F-Actin Structure Destabilization and DNase I Binding Loop Fluctuations: Mutational Cross-Linking and Electron Microscopy Analysis of Loop States and Effects on F-Actin

The conformational dynamics of filamentous actin (F-actin) is essential for the regulation and functions of cellular actin networks. The main contribution to F-actin dynamics and its multiple conformational states arises from the mobility and flexibility of the DNase I binding loop (D-loop; residues 40-50) on subdomain 2. Therefore, we explored the structural constraints on D-loop plasticity at the F-actin interprotomer space by probing its dynamic interactions with the hydrophobic loop (H-loop), the C-terminus, and the W-loop via mutational disulfide cross-linking. To this end, residues of the D-loop were mutated to cysteines on yeast actin with a C374A background. These mutants showed no major changes in their polymerization and nucleotide exchange properties compared to wild-type actin. Copper-catalyzed disulfide cross-linking was investigated in equimolar copolymers of cysteine mutants from the D-loop with either wild-type (C374) actin or mutant S265C/C374A (on the H-loop) or mutant F169C/C374A (on the W-loop). Remarkably, all tested residues of the D-loop could be cross-linked to residues 374, 265, and 169 by disulfide bonds, demonstrating the plasticity of the interprotomer region. However, each cross-link resulted in different effects on the filament structure, as detected by electron microscopy and light-scattering measurements. Disulfide cross-linking in the longitudinal orientation produced mostly no visible changes in filament morphology, whereas the cross-linking of D-loop residues > 45 to the H-loop, in the lateral direction, resulted in filament disruption and the presence of amorphous aggregates on electron microscopy images. A similar aggregation was also observed upon cross-linking the residues of the D-loop (> 41) to residue 169. The effects of disulfide cross-links on F-actin stability were only partially accounted for by the simulations of current F-actin models. Thus, our results present evidence for the high level of conformational plasticity in the interprotomer space and document the link between D-loop interactions and F-actin stability.     

Myosin binding surface on actin probed by hydroxyl radical footprinting and site-directed labels

Actin and myosin are the two main proteins required for cell motility and muscle contraction. The structure of their strongly bound complex—rigor state—is a key for delineating the functional mechanism of actomyosin motor. Current knowledge of that complex is based on models obtained from the docking of known atomic structures of actin and myosin subfragment 1 (S1; the head and neck region of myosin) into low-resolution electron microscopy electron density maps, which precludes atomic- or side-chain-level information. Here, we use radiolytic protein footprinting for global mapping of sites across the actin molecules that are impacted directly or allosterically by myosin binding to actin filaments. Fluorescence and electron paramagnetic resonance spectroscopies and cysteine actin mutants are used for independent, residue-specific probing of S1 effects on two structural elements of actin. We identify actin residue candidates involved in S1 binding and provide experimental evidence to discriminate between the regions of hydrophobic and electrostatic interactions. Focusing on the role of the DNase I binding loop (D-loop) and the W-loop residues of actin in their interactions with S1, we found that the emission properties of acrylodan and the mobility of electron paramagnetic resonance spin labels attached to cysteine mutants of these residues change strongly and in a residue-specific manner upon S1 binding, consistent with the recently proposed direct contacts of these loops with S1. As documented in this study, the direct and indirect changes on actin induced by myosin are more extensive than known until now and attest to the importance of actin dynamics to actomyosin function.     

Replacement of ATP with ADP affects the dynamic and conformational properties of actin monomer.

The effect of the replacement of ATP with ADP on the conformational and dynamic properties of the actin monomer was investigated, by means of electron paramagnetic resonance (EPR) and fluorescence spectroscopic methods. The measurement of the ATP concentration during these experiments provided the opportunity to estimate the time dependence of ADP-Mg-G-actin concentration in the samples. According to the results of the fluorescence resonance energy transfer experiments, the Gln-41 and Cys-374 residues are closer to each other in the ADP-Mg-G-actin than in the ATP-Mg-G-actin. The fluorescence resonance energy transfer efficiency increased simultaneously with the ADP-G-actin concentration and reached its maximum value within 30 min at 20 degrees C. The EPR data indicate the presence of an ADP-Mg-G-actin population that can be characterized by an increased rotational correlation time, which is similar to the one observed in actin filaments, and exists only transiently. We suggest that the conformational transitions, which were reflected by our EPR data, were coupled with the transient appearance of short actin oligomers during the nucleotide exchange. Besides these relatively fast conformational changes, there is a slower conformational transition that could be detected several hours after the initiation of the nucleotide exchange.     

