Bifunctional Rhodamine Research Articles

Changes in the Conformation of Troponin C on Activation of Skeletal Muscle

Skeletal muscle contraction is regulated by calcium-dependent changes in the structure and thin-filament location of troponin and tropomyosin. The structural changes in the isolated calcium-binding subunit of troponin (TnC) are well characterized, but those of TnC in the native thin filament are much less clear. We measured the in situ orientation of the C-terminal lobe of TnC (CTnC) by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteines at TnC residues 96-103, 116-123, 132-139, and 119-135. Each BR-labeled TnC was exchanged into single permeablized fibers from rabbit psoas muscle, and polarized fluorescence from the BR-TnCs was measured during relaxation and maximal calcium activation. The orientation distribution of CTnC with respect to the thin filament axis was calculated by maximum entropy analysis using the in vitro structure of CTnC in the troponin core complex (Vinogradova et al. (2005) PNAS102:5038-5043). The peak angle between E helix of CTnC and the filament axis was 49° in relaxed muscle and 64° during maximal activation. Comparison with the results of our previous study of the orientation of the N-terminal lobe of TnC (Ferguson et al. (2003) Mol. Cell11: 865-874) suggests that the central D/E helix of TnC is bent by about 30° in relaxed muscle and becomes straight during maximal activation.Supported by MRC (UK).

Myosin VI undergoes a 180° power stroke implying an uncoupling of the front lever arm

We simultaneously measure both the step size, via FIONA, and the 3-D orientation, via DOPI, of the light-chain domain of individual dimeric myosin VIs. This allows for the correlation of the change in orientation of the light chain domain to the stepping of the motor. Three different pairs of positions were tested using a rigid bifunctional rhodamine on the calmodulin of the IQ domain. The data for all three labeling positions support the model that the light chain domain undergoes a significant rotation of approximately 180 degrees . Contrary to an earlier study [Sun, Y. et al. (2007) Mol Cell 28, 954-964], our data does not support a model of multiple angles of the lever arm of the lead head, nor "wiggly" walking on actin. Instead, we propose that for the two heads of myosin VI to coordinate their processive movement, the lever arm of the lead head must be uncoupled from the converter until the rear head detaches. More specifically, intramolecular strain causes the myosin VI lever arm of the lead head to uncouple from the motor domain, allowing the motor domain to go through its product-release (phosphate and ADP) steps at an unstrained rate. The lever arm of the lead head rebinds to the motor and attains a rigor conformation when the rear head detaches. By coupling the orientation and position information with previously described kinetics, this allows us to explain how myosin VI coordinates its heads processively while maintaining the ability to move under load with a (semi-) rigid lever arm.

Structural Dynamics of SERCA and Phospholamban by Fluorescence and Phosphorescence

We used fluorescence and phosphorescence to investigate the structure and dynamics of phospholamban (PLB), and its regulation of its inhibited target, sarcoplasmic reticulum calcium ATPase (SERCA). Polarized TIRF of PLB, labeled in the cytoplasmic domain helix with bifunctional rhodamine (BFR), shows that this domain lies parallel to the membrane surface. The structural and functional effects of PLB phosphorylation and mutation are under investigation. Single molecular TIRF microscopy was used to measure the translational diffusional coefficient of Alexa488 labeled monomeric PLB reconstituted into a supported lipid bilayer. The diffusional coefficient of monomeric PLB is 0.7 μm2/s, which is consistent with its molecular weight. Time-resolved phosphorescence anisotropy of erythrosin iodoacetamide (ErIA) labeled SERCA in cardiac sarcoplasmic reticulum (SR) was measured with and without phosphorylation of PLB in presence of high and low Ca concentrations. Phosphorylation of PLB decreased the final anisotropy of ErIA labeled SERCA at low Ca, indicating decreased SERCA self-association. This supports the proposal that PLB inhibits SERCA by inducing SERCA-SERCA association, which is relieved by phosphorylation.

