Fluorescence Spectroscopic Approach Research Articles

Electrochemical Proximity Assay of Platelet-Derived Growth Factor through Combined Surface Plasmon Resonance, Electrochemistry, and Fluorescence Spectroscopy Approach

Platelet-derived growth factor (PDGF) is a potential cancer marker, which plays an important role in early detection of cancers. Due to its trace amounts in blood sample, it is difficult to be detected. Electrochemical Proximity Assay (ECPA) is an extremely flexible and sensitive technique with high potential for quantitation of a variety of proteins in clinical laboratory settings or at the point-of-care systems. In this study, we aim to use ECPA strategy on the PDGF recognition to achieve high sensitivity and specificity. In contrast to previous ECPA approach in which signals are only read out by electrochemical measurement, three characterization techniques including surface plasmon resonance, electrochemistry and fluorescence spectroscopy are combined together here so that these techniques are capable of cross-validating and complementing each other.

Electrochemical Proximity Assay of Picomolar Platelet-Derived Growth Factor through Combined Surface Plasmon Resonance, Electrochemistry, and Fluorescence Spectroscopy Approach

Platelet-derived growth factor (PDGF) is a potential cancer marker, which will play an important role in early detection of cancers. Due to its trace amounts in contrast to abundant proteins and other interfering species in blood sample, it is difficult to be detected. Electrochemical Proximity Assay (ECPA) is an extremely flexible and sensitive technique with high potential for quantitation of a variety of proteins in clinical laboratory settings or at the point-of-care systems. In this study, we applied ECPA strategy on the PDGF recognition to achieve high sensitivity and specificity. Different with previous ECPA approach in which signals are only read out by electrochemical measurement, three characterization techniques including surface plasmon resonance, electrochemistry and fluorescence spectroscopy are combined together here so that these techniques are capable of cross-validating and complementing each other. With the combined advantages of these techniques, the limit of detection of PDGF quantification reaches as low as picomolar level. More importantly, the cross-validation of signals contributes to identification of the interference from non-specific binding, and further improvement of the detection accuracy. In this research, the influence factors such as concentration of biosensor’s components, injection time and flow rate were studies, the response linear range, sensitivity and selectivity of PDGF detection were obtained. Furthermore, the mechanisms of action and dynamics of sensing and protein recognition in the process of PDGF recognition was investigated. This work not only provides an accurate quantitation information for biomarker PDGF, but allows a fundamental understanding of the biosensor formation with an unprecedented level, which can overcome several key limitations of current, widely-used surface characterization technologies.

Insights into the interaction of methotrexate and human serum albumin: A spectroscopic and molecular modeling approach.

In this study, fluorescence spectroscopy and molecular modeling approaches were employed to investigate the binding of methotrexate to human serum albumin (HSA) under physiological conditions. From the mechanism, it was demonstrated that fluorescence quenching of HSA by methotrexate results from the formation of a methotrexate/HSA complex. Binding parameters calculated using the Stern-Volmer method and the Scatchard method showed that methotrexate binds to HSA with binding affinities in the order 104 L·mol-1 . Thermodynamic parameter studies revealed that the binding reaction is spontaneous, and that hydrogen bonds and van der Waals interactions play a major role in the reaction. Site marker competitive displacement experiments and a molecular modeling approach demonstrated that methotrexate binds with appropriate affinity to site I (subdomain IIA) of HSA. Furthermore, we discuss some factors that influence methotrexate binding to HSA.

Exploring the Binding Mechanism of Flavonoid Quercetin to Phospholipase A2: Fluorescence Spectroscopy and Computational Approach

The interaction of flavonoid Quercetin with Phospholipase A2 isolated from snake venom Bothrops brazili (MTX-II) was investigated by fluorescence spectroscopy and molecular modeling. The fluorimetric titrations were conducted at 288, 298 and 308 K and at pH 8.0. Stern-Volmer quenching constant (KSV) and binding constant (Kb) were calculated along with the corresponding thermodynamic parameters ΔG, ΔH and ΔS at 288 and 298 K. From these analysis evidences of complex formation in between MTX-II and QCT are found. Besides that modified Stern-Volmer plot show evidences for two types of intrinsic fluorophores with different accessibilities at 308 K. The mean distance between the donor (MTX-II) and acceptor (QCT) was determined by fluorescence resonance energy transfer (FRET). The optimized structure of QCT was obtained by ab initio calculation, which geometry was performed in its ground states by using DFT/B3LYP functional with 6-311+G (d,p) basis set. The molecular docking analysis show that QCT may be localized at two main clusters, the first is at the dimer interface and the second at the active site like region. The clusters positions and binding energies reinforce the experimental data.