Spontaneous Structural Changes in Actin Regulate G-F Transformation

Transformations between G- (monomeric) and F-actin (polymeric) are important in cellular behaviors such as migration, cytokinesis, and morphing. In order to understand these transitions, we combined single-molecule Förster resonance energy transfer with total internal reflection fluorescence microscopy to examine conformational changes of individual actin protomers. We found that the protomers can take different conformational states and that the transition interval is in the range of hundreds of seconds. The distribution of these states was dependent on the environment, suggesting that actin undergoes spontaneous structural changes that accommodate itself to polymerization.     

Structural dynamics of the DnaK-peptide complex

The molecular chaperone DnaK recognizes and binds substrate proteins via a stretch of seven amino acid residues that is usually only exposed in unfolded proteins. The binding kinetics are regulated by the nucleotide state of DnaK, which alternates between DnaK.ATP (fast exchange) and DnaK.ADP (slow exchange). These two forms cycle with a rate mainly determined by the ATPase activity of DnaK and nucleotide exchange. The different substrate binding properties of DnaK are mainly attributed to changes of the position and mobility of a helical region in the C-terminal peptide-binding domain, the so-called LID. It closes the peptide-binding pocket and thus makes peptide binding less dynamic in the ADP-bound state, but does not (strongly) interact with peptides directly. Here, we address the question if nucleotide-dependent structural changes may be observed in the peptide-binding region that could also be connected to peptide binding kinetics and more importantly could induce structural changes in peptide stretches using the energy available from ATP hydrolysis. Model peptides containing two cysteine residues at varying positions were derived from the structurally well-documented peptide NRLLLTG and labelled with electron spin sensitive probes. Measurements of distances and mobilities of these spin labels by electron paramagnetic resonance spectroscopy (EPR) of free peptides or peptides bound to the ATP and ADP-state of DnaK, respectively, showed no significant changes of mobility nor distance of the two labels. This indicates that no structural changes that could be sensed by the probes at the position of central leucine residues located in the center of the binding region occur due to different nucleotide states. We conclude from these studies that the ATPase activity of DnaK is not connected to structural changes of the peptide-binding pocket but rather only has an effect on the LID domain or other further remote residues.     

Conformational ensemble of a multidomain protein explored by Gd3+ electron paramagnetic resonance

Despite their importance in function, the conformational state of proteins and its changes are often poorly understood, mainly because of the lack of an efficient tool. MurD, a 47-kDa protein enzyme responsible for peptidoglycan biosynthesis, is one of those proteins whose conformational states and changes during their catalytic cycle are not well understood. Although it has been considered that MurD takes a single conformational state in solution as shown by a crystal structure, the solution nuclear magnetic resonance (NMR) study suggested the existence of multiple conformational state of apo MurD in solution. However, the conformational distribution has not been evaluated. In this work, we investigate the conformational states of MurD by the use of electron paramagnetic resonance (EPR), especially intergadolinium distance measurement using double electron-electron resonance (DEER) measurement. The gadolinium ions are fixed on specific positions on MurD via a rigid double-arm paramagnetic lanthanide tag that has been originally developed for paramagnetic NMR. The combined use of NMR and EPR enables accurate interpretation of the DEER distance information to the structural information of MurD. The DEER distance measurement for apo MurD shows a broad distance distribution, whereas the presence of the inhibitor narrows the distance distribution. The results suggest that MurD exists in a wide variety of conformational states in the absence of ligands, whereas binding of the inhibitor eliminates variation in conformational states. The multiple conformational states of MurD were previously implied by NMR experiments, but our DEER data provided structural characterization of the conformational variety of MurD.     