Lever Arm Length Determines The Azimuthal But Not The Axial Orientation Of Myosin V During Processive Motility

In order to assess how lever arm length affects the three-dimensional motions of myosin V during processive motility, two constructs were studied using single molecule polarized total internal reflection fluorescence (polTIRF) microscopy. MyoV6IQ and MyoV4IQ contain 6 and 4 calmodulin (CaM) binding IQ motifs, and otherwise consist of the native myosin V excluding the tail domain. Bifunctional rhodamine labeled CaM replaced a native CaM, giving probe angles βP relative to the actin axis and αP, the azimuth around actin. As with other processive myosins, αP and βP exhibited tilting of the probe with each step. With MyoV6IQ, αP often returned to its initial value after two steps, as expected for nearly straight walking. This behavior enabled us to determine the orientation of the lever arm, αL and βL, as well as θL and fL, the probe angles relative to CaM. βL was 100° and 40° in the leading and trailing heads, respectively. In MyoV4IQ, βP was similar to 6IQ, but αP seldom returned to its earlier value after two steps. This indicates considerable net azimuthal rotation, as expected for smaller step sizes. Thus, lever arm length determines the azimuthal angular path, whereas the axial orientation is likely determined by structural constraints in the motor domain. Modified gliding filament assays were performed using polTIRF to detect twirling of actin about its axis during motility. MyoV6IQ twirled almost exclusively left-handed with a pitch of 1.4 μm. MyoV4IQ twirled with both right- and left-handed pitches of 1.0 and 1.2 μm, respectively. Bidirectional twirling of MyoV4IQ contrasts with every isoform of myosin previously tested (II, native V, VI and X) all of which twirled with a single handedness. This work was supported by NIH grant AR05117.

Calcium‐ and myosin‐dependent changes in troponin structure during activation of heart muscle

Each heartbeat is triggered by a pulse of intracellular calcium ions which bind to troponin on the actin-containing thin filaments of heart muscle cells, initiating a change in filament structure that allows myosin to bind and generate force. We investigated the molecular mechanism of calcium regulation in demembranated trabeculae from rat ventricle using polarized fluorescence from probes on troponin C (TnC). Native TnC was replaced by double-cysteine mutants of human cardiac TnC with bifunctional rhodamine attached along either the C helix, adjacent to the regulatory Ca(2+)-binding site, or the E helix in the IT arm of the troponin complex. Changes in the orientation of both troponin helices had the same steep Ca(2+) dependence as active force production, with a Hill coefficient (n(H)) close to 3, consistent with a single co-operative transition controlled by Ca(2+) binding. Complete inhibition of active force by 25 microM blebbistatin had very little effect on the Ca(2+)-dependent structural changes and in particular did not significantly reduce the value of n(H). Binding of rigor myosin heads to thin filaments following MgATP depletion in the absence of Ca(2+) also changed the orientation of the C and E helices, and addition of Ca(2+) in rigor produced further changes characterized by increased Ca(2+) affinity but with n(H) close to 1. These results show that, although myosin binding can switch on thin filaments in rigor conditions, it does not contribute significantly under physiological conditions. The physiological mechanism of co-operative Ca(2+) regulation of cardiac contractility must therefore be intrinsic to the thin filaments.

Conformation and Dynamics of a Rhodamine Probe Attached at Two Sites on a Protein: Implications for Molecular Structure Determination in situ

Replica exchange molecular dynamics (REMD) calculations were used to determine the conformation and dynamics of bifunctional rhodamine probes attached to pairs of cysteines in three model systems: (a) a polyalanine helix, (b) the isolated C helix (residues 53-66) of troponin C, and (c) the C helix of the N-terminal region (residues 1-90) of troponin C (sNTnC). In each case, and for both diastereoisomers of each probe-protein complex, the hydrophobic face of the probe is close to the protein surface, and its carboxylate group is highly solvated. The visible-range fluorescence dipole of the probe is approximately parallel to the line joining the two cysteine residues, as assumed in previous in situ fluorescence polarization studies. The independent rotational motion of the probe with respect to the protein on the nanosecond time scale is highly restricted, in agreement with data from fluorescence polarization and NMR relaxation studies. The detailed interaction of the probe with the protein surface depends on steric factors, electrostatic and hydrophobic interactions, hydrogen bonds, and hydration effects. The interaction is markedly different between diastereoisomers, and multiple preferred conformations exist for a single diasteroisomer. These results show that the combination of the hydrophobic xanthylium moiety of bifunctional rhodamine with the carboxylate substitution in its pendant phenyl ring causes the probe to be immobilized on the protein surface, while the two-site cysteine attachment defines the orientation of its fluorescence dipole. These features allow the orientation of protein components to be accurately determined in situ by polarized fluorescence measurements from bifunctional rhodamine probes.