Localization and dynamics of the anticarcinogenic curcumin with GM1 and other miceller assemblies.

Structural transitions involving shape changes play an important role in cellular physiology and enhance the bioavailability of the natural food like curcumin in surfactant aggregates. In this work, we have studied the localization, dynamics and stability of curcumin in various miceller assemblies using a combination of absorbance and fluorescence spectroscopic approaches. The measurements of absorption and fluorescence spectra of curcumin revealed that the nature of interactions of ionic and nonionic surfactants and the glycosphingolipid, GM1 with curcumin is significantly different with surfactant concentrations. At low concentrations of SDS and the GM1 the head group of SDS and GM1 binds to the central β-diketone group of curcumin to form SDS-curcumin or GM1-curcumin complexes. At high concentrations, both formed micelles with curcumin completely solubilized inside. Cucurmin is solubilized in the stern layer of SDS micelles. Compared to spherical micelles, rod shaped micelles allow more curcumin to bind through hydrophobic interactions indicated by higher absorption and fluorescence, enhanced partition coefficient and stability. Whereas curcumin associates with GM1 micelles with lower partition coefficient, solubility and remain closer to aqueous phase decreasing its bioavailability and stability. While cucurmin is solubilized in the palisade layer of deoxycholate and octyl glucopyranoside micelles through the alkyl chains providing more hydrophobic microenvironment to curcumin with enhanced stability and bioavailability. Graphical abstract Schematic diagram of the two different types of detergent micelles and larger GM1 micelles.

A Simple Fluorescence Spectroscopic Approach for Simultaneous and Rapid Detection of Four Polycyclic Aromatic Hydrocarbons (PAH4) in Vegetable Oils

Based on the conclusions of the European Food Safety Authority (EFSA), the four polycyclic aromatic hydrocarbons (PAH4) chrysene (Chr), benzo(a)anthracene (BaA), benzo(a)pyrene (BaP), and benzo(b)fluoranthene (BbF) are new markers for the occurrence of PAHs in food. Vegetable oils are extremely easily contaminated by PAHs, due to the raw material pollution and the imperfections of processing techniques. Therefore, it is of great importance to establish an efficient method for the rapid determination of PAH4 in vegetable oils. In this research, second-derivative nonlinear variable-angle-matrix isopotential synchronous fluorescence spectroscopic approach has been proposed for the simultaneous and rapid determination of PAH4 in vegetable oils with ultrasonic-assisted extraction. This method is capable of resolving the spectra of these four PAHs well. The limits of detection (LODs) for Chr, BaA, BaP, and BbF were 13.0, 8.64, 0.16, and 7.68 μg kg−1, respectively. The recoveries in various oil samples for Chr, BaA, BaP, and BbF were 86.2 ± 15.3, 93.8 ± 11.7, 93.9 ± 12.1, and 94.3 ± 7.4 %, respectively, at the spiked levels of 10∼400 μg kg−1. The relative standard deviations (RSDs) achieved for Chr, BaA, BaP, and BbF were 3.0, 8.4, 6.3, and 7.2 %, respectively. The established method is simple, economical, and reliable, as well as provides an attractive alternative for the simultaneous and rapid determination of PAH4 in vegetable oils.

Activity of α‐Chymotrypsin in Cationic and Nonionic Micellar Media: Ultraviolet and Fluorescence Spectroscopic Approach