Crystal structures of expressed non-polymerizable monomeric actin in the ADP and ATP states

Actin filament growth and disassembly, as well as affinity for actin-binding proteins, is mediated by the nucleotide-bound state of the component actin monomers. The structural differences between ATP-actin and ADP-actin, however, remain controversial. We expressed a cytoplasmic actin in Sf9 cells, which was rendered non-polymerizable by virtue of two point mutations in subdomain 4 (A204E/P243K). This homogeneous monomer, called AP-actin, was crystallized in the absence of toxins, binding proteins, or chemical modification, with ATP or ADP at the active site. The two surface mutations do not perturb the structure. Significant differences between the two states are confined to the active site region and sensor loop. The active site cleft remains closed in both states. Minor structural shifts propagate from the active site toward subdomain 2, but dissipate before reaching the DNase binding loop (D-loop), which remains disordered in both the ADP and ATP states. This result contrasts with previous structures of actin made monomeric by modification with tetramethylrhodamine, which show formation of an alpha-helix at the distal end of the D-loop in the ADP-bound but not the ATP-bound form (Otterbein, L. R., Graceffa, P., and Dominguez, R. (2001) Science 293, 708-711). Our reanalysis of the TMR-modified actin structures suggests that the nucleotide-dependent formation of the D-loop helix may result from signal propagation through crystal packing interactions. Whereas the observed nucleotide-dependent changes in the structure present significantly different surfaces on the exterior of the actin monomer, current models of the actin filament lack any actin-actin interactions that involve the region of these key structural changes.     

Reversibly bound and covalently attached ligands induce conformational changes in the omega loop, Cys69-Cys96, of mouse acetylcholinesterase

We have used a combination of cysteine substitution mutagenesis and site-specific labeling to characterize the structural dynamics of mouse acetylcholinesterase (mAChE). Six cysteine-substituted sites of mAChE (Leu(76), Glu(81), Glu(84), Tyr(124), Ala(262), and His(287)) were labeled with the environmentally sensitive fluorophore, acrylodan, and the kinetics of substrate hydrolysis and inhibitor association were examined along with spectroscopic characteristics of the acrylodan-conjugated, cysteine-substituted enzymes. Residue 262, being well removed from the active center, appears unaffected by inhibitor binding. Following the binding of ligand, hypsochromic shifts in emission of acrylodan at residues 124 and 287, located near the perimeter of the gorge, reflect the exclusion of solvent and a hydrophobic environment created by the associated ligand. By contrast, the bathochromic shifts upon inhibitor binding seen for acrylodan conjugated to three omega loop (Omega loop) residues 76, 81, and 84 reveal that the acrylodan side chains at these positions are displaced from a hydrophobic environment and become exposed to solvent. The magnitude of fluorescence emission shift is largest at position 84 and smallest at position 76, indicating that a concerted movement of residues on the Omega loop accompanies gorge closure upon ligand binding. Acrylodan modification of substituted cysteine at position 84 reduces ligand binding and steady-state kinetic parameters between 1 and 2 orders of magnitude, but a similar substitution at position 81 only minimally alters the kinetics. Thus, combined kinetic and spectroscopic analyses provide strong evidence that conformational changes of the Omega loop accompany ligand binding.     

Domain motions accompanying Tet repressor induction defined by changes of interspin distances at selectively labeled sites.

To investigate internal movements in Tet repressor (TetR) during induction by tetracycline (tc) we determined the interspin distances between pairs of nitroxide spin labels attached to specific sites by electron paramagnetic resonance (EPR) spectroscopy. For this purpose, we constructed six TetR variants with engineered cysteine pairs located in regions with presumed conformational changes. These are I22C and N47C in the DNA reading head, T152C/Q175C, A161C/Q175C and R128C/D180C near the tc-binding pocket, and T202C in the dimerization surface. All TetR mutants show wild-type activities in vivo and in vitro. The binding of tc results in a considerable decrease of the distance between the nitroxide groups attached to both I22C residues in the TetR dimer and an increase of the distance between the N47C residues. These opposite effects are consistent with a twisting motion of the DNA reading heads. Changes of the spin-spin interactions between nitroxide groups attached to residues near the tc-binding pocket demonstrate that the C-terminal end of alpha-helix 9 moves away from the protein core upon DNA binding. Alterations of the dipolar interaction between nitroxide groups at T202C indicate different conformations for tc and DNA-bound repressor also in the dimerization area. These results are used to model structural changes of TetR upon induction.     