NMR Studies of the Dynamics of a Bifunctional Rhodamine Probe Attached to Troponin C

Fluorescence polarization measurements of bifunctional rhodamine (BR) probes provide a powerful approach to determine the in situ orientation of proteins within ordered complexes such as muscle fibers. For accurate interpretation of fluorescence measurements, it is important to understand the probe dynamics relative to the protein to which it is attached. We previously determined the structure of the N-domain of chicken skeletal troponin C, BR-labeled on the C helix, in complex with the switch region of troponin I, and demonstrated that the probe does not perturb the structure or dynamics of the protein. In this study, the motion of the fluorescence label relative to the protein has been characterized using NMR relaxation measurements of 13C-labeled methyl groups on the BR probe and 15N-labeled backbone amides of the protein. Probe dynamics were monitored using off-resonance 13C-R(1rho), 13C-R(1) and {1H}-13C NOE at magnetic field strengths of 500, 600, and 800 MHz. Relaxation data were interpreted in terms of the overall rotational correlation time of the protein and a two-time scale model for internal motion of the BR methyl groups, using a numerical optimization with Monte Carlo parameter error estimation. The analysis yields a 1.5 +/- 0.4 ps correlation time for rotation around the three-fold methyl symmetry axis, and a 0.8 +/- 0.4 ns rotational correlation time for reorientation of the 13C-14N bond with an associated S2s of 0.79 +/- 0.03. Order parameters of the backbone NH vectors in the helix to which the probe is attached average S2 approximately 0.85, implying that the amplitude of independent reorientation of the BR probe is small in magnitude, consistent with results from fluorescence polarization measurements in reconstituted muscle fibers.

Toward Protein Structure In Situ: Comparison of Two Bifunctional Rhodamine Adducts of Troponin C

As part of a program to develop methods for determining protein structure in situ, sTnC was labeled with a bifunctional rhodamine (BR or BSR), cross-linking residues 56 and 63 of its C-helix. NMR spectroscopy of the N-terminal domain of BSR-labeled sTnC in complex with Ca 2+ and the troponin I switch peptide (residues 115–131) showed that BSR labeling does not significantly affect the secondary structure of the protein or its dynamics in solution. BR-labeling was previously shown to have no effect on the solution structure of this complex. Isometric force generation in isolated demembranated fibers from rabbit psoas muscle into which BR- or BSR-labeled sTnC had been exchanged showed reduced Ca 2+-sensitivity, and this effect was larger with the BSR label. The orientation of rhodamine dipoles with respect to the fiber axis was determined by polarized fluorescence. The mean orientations of the BR and BSR dipoles were almost identical in relaxed muscle, suggesting that both probes accurately report the orientation of the C-helix to which they are attached. The BSR dipole had smaller orientational dispersion, consistent with less flexible linkers between the rhodamine dipole and cysteine-reactive groups.

Structural changes in troponin in response to Ca 2+ and myosin binding to thin filaments during activation of skeletal muscle

Contraction of skeletal and cardiac muscle is regulated by Ca2+ -dependent structural changes in troponin that control the interaction between myosin and actin. We measured the orientations of troponin domains in skeletal muscle fibers using polarized fluorescence from bifunctional rhodamine probes on the C and E helices of troponin C. The C helix, in the regulatory head domain, tilts by approximately 30 degrees when muscle is activated in physiological conditions, with a Ca2+ -sensitivity similar to that of active force. Complete inhibition of active force did not affect C-helix orientation, and binding of rigor myosin heads did not affect its orientation at saturating [Ca2+]. The E helix, in the IT arm of troponin, tilted by approximately 10 degrees on activation, and this was reduced to only 3 degrees when active force was inhibited. Binding of rigor myosin heads produced a larger tilt of the E helix. Thus, in situ, the regulatory head acts as a pure Ca2+ -sensor, whereas the IT arm is primarily sensitive to myosin head binding. The polarized fluorescence data from active muscle are consistent with an in vitro structure of the troponin core complex in which the D and E helices of troponin C are collinear. The present data were used to orient this structure in the fiber and suggest that the IT arm is at approximately 30 degrees to the filament axis in active muscle. In relaxed muscle, the IT arm tilts to approximately 40 degrees but the D/E helix linker melts, allowing the regulatory head to tilt through a larger angle.

Defocused orientation and position imaging (DOPI) of myosin V.

The centroid of a fluorophore can be determined within approximately 1.5-nm accuracy from its focused image through fluorescence imaging with one-nanometer accuracy (FIONA). If, instead, the sample is moved away from the focus, the point-spread-function depends on both the position and 3D orientation of the fluorophore, which can be calculated by defocused orientation and position imaging (DOPI). DOPI does not always yield position accurately, but it is possible to switch back and forth between focused and defocused imaging, thereby getting the centroid and the orientation with precision. We have measured the 3D orientation and stepping behavior of single bifunctional rhodamine probes attached to one of the calmodulins of the light-chain domain (LCD) of myosin V as myosin V moves along actin. Concomitant with large and small steps, the LCD rotates and then dwells in the leading and trailing position, respectively. The probe angle relative to the barbed end of the actin (beta) averaged 128 degrees while the LCD was in the leading state and 57 degrees in the trailing state. The angular difference of 71 degrees represents rotation of LCD around the bound motor domain and is consistent with a 37-nm forward step size of myosin V. When beta changes, the probe rotates +/-27 degrees azimuthally around actin and then rotates back again on the next step. Our results remove degeneracy in angles and the appearance of nontilting lever arms that were reported.