ABSTRACTα‐Chymotrypsin (α‐CT) activity was measured in aqueous buffer with the following alkyltriphenylphosphonium bromide surfactants in the series cetyl, tetradecyl, and dodecyl as a tail length. For the sake of comparison with mixed micellar investigation on activity of α‐CT, cationic cetyltriphenylphosphonium bromide (CTPB) and nonionic surfactant Triton X‐100, Brij‐56, Brij‐35, Tween 20, and Igepal Co‐210 have been used. The p‐nitrophenyl acetate (PNPA) hydrolysis rate was determined at the surfactant concentration of both cationic and mixed micellar systems by a UV–vis spectrophotometer. The catalytic reaction follows the Michaelis–Menten mechanism, and the catalytic efficiency (kcat/KM) was evaluated for both homogeneous and mixed‐micellar media. The maximum catalytic efficiency was observed at 5 mM concentration of CTPB, but the highest catalytic efficiency, 572 M−1 s−1, was measured in the presence of mixed micellar (7.5 mM CTPB + 2.5 mM Tween‐20). The fluorescence (FL) spectra showed the differences of α‐CT conformations in the presence of cationic surfactants. The FL results suggest that the influence of cationic surfactant on proteolysis arises from the interaction with the α‐CT. The binding constant, ksv, of α‐CT with cationic aggregates was determined in the buffer using the Stern–Volmer equation by the fluorescence spectroscopic approach.

Real-Time Drug Release Analysis of Enzyme and pH Responsive Polysaccharide Nanovesicles.

The accurate estimation of drug release kinetics of polymeric vehicles is an indispensable prerequisite for the developments of successful drug carriers for cancer therapy. The present investigation reports the development of time-resolved fluorescence spectroscopic approach for the real-time release kinetics of fluorophore loaded polysaccharide vesicles that are potential vectors in cancer treatment. The polysaccharide vesicles were custom designed with appropriate enzyme and pH responsiveness and loaded with water-soluble biocompatible fluorophore Rhodamine B (Rh-B). The semipermeable membrane dialysis method along with steady state absorbance spectroscopic technique was found to be inaccurate for the estimation of drug release. Time correlated single photon counting (TCSPC) technique was found to exhibit significant difference in excited state decay profiles and fluorescent lifetime of Rh-B in the free and polymer bound states. This enabled the establishment of real-time drug release protocols by TCSPC method for polysaccharide vesicles that are responsible to pH and enzyme with respect to intracellular compartments. Real-time analysis predicted the release kinetics 20-25% higher accuracy when compared to the dialysis method under in vitro conditions. Moreover, the ability of enzyme to cleave the polysaccharide vesicles was further validated by docking studies. The positioning of the molecules in active site of enzyme and the binding energy data were generated using AUTODOCK program to study the rupture of polysaccharide vesicles. This new TCSPC technique could be very useful for studying the drug release pattern of synthetic polymer vesicles loaded with Rh-B fluorophore.

Cardiolipin's propensity for phase transition and its reorganization by dynamin-related protein 1 form a basis for mitochondrial membrane fission.

Cardiolipin (CL) is an atypical, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a cytosolic member of the dynamin superfamily of large GTPases, interacts with CL and functions to sustain the balance of mitochondrial division and fusion by catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly and GTPase activity at the membrane surface, the mechanism by which CL functions in membrane fission, if at all, remains unclear. Here, using a variety of fluorescence spectroscopic and imaging approaches together with model membranes, we demonstrate that Drp1 and CL function cooperatively in effecting membrane constriction toward fission in three distinct steps. These involve 1) the preferential association of Drp1 with CL localized at a high spatial density in the membrane bilayer, 2) the reorganization of unconstrained, fluid-phase CL molecules in concert with Drp1 self-assembly, and 3) the increased propensity of CL to transition from a lamellar, bilayer arrangement to an inverted hexagonal, nonbilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. Thus we propose that Drp1 and CL function in concert to catalyze mitochondrial division.

Study of interaction between human serum albumin and three phenanthridine derivatives: Fluorescence spectroscopy and computational approach

Over the past decades, phenanthridine derivatives have captured the imagination of many chemists due to their wide applications. In the present work, the interaction between phenanthridine derivatives benzo [4,5]imidazo[1,2-a]thieno[2,3-c]quinoline (BTQ), benzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (BFQ), 5,6-dimethylbenzo[4,5]imidazo[1,2-a]furo[2,3-c]quinoline (DFQ) and human serum albumin (HSA) were investigated by molecular modeling techniques and spectroscopic methods. The results of molecular modeling simulations revealed that the phenanthridine derivatives could bind on both site I in HSA. Fluorescence data revealed that the fluorescence quenching of HSA by phenanthridine derivatives were the result of the formation of phenanthridine derivatives–HSA complex, and the binding intensity between three phenanthridine derivatives and HSA was BTQ>BFQ>DFQ. Thermodynamics confirmed that the interaction were entropy driven with predominantly hydrophobic forces. The effects of some biological metal ions and toxic ions on the binding affinity between phenanthridine derivatives and HSA were further examined.