Unraveling photoexcited conformational changes of bacteriorhodopsin by time resolved electron paramagnetic resonance spectroscopy

By means of time-resolved electron paramagnetic resonance (EPR) spectroscopy, the photoexcited structural changes of site-directed spin-labeled bacteriorhodopsin are studied. A complete set of cysteine mutants of the C-D loop, positions 100-107, and of the E-F loop, including the first alpha-helical turns of helices E and F, positions 154-171, was modified with a methanethiosulfonate spin label. The EPR spectral changes occurring during the photocycle are consistent with a small movement of helix C and an outward tilt of helix F. These helix movements are accompanied by a rearrangement of the E-F loop and of the C-terminal turn of helix E. The kinetic analysis of the transient EPR data and the absorbance changes in the visible spectrum reveals that the conformational change occurs during the lifetime of the M intermediate. Prominent rearrangements of nitroxide side chains in the vicinity of D96 may indicate the preparation of the reprotonation of the Schiff base. All structural changes reverse with the recovery of the bacteriorhodopsin initial state.     

A Nucleotide State-sensing Region on Actin

The nucleotide state of actin (ATP, ADP-P_i, or ADP) is known to impact its interactions with other actin molecules upon polymerization as well as with multiple actin binding proteins both in the monomeric and filamentous states of actin. Recently, molecular dynamics simulations predicted that a sequence located at the interface of subdomains 1 and 3 (W-loop; residues 165–172) changes from an unstructured loop to a β-turn conformation upon ATP hydrolysis (Zheng, X., Diraviyam, K., and Sept, D. (2007) Biophys. J. 93, 1277–1283). This region participates directly in the binding to other subunits in F-actin as well as to cofilin, profilin, and WH2 domain proteins and, therefore, could contribute to the nucleotide sensitivity of these interactions. The present study demonstrates a reciprocal communication between the W-loop region and the nucleotide binding cleft on actin. Point mutagenesis of residues 167, 169, and 170 and their site-specific labeling significantly affect the nucleotide release from the cleft region, whereas the ATP/ADP switch alters the fluorescence of probes located in the W-loop. In the ADP-P_i state, the W-loop adopts a conformation similar to that in the ATP state but different from the ADP state. Binding of latrunculin A to the nucleotide cleft favors the ATP-like conformation of the W-loop, whereas ADP-ribosylation of Arg-177 forces the W-loop into a conformation distinct from those in the ADP and ATP-states. Overall, our experimental data suggest that the W-loop of actin is a nucleotide sensor, which may contribute to the nucleotide state-dependent changes in F-actin and nucleotide state-modulated interactions of both G- and F-actin with actin-binding proteins.     

Detection of drug‐induced conformational change of a transmembrane protein in lipid bilayers using site‐directed spin labeling

As a target of antiviral drugs, the influenza A M2 protein has been the focus of numerous structural studies and has been extensively explored as a model ion channel. In this study, we capitalize on the expanding body of high‐resolution structural data available for the M2 protein to design and interpret site‐directed spin‐labeling electron paramagnetic resonance spectroscopy experiments on drug‐induced conformational changes of the M2 protein embedded in lipid bilayers. We obtained data in the presence of adamantane drugs for two different M2 constructs (M2TM 22–46 and M2TMC 23–60). M2TM peptides were spin labeled at the N‐terminal end of the transmembrane domain. M2TMC peptides were spin labeled site specifically at cysteine residues substituted for amino acids within the transmembrane domain (L36, I39, I42, and L43) and the C‐terminal amphipathic helix (L46, F47, F48, C50, I51, Y52, R53, F54, F55, and E56). Addition of adamantane drugs brought about significant changes in measured electron paramagnetic resonance spectroscopy environmental parameters consistent with narrowing of the transmembrane channel pore and closer packing of the C‐terminal amphipathic helices.     