Adaptability of myosin V studied by simultaneous detection of position and orientation

We studied the structural dynamics of chicken myosin V by combining the localization power of fluorescent imaging with one nanometer accuracy (FIONA) with the ability to detect angular changes of a fluorescent probe. The myosin V was labeled with bifunctional rhodamine on one of its calmodulin light chains. For every 74 nm translocation, the probe exhibited two reorientational motions, associated with alternating smaller and larger translational steps. Molecules previously identified as stepping alternatively 74-0 nm were found to actually step 64-10 nm. Additional tilting often occurred without full steps, possibly indicating flexibility of the attached myosin heads or probing of their vicinity. Processive myosin V molecules sometimes shifted from the top to the side of actin, possibly to avoid an obstacle. The data indicate marked adaptability of this molecular motor to a nonuniform local environment and provide strong support for a straight-neck model of myosin V in which the lever arm of the leading head is tilted backwards at the prepowerstoke angle.

Bifunctional Rhodamine Probes of Myosin Regulatory Light Chain Orientation in Relaxed Skeletal Muscle Fibers

The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence from bifunctional rhodamine (BR) probes cross-linking pairs of cysteine residues introduced into the RLC. Pure 1:1 BR-RLC complexes were exchanged into single muscle fibers in EDTA rigor solution for 30 min at 30°C; ∼60% of the native RLC was removed and stoichiometrically replaced by BR-RLC, and >85% of the BR-RLC was located in the sarcomeric A-bands. The second- and fourth-rank order parameters of the orientation distributions of BR dipoles linking RLC cysteine pairs 100-108, 100-113, 108-113, and 104-115 were calculated from polarized fluorescence intensities, and used to determine the smoothest RLC orientation distribution—the maximum entropy distribution—consistent with the polarized fluorescence data. Maximum entropy distributions in relaxed muscle were relatively broad. At the peak of the distribution, the “lever” axis, linking Cys 707 and Lys 843 of the myosin heavy chain, was at 70–80° to the fiber axis, and the “hook” helix (Pro 830–Lys 843) was almost coplanar with the fiber and lever axes. The temperature and ionic strength of the relaxing solution had small but reproducible effects on the orientation of the RLC region.

The NMR Structure of a Bifunctional Rhodamine Labeled N-domain of Troponin C inComplex with the Regulatory 'Switch' Peptide from Troponin I

The NMR Structure of a Bifunctional Rhodamine Labeled N-domain of Troponin C inComplex with the Regulatory 'Switch' Peptide from Troponin I

In Situ Orientations of Protein Domains: Troponin C in Skeletal Muscle Fibers

A recently developed approach for mapping protein-domain orientations in the cellular environment was used to investigate the Ca(2+)-dependent structural changes in the tropomyosin/troponin complex on the actin filament that regulate muscle contraction. Polarized fluorescence from bifunctional rhodamine probes attached along four alpha helices of troponin C (TnC) was measured in permeabilized skeletal muscle fibers. In relaxed muscle, the N-terminal lobe of TnC is less closed than in crystal structures of the Ca(2+)-free domain, and its D helix is approximately perpendicular to the actin filament. In contrast to crystal structures of isolated TnC, the D and E helices are not collinear. On muscle activation, the N lobe orientation becomes more disordered and the average angle between the C helix and the filament changes by 32 degrees +/- 5 degrees. These results illustrate the potential of in situ measurements of helix and domain orientations for elucidating structure-function relations in native macromolecular complexes.

NMR structure of a bifunctional rhodamine labeled N-domain of troponin C complexed with the regulatory "switch" peptide from troponin I: implications for in situ fluorescence studies in muscle fibers.