Cardiolipin Reorganization and Phase Transition Induced by Dynamin-Related Protein 1 Facilitates Mitochondrial Membrane Fission

Cardiolipin (CL) is a unique, dimeric phospholipid essential for mitochondrial dynamics in eukaryotic cells. Dynamin-related protein 1 (Drp1), a member of the dynamin superfamily of large GTPases, maintains the balance of mitochondrial division and fusion by rapidly catalyzing mitochondrial fission. Although recent studies have indicated a role for CL in stimulating Drp1 self-assembly as well as GTPase activity on the mitochondrial surface, the exact mechanism by which CL functions in membrane fission remains unclear. Here we use a variety of fluorescence spectroscopic and imaging approaches, together with model membranes, to demonstrate that Drp1 and CL function cooperatively in effecting membrane fission in three distinct steps: (i) Drp1's preferential association with unconstrained, fluid-phase CL molecules located at a high spatial density in the membrane bilayer, (ii) CL's reorganization in concert with Drp1 self-assembly, and (iii) CL's rapid phase transition from a lamellar, bilayer structure to an inverted hexagonal, non-bilayer configuration in the presence of Drp1 and GTP, resulting in the creation of localized membrane constrictions that are primed for fission. We propose that Drp1 thus catalyzes mitochondrial division.

Ruthenium (II) Complexes Interact with Human Serum Albumin and Induce Apoptosis of Tumor Cells

The interaction of ruthenium (II) complex [Ru(bpy)2(mal)](2+) (RBM) and [Ru(phen)2(mal)](2+) (RPM) (bpy = 2, 2-bipyridine, phen = 1,10-phenanthroline, mal = malonyl carboxylate) with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and circular dichroism (CD) spectroscopy approaches. A strong fluorescence quenching reaction of complexes to HSA was observed and the quenching mechanism was suggested as static quenching according to the Stern-Volmer (S-V) equation. The number of binding sites n and observed binding constant Kb was measured by fluorescence quenching method. The thermodynamic parameters ΔH, ΔS, and ΔG at different temperatures were calculated and the results indicate the binding reaction is mainly entropy-driven and Vander Waals force played a major role in the reaction. The result of CD showed that the secondary structure of HSA molecules was changed in the presence of the ruthenium (II) complexes. Furthermore, the cell viability of ruthenium (II) complexes was evaluated by MTT and complex RPM has shown significant higher anticancer potency than RBM against all the cell lines screened. RPM showed a significant antitumor activity through induction of apoptosis in A549 cells.

A pyrene based fluorescence approach to study conformation of apolipoprotein E3 in macrophage-generated nascent high density lipoprotein

Apolipoprotein E3 (apoE3) is an anti-atherogenic apolipoprotein with the ability to exist in lipid-free and lipoprotein-associated states. During atherosclerosis, its function in promoting cholesterol efflux from macrophages via the ATP-binding cassette transporter A1 (ABCA1) takes a prominent role, leading to generation of nascent high density lipoprotein (nHDL) particles. The objective of this study is to understand the conformation adopted by apoE3 in macrophage-generated nHDL using a fluorescence spectroscopic approach involving pyrene. Pyrene-labeled recombinant human apoE3 displayed a robust ability to stimulate ABCA1-mediated cholesterol efflux from cholesterol-loaded J774 macrophages (which do not express apoE), comparable to that elicited by unlabeled apoE3. The nHDL recovered from the conditioned medium revealed the presence of apoE3 by immunoblot analysis. A heterogeneous population of nHDL bearing exogenously added apoE3 was generated with particle size varying from ∼12 to ∼19nm in diameter, corresponding to molecular mass of ∼450 to ∼700kDa. The lipid: apoE3 ratio varied from ∼60:1 to 10:1. A significant extent of pyrene excimer emission was noted in nHDL, indicative of spatial proximity between Cys112 on neighboring apoE3 molecules similar to that noted in reconstituted HDL. Cross-linking analysis using Cys-specific cross-linkers revealed the predominant presence of dimers. Taken together the data indicate a double belt arrangement of apoE molecules on nHDL. A similar organization of the C-terminal tail of apoE on nHDL was noted when pyrene-apoEA277C(201–299) was used as the cholesterol acceptor. These studies open up the possibility of using exogenously labeled apoE3 to generate nHDL for structural and conformational analysis.