Characterization of apolipoprotein A-I structure using a cysteine-specific fluorescence probe

Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created and expressed in Escherichia coli systems. Specific labeling with the thiol-reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), was used to study the structural organization and dynamic properties of the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound states. Spectroscopic approaches, including circular dichroism and various fluorescence methods, were used to examine the properties of the mutant proteins and of their covalent adducts with the fluorescence probe. The mutations themselves had no effect on the structure and stability of apoA-I in the lipid-free state and in reconstituted HDL (rHDL) complexes. Furthermore, covalent modification with acrylodan did not alter the properties of the apoA-I variants in the lipid-bound state nor in the lipid-free A232C mutant, but it affected the structure and local stability of the lipid-free protein in the D9C mutant. Fluorescence results using the acrylodan probe confirmed a well-organized structure in the N-terminal region of apoA-I. Also, they suggested a three-dimensional structure in the C-terminal region, stabilized by protein-protein contacts. When Trp residues and acrylodan were used as donor-acceptor pairs for fluorescence resonance energy transfer (FRET), average distances could be measured. Both intensity and lifetime changes of the Trp emission indicated a protein folding in solution that brings the C-terminus of the protein near the Trp residues in the N-terminal half of the sequence. Also, the N- and C-terminal domains of apoA-I appeared to be near each other in rHDL having two apoA-I per particle.     

Functional implications of the propionate 7-arginine 220 interaction in the FixLH oxygen sensor from Bradyrhizobium japonicum

BjFixL from Bradyrhizobium japonicum is a heme-based oxygen sensor implicated in the signaling cascade that enables the bacterium to adapt to fluctuating oxygen levels. Signal transduction is initiated by the binding of O(2) to the heme domain of BjFixL, resulting in protein conformational changes that are transmitted to a histidine kinase domain. We report structural changes of the heme and its binding pocket in the Fe(II) deoxy and Fe(III) met states of the wild-type BjFixLH oxygen sensor domain and four mutants of the highly conserved residue arginine 220. UV-visible, electron paramagnetic resonance, and resonance Raman spectroscopies all showed that the heme iron of the R220H mutant is unexpectedly six-coordinated at physiological pH in the Fe(III) state but undergoes pH- and redox-dependent coordination changes. This behavior is unprecedented for FixL proteins, but is reminiscent of another oxygen sensor from E. coli, EcDos. All mutants in their deoxy states are five-coordinated Fe(II), although we report rupture of the residue 220-propionate 7 interaction and structural modifications of the heme conformation as well as propionate geometry and flexibility. In this work, we conclude that part of the structural reorganization usually attributed to O(2) binding in the wild-type protein is in fact due to rupture of the Arg220-P7 interaction. Moreover, we correlate the structural modifications of the deoxy Fe(II) states with k(on) values and conclude that the Arg220-P7 interaction is responsible for the lower O(2) and CO k(on) values reported for the wild-type protein.     

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.     

Nucleotide binding induces global and local structural changes of myosin head in muscle fibres.

Thermal stability and internal dynamics of myosin heads in fiber bundles from rabbit psoas muscle has been studied by electron paramagnetic resonance (EPR) spectroscopy and differential scanning calorimetry (DSC). Using ADP, ATP and orthovanadate (V(i)), three intermediate states of the ATP hydrolysis cycle were simulated in glycerinated muscle fibers. DSC transitions contained three overlapping endotherms in each state. Deconvolution showed that the transition temperature of 58.4 degrees C was almost independent of the intermediate state of myosin, while nucleotide binding shifted the melting temperatures of 54.0 and 62.3 degrees C, and changed the enthalpies. These changes suggest global rearrangements of the internal structure in myosin head. In the presence of ADP and ADP plus V(i), the conventional EPR spectra showed changes in the ordering of the probe molecules, suggesting local conformational and motional changes in the internal structure of myosin heads. Saturation transfer EPR measurements reported increased rotational mobility of spin labels in the presence of ATP plus orthovanadate corresponding to a weakly binding state of myosin to actin.     

