with different techniques of fluorescence spectroscopy. Fluorescence polarization data of the thermal Perrin plot indicate some mobility in the cofactor binding site, while the isothermic measurements (at 20 degrees C, in high-viscosity solvents) demonstrate that the mobile unit carrying the emission oscillator is practically insensitive to the external viscosity. Characteristics of the thermal Perrin plots obtained for both native and reduced phosphorylase b can be interpreted either as a freely moving cofactor in a medium of high viscosity (0.3 P) or as the motion of a unit larger than a lysine-bonded pyridoxal 5'-phosphate in a medium with the viscosity of water. Data for acrylamide quenching and time-resolved fluorescence measurements suggest that the latter interpretation should valid. These data also suggest a tightly packed microenvironment around the pyridoxal moiety.     

A potent fluorescent ATP-like inhibitor of cAMP-dependent protein kinase

The fluorescent ATP analogue 8-azido-2'-O-[14C]dansyl-ATP ([ 14C]AD-ATP) was used to probe the ATP-binding site in the catalytic (C) subunit of cAMP-dependent protein kinase. AD-ATP was found to inhibit the phosphotransferase activity of C subunit with extremely high specificity. Complete inhibition was observed when each mol of C subunit was covalently labeled with 1 mol of this fluorescent ATP analogue. The labeling can be accelerated by the presence of Mg2+ or Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly), whereas high concentrations of ATP can almost completely protect the enzyme from AD-ATP. Detailed studies indicated that AD-ATP competes with ATP for binding to C subunit. Analysis of the kinetic data gave dissociation constants of 2.9 and 13 microM for AD-ATP and ATP bound to C subunit, respectively. AD-ATP has a fluorescence emission peak at 510 nm in pH 7.0 aqueous buffer containing 25% glycerol. After covalent binding to C subunit this emission peak shifts to 455 nm, which suggests that the label at ATP site is in an endogenous hydrophobic environment. Upon the binding of Mg2+ or Kemptide, the fluorescence of AD-ATP-labeled C subunit can be enhanced by 50 and 45%, respectively. This enhancement suggests that the binding of either the peptide substrate or Mg2+ induces conformational change at the active site of C subunit. Analysis of the fluorescence data shows that the values of Kd for Mg2+ and Kemptide bound to AD-ATP-labeled C subunit are 0.2 mM and 2.1 microM, respectively. The normal procedure for the preparation of the C subunit from the bovine heart muscle has been simplified to require only one-fifth of the usual working time to obtain the homogeneous enzyme with 70% yield from the crude extract.     

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. IV. Conformational changes in the region containing fluorescence-labeled sulfhydryl group(s) of troponin.

Conformational changes associated with the functional states of the molecule of troponin were studied using SH-direct fluorogenic reagents, N-(p-(2-benzimidazolyl)phenyl) maleimide (BIPM) and N-(1-anilinonaphthyl-4) maleimide (ANM). 1. The fluorescence parameters of ANM-troponin, intensity, and polarization, did not change on combining it with tropomyosin alone, but markedly changed when F-actin was further added to the system. 2. The conformation around the dye-labeled sulfhydryl group(s) was shown to be susceptible to Ca2+ in terms of fluorescence intensity of the label, thermal transition of the conformation, and the microenvironment near the label. 3. On addition of Ca2+, the fluorescence characteristics of the two systems, ANM-troponin . tropomyosin and ANM-troponin . tropomyosin . F-actin complexes, were altered in opposite directions. When BIPM was used in place of ANM, similar changes were observed: a simple decrease in the intensity when pCa was decreased from 7.4 to 5.5 in the system without F-actin and a sigmoidal increase in the range from pCa 7 to 6 in the system with F-actin. Heavy meromyosin, when added to the latter complex (the reconstituted thin filaments), made the profile of its Ca2+ concentration dependence of fluorescence similar to that of the former complex. When tropomyosin was labeled in place of troponin, similar results were obtained. The data obtained imply that the Ca2+-induced conformational changes of troponin are markedly modified when detached from actin, and that heavy meromyosin weakens the interaction of the troponin . tropomyosin complex with F-actin.     

Tropomyosin and myosin subfragment 1 induce in thin muscle fiber filaments differing conformational changes in the C-terminal portion of the polypeptide chain of actin

Muscle fibres, free of myosin, troponin and tropomyosin, containing thin filaments reconstructed from G-actin and modified by fluorescent label 1,5-IAEDANS were used for polarized microfluorimetric studies of the effect of tropomyosin (TM) from smooth muscles, and of subfragment 1 (S1) from skeletal muscles on the structural state of F-actin. TM and S1 were shown to initiate different changes in polarized fluorescence of 1,5-IAEDANS of F-actin: TM increases, whereas S1 decreases fluorescent anisotropy. It was suggested that the structural state of F-actin may differ in the C-terminal of polypeptide chain of actin.     