Spectroscopic studies of the unfolding of a multimeric protein α‐crystallin

α-Crystallin is a multimeric eye lens protein having molecular chaperone-like function which is crucial for lens transparency. The stability and unfolding-refolding properties of α-crystallin plays important roles for its function. We undertook a multi probe based fluorescence spectroscopic approach to explore the changes in the various levels of organization of this protein at different urea concentration. Steady state fluorescence studies reveal that at 0.6M urea a compact structural intermediate is formed which has a native-like secondary structure with enhanced surface exposure of hydrophobic groups. At 2.8M urea the tertiary interactions are largely collapsed with partial collapse of secondary and quaternary structure. The surface solvation probed by picosecond time resolved fluorescence of acrylodan labeled α-crystallin revealed dry native-like core of α-crystallin at 0.6M urea compared to enhanced water penetration at 2.8M urea and extensive solvation at 6M urea. Activation energy for the subunit exchange decreased by 22 kJ mol(-1) on changing urea concentration from 0 to 0.6M compared with over 75 kJ mol(-1) on changing urea concentration from 0 to 2.8M. Light scattering and analytical ultracentrifugation techniques were used to determine size and oligomerization of the unfolding intermediates. The data indicated swelling but no oligomer breakdown at 0.6M urea. At 2.8M urea the oligomeric size is considerably reduced and a monomer is produced at 6M urea. The data clearly reveals that structural breakdown of α-crystallin does not follow hierarchical sequence as tertiary structure dissolution takes place before complete oligomeric dissociation.

Characterization of Two Quinone Radicals in the NADH:Ubiquinone Oxidoreductase from Escherichia coli by a Combined Fluorescence Spectroscopic and Electrochemical Approach

The NADH:ubiquinone oxidoreductase (complex I) couples the transfer of electrons from NADH to ubiquinone with the translocation of protons across the membrane. It was proposed that the electron transfer involves quinoid groups localized at the end of the electron transfer chain. To identify these groups, fluorescence excitation and emission spectra of Escherichia coli complex I and its fragments, namely, the NADH dehydrogenase fragment containing the flavin mononucleotide and six iron-sulfur (Fe-S) clusters, and the quinone reductase fragment containing three Fe-S clusters were measured. Signals sensitive to reduction by either NADH or dithionite were detected within the complex and the quinone reductase fragment and attributed to the redox transition of protonated ubiquinone radicals. A fluorescence spectroscopic electrochemical redox titration revealed midpoint potentials of -37 and- 235 mV (vs the standard hydrogen electrode) for the redox transitions of the quinone radicals in complex I at pH 6 with an absorption around 325 nm and a fluorescence emission at 460/475 nm. The role of these cofactor(s) for electron transfer is discussed.

Interaction of Sulfadiazine with Model Water Soluble Proteins: A Combined Fluorescence Spectroscopic and Molecular Modeling Approach

The binding behavior of antibacterial drug sulfadiazine (SDZ) with water soluble globular proteins like bovine as well as human serum albumin (BSA and HSA, respectively) and lysozyme (LYS) was monitored by fluorescence titration and molecular docking calculations. The experimental data reveal that the quenching of the intrinsic protein fluorescence in presence of SDZ is due to the strong interaction in the drug binding site of the respective proteins. The Stern-Volmer plot shows positive deviation at higher quencher concentration for all the proteins and was explained in terms of a sphere of action model. The calculated fluorophore-quencher distances vary within 4 ~ 11Å in different cases. Fluorescence experiments at different temperature indicate thermodynamically favorable binding of SDZ with the proteins with apparently strong association constant (~10(4)-10(5)M(-1)) and negative free energy of interaction within the range of -26.0 ~ -36.8kJmol(-1). The experimental findings are in good agreement with the respective parameters obtained from best energy ranked molecular docking calculation results of SDZ with all the three proteins.

Perspective: Reaches of chemical physics in biology.