Hydrophobic loop dynamics and actin filament stability

It has been postulated that the hydrophobic loop of actin (residues 262-274) swings out and inserts into the opposite strand in the filament, stabilizing the filament structure. Here, we analyzed the hydrophobic loop dynamics utilizing four mutants that have cysteine residues introduced at a single location along the yeast actin loop. Lateral, copper-catalyzed disulfide cross-linking of the mutant cysteine residues to the native C374 in the neighboring strand within the filament was fastest for S265C, followed by V266C, L267C, and then L269C. Site-directed spin labeling (SDSL) studies revealed that C265 lies closest to C374 within the filament, followed by C266, C267, and then C269. These results are not predicted by the Holmes extended loop model of F-actin. Furthermore, we find that disulfide cross-linking destroys L267C and L269C filaments; only small filaments are observed via electron microscopy. Conversely, phalloidin protects the L267C and L269C filaments and inhibits their disulfide cross-linking. Combined, our data indicate that, in solution, the loop resides predominantly in a "parked" position within the filament but is able to dynamically populate other conformational states which stabilize or destabilize the filament. Such states may be exploited within a cell by filament-stabilizing and -destabilizing factors.     

The Uncoupling of the Effects of Formins on the Local and Global Dynamics of Actin Filaments

In this study, experiments were carried out in the conventional and saturation-transfer electron paramagnetic resonance (EPR) time domains to explore the effect of mDia1-FH2 formin fragments on the dynamic and conformational properties of actin filaments. Conventional EPR measurements showed that addition of formin to actin filaments produced local conformational changes in the vicinity of Cys-374 by increasing the flexibility of the protein matrix in the environment of the label. The results indicated that it was the binding of formin to the barbed end that resulted in these conformational changes. The conventional EPR results obtained with actin labeled on the Lys-61 site showed that the binding of formins could only slightly affect the structure of the subdomain 2 of actin, reflecting the heterogeneity of the formin-induced conformational changes. Saturation transfer EPR measurements revealed that the binding of formins decreased the torsional flexibility of the actin filaments in the microsecond time range. We concluded that changes in the local and the global conformational fluctuations of the actin filaments are associated with the binding of formins to actin. The results on the two EPR time domains showed that the effects of formins on the substantially different types of motions were uncoupled.     

Molecular motion of spin labeled side chains in alpha-helices: analysis by variation of side chain structure

Two single cysteine substitution mutants at helix surface sites in T4 lysozyme (D72C and V131C) have been modified with a series of nitroxide methanethiosulfonate reagents to investigate the structural and dynamical origins of their electron paramagnetic resonance spectra. The novel reagents include 4-substituted derivatives of either the pyrroline or pyrrolidine series of nitroxides. The spectral line shapes were analyzed as a function of side chain structure and temperature using a simulation method with a single order parameter and diffusion rates about three orthogonal axes as parameters. Taken together, the results provide strong support for an anisotropic motional model of the side chain, which was previously proposed from qualitative features of the spectra and crystal structures of spin labeled T4 lysozyme. Site-specific differences in apparent order parameter are interpreted in terms of backbone dynamics modes with characteristic correlation times in the nanosecond or faster time scale. The saturated 4-substituted pyrrolidine nitroxides are shown to be a suitable template for novel "functionalized" side chains designed to mimic salient features of the native side chains they replace.     

Conformational changes of actin induced by calponin.

Calponin, an actin-linked regulatory protein in smooth muscle, caused a remarkable change in the fluorescence intensity of pyrene-labeled actin in the filamentous form. Calponin, an equimolar ratio to actin, decreased the fluorescence intensity of pyrene-labeled F-actin by some 60% to the level near monomeric actin. This change was partially reversed by Ca2+, when calmodulin was present. Thus it appears that calponin causes conformational changes in actin molecules in an actin filament so as to inhibit their interactions with myosin.