Interaction of fluorescently-labeled contractile proteins with the cytoskeleton in cell models

To determine if a living cell is necessary for the incorporation of actin, alpha-actinin, and tropomyosin into the cytoskeleton, we have exposed cell models to fluorescently labeled contractile proteins. In this in vitro system, lissamine rhodamine-labeled actin bound to attachment plaques, ruffles, cleavage furrows and stress fibers, and the binding could not be blocked by prior exposure to unlabeled actin. Fluorescently labeled alpha-actinin also bound to ruffles, attachment plaques, cleavage furrows, and stress fibers. The periodicity of fluorescent alpha-actinin along stress fibers was wider in gerbil fibroma cells than it was in PtK2 cells. The fluorescent alpha-actinin binding in cell models could not be blocked by the prior addition of unlabeled alpha-actinin suggesting that alpha-actinin was binding to itself. While there was only slight binding of fluorescent tropomyosin to the cytoskeleton of interphase cells, there was stronger binding in furrow regions of models of dividing cells. The binding of fluorescently labeled tropomyosin could be blocked by prior exposure of the cell models to unlabeled tropomyosin. If unlabeled actin was permitted to polymerize in the stress fibers in cell models, fluorescently labeled tropomyosin stained the fibers. In contrast to the labeled contractile proteins, fluorescently labeled ovalbumin and BSA did not stain any elements of the cytoskeleton. Our results are discussed in terms of the structure and assembly of stress fibers and cleavage furrows.     

Studies on calcium ion-induced conformation changes in the actin-tropomyosin-troponin system by fluorimetry. III. Changes in the conformation of tropomyosin associated with functional states.

The local conformational changes in the tropomyosin molecule under various conditions were studied by means of fluorimetry using SH-directed fluorescent dyes, N-(1-anilinonaphthyl-4)maleimide (ANM) and N-(3-pyrene)maleimide (PRM). 1. The fluorescence intensity, polarization and the emmission maximum of ANM-tropomyosin were found to be susceptible to ionic strength, but in different ways. The changes in these parameters suggest that the fluorescence-labeled sulfhydryl group or groups become more buried in a hydrophobic internal region by salt-induced depolymerization of aggregate and by adding F-actin to tropomyosin. 2. Titration of the labeled tropomyosin with F-actin revealed a cooperative nature in ANM labeling and a simple saturation kinetics in PRM labeling. The dissociation constant of F-actin to PRM-tropomyosin was calculated to be 5.8-10(-6) M. 3. Temperature dependence of the fluorescence polarization showed a thermal transition in the conformation of ANM- or PRM-tropomyosin at around 30 degrees C. Flexibility or segmental motion of the region containing the fluorophore was suppressed significantly on adding troponin and markedly on adding F-actin. 4. Measurements of the quantum yield and polarization of the ANM-tropomyosin-F-actin complex suggested that troponin strengthened the binding between the two proteins and that Ca2+ reversed this effect.     

A comparative study of electrochemically and fluorometrically addressed molecular reporter groups: effects of protein microenvironment

To probe the effects of protein microenvironment on electrochemically and fluorometrically addressed molecular reporter groups, genetically engineered apo-cytochrome c peroxidase derivatives W51C, A174C, K243C, and S246C, each containing a single cysteine residue, were labeled at identical sites with two kinds of microenvironment sensitive reporters, either an electrochemically active sulfhydryl-reactive reagent, [Ru(II)(NH(3))(4)(1,10-phenanthroline-5-maleimide)](PF(6))(2) [RuPA4] or a fluorescent 6-acryloyl-2-dimethylaminonaphthalene [acrylodan] probe. Two types of sites were labeled with each probe based on their predicted solvent accessibilities from the known structure for holo-cytochrome c peroxidase. One set of sites (K243C and S246C) was selected to be completely solvent exposed, while the other two sites (W51C and A174C) were less accessible, residing in or near the heme binding site. Spectroscopic properties of the fluorescent probe were consistent with predictions for relative solvent accessibilities; however, even the less solvent accessible probes reported a quite polar environment, suggesting that this region of the apo-protein is either substantially solvent exposed or undergoes significant dynamic motion. A linear correlation was observed between the lambda(max) of the metal to ligand charge-transfer (MLCT) absorption band of the RuPA4 complex and the acrylodan emission maximum for the four labeled apo-protein variants. The same trend occurred for the formal potential of RuPA4 versus the acrylodan emission maximum, with the exception of electrochemical probe behavior at position 174, possibly due to specific probe-protein interactions.