Chemical physics as a discipline contributes many experimental tools, algorithms, and fundamental theoretical models that can be applied to biological problems. This is especially true now as the molecular level and the systems level descriptions begin to connect, and multi-scale approaches are being developed to solve cutting edge problems in biology. In some cases, the concepts and tools got their start in non-biological fields, and migrated over, such as the idea of glassy landscapes, fluorescence spectroscopy, or master equation approaches. In other cases, the tools were specifically developed with biological physics applications in mind, such as modeling of single molecule trajectories or super-resolution laser techniques. In this introduction to the special topic section on chemical physics of biological systems, we consider a wide range of contributions, all the way from the molecular level, to molecular assemblies, chemical physics of the cell, and finally systems-level approaches, based on the contributions to this special issue. Chemical physicists can look forward to an exciting future where computational tools, analytical models, and new instrumentation will push the boundaries of biological inquiry.

Forest soil organic carbon dynamics as affected by plant species and their corresponding litters: a fluorescence spectroscopy approach

Background and aims The effect of forest cover distribution and plant litter input on soil organic carbon were analyzed to better understand the dynamics of carbon cycling across ecosystems on the “Natural Oriented Reserve Bosco delle Pianelle”. Fluorescence spectroscopy represents a very useful tool to characterize soil organic matter properties, since it allows to directly monitor the molecular status of a fluorophore depending on its chemical environment, as well as on its structure, substituents of the aromatic moieties, and molecular weight. Here, fluorescence analysis was performed on humic acids isolated from four litters (HALs) and their underlying soils (HAs) at three depths.

A combined fluorescence spectroscopy, confocal and 2-photon microscopy approach to re-evaluate the properties of sphingolipid domains

The aim of this study is to provide further insight about the interplay between important signaling lipids and to characterize the properties of the lipid domains formed by those lipids in membranes containing distinct composition. To this end, we have used a combination of fluorescence spectroscopy, confocal and two-photon microscopy and a stepwise approach to re-evaluate the biophysical properties of sphingolipid domains, particularly lipid rafts and ceramide (Cer)-platforms. By using this strategy we were able to show that, in binary mixtures, sphingolipids (Cer and sphingomyelin, SM) form more tightly packed gel domains than those formed by phospholipids with similar acyl chain length. In more complex lipid mixtures, the interaction between the different lipids is intricate and is strongly dictated by the Cer-to-Chol ratio. The results show that in quaternary phospholipid/SM/Chol/Cer mixtures, Cer forms gel domains that become less packed as Chol is increased. Moreover, the extent of gel phase formation is strongly reduced in these mixtures, even though Cer molar fraction is increased. These results suggest that in biological membranes, lipid domains such as rafts and ceramide platforms, might display distinctive biophysical properties depending on the local lipid composition at the site of the membrane where they are formed, further highlighting the potential role of membrane biophysical properties as an underlying mechanism for mediating specific biological processes.

Myosin and Tropomyosin Stabilize the Conformation of Formin-nucleated Actin Filaments

The conformational elasticity of the actin cytoskeleton is essential for its versatile biological functions. Increasing evidence supports that the interplay between the structural and functional properties of actin filaments is finely regulated by actin-binding proteins; however, the underlying mechanisms and biological consequences are not completely understood. Previous studies showed that the binding of formins to the barbed end induces conformational transitions in actin filaments by making them more flexible through long range allosteric interactions. These conformational changes are accompanied by altered functional properties of the filaments. To get insight into the conformational regulation of formin-nucleated actin structures, in the present work we investigated in detail how binding partners of formin-generated actin structures, myosin and tropomyosin, affect the conformation of the formin-nucleated actin filaments using fluorescence spectroscopic approaches. Time-dependent fluorescence anisotropy and temperature-dependent Förster-type resonance energy transfer measurements revealed that heavy meromyosin, similarly to tropomyosin, restores the formin-induced effects and stabilizes the conformation of actin filaments. The stabilizing effect of heavy meromyosin is cooperative. The kinetic analysis revealed that despite the qualitatively similar effects of heavy meromyosin and tropomyosin on the conformational dynamics of actin filaments the mechanisms of the conformational transition are different for the two proteins. Heavy meromyosin stabilizes the formin-nucleated actin filaments in an apparently single step reaction upon binding, whereas the stabilization by tropomyosin occurs after complex formation. These observations support the idea that actin-binding proteins are key elements of the molecular mechanisms that regulate the conformational and functional diversity of actin filaments in living cells.