The structure of the calcium-saturated regulatory domain of skeletal troponin C (sNTnC) complexed with the switch peptide comprising residues 115-131 of troponin I (TnI), and with a bifunctional rhodamine fluorescent label attached to residues 56 (E56C) and 63 (E63C) on the C helix of sNTnC, has been determined using nuclear magnetic resonance (NMR) spectroscopy. The structure shows that the integrity of the C helix is not altered by the E(56,63)C mutations or by the presence of the bifunctional rhodamine and that the label does not interact with the hydrophobic cleft of sNTnC. Moreover, the overall fold of the protein and the position of the TnI peptide are similar to those observed previously with related cardiac NTnC complexes with residues 147-163 of cardiac TnI [Li et al. (1999) Biochemistry 38, 8289-8298] and including the drug bepridil [Wang et al. (2002) J. Biol. Chem. 277, 31124-31133]. The degree of opening of the structure is reduced as compared to that of calcium-saturated sNTnC in the absence of the switch peptide [Gagné et al. (1995) Nat. Struct. Biol. 2, 784-789]. The switch peptide is bound in a shallow and complementary hydrophobic surface cleft largely defined by helices A and B and also has key ionic interactions with sNTnC. These results show that bifunctional rhodamine probes can be attached to surface helices via suitable pairs of solvent-accessible residues that have been mutated to cysteines, without altering the conformation of the labeled domain. A set of such probes can be used to determine the orientation and motion of the target domain in the cellular environment [Corrie et al. (1999) Nature 400, 425-430; Ferguson et al. (2003) Mol. Cell 11(4), in press].

Orientation Changes of the Myosin Light Chain Domain During Filament Sliding in Active and Rigor Muscle

Structural changes in myosin power many types of cell motility including muscle contraction. Tilting of the myosin light chain domain (LCD) seems to be the final step in transducing the energy of ATP hydrolysis, amplifying small structural changes near the ATP binding site into nanometer-scale motions of the filaments. Here we used polarized fluorescence measurements from bifunctional rhodamine probes attached at known orientations in the LCD to describe the distribution of orientations of the LCD in active contraction and rigor. We applied rapid length steps to perturb the orientations of the population of myosin heads that are attached to actin, and thereby characterized the motions of these force-bearing myosin heads. During active contraction, this population is a small fraction of the total. When the filaments slide in the shortening direction in active contraction, the long axis of LCD tilts towards its nucleotide-free orientation with no significant twisting around this axis. In contrast, filament sliding in rigor produces coordinated tilting and twisting motions.

A Maximum Entropy Analysis of Protein Orientations Using Fluorescence Polarization Data from Multiple Probes

Techniques have recently become available to label protein subunits with fluorescent probes at predetermined orientation relative to the protein coordinates. The known local orientation enables quantitative interpretation of fluorescence polarization experiments in terms of orientation and motions of the protein within a larger macromolecular assembly. Combining data obtained from probes placed at several distinct orientations relative to the protein structure reveals functionally relevant information about the axial and azimuthal orientation of the labeled protein segment relative to its surroundings. Here we present an analytical method to determine the protein orientational distribution from such data. The method produces the broadest distribution compatible with the data by maximizing its informational entropy. The key advantages of this approach are that no a priori assumptions are required about the shape of the distribution and that a unique, exact fit to the data is obtained. The relative orientations of the probes used for the experiments have great influence on information content of the maximum entropy distribution. Therefore, the choice of probe orientations is crucial. In particular, the probes must access independent aspects of the protein orientation, and two-fold rotational symmetries must be avoided. For a set of probes, a “figure of merit” is proposed, based on the independence among the probe orientations. With simulated fluorescence polarization data, we tested the capacity of maximum entropy analysis to recover specific protein orientational distributions and found that it is capable of recovering orientational distributions with one and two peaks. The similarity between the maximum entropy distribution and the test distribution improves gradually as the number of independent probe orientations increases. As a practical example, ME distributions were determined with experimental data from muscle fibers labeled with bifunctional rhodamine at known orientations with respect to the myosin regulatory light chain (RLC). These distributions show a complex relationship between the axial orientation of the RLC relative to the fiber axis and the azimuthal orientation of the RLC about its own axis. Maximum entropy analysis reveals limitations in available experimental data and supports the design of further probe angles to resolve details of the orientational distribution.

Dynamic measurement of myosin light-chain-domain tilt and twist in muscle contraction.

A new method is described for measuring motions of protein domains in their native environment on the physiological timescale. Pairs of cysteines are introduced into the domain at sites chosen from its static structure and are crosslinked by a bifunctional rhodamine. Domain orientation in a reconstituted macromolecular complex is determined by combining fluorescence polarization data from a small number of such labelled cysteine pairs. This approach bridges the gap between in vitro studies of protein structure and cellular studies of protein function and is used here to measure the tilt and twist of the myosin light-chain domain with respect to actin filaments in single muscle cells. The results reveal the structural basis for the lever-arm action of the light-chain domain of the myosin motor during force generation in muscle.

A homobifunctional rhodamine for labeling proteins with defined orientations of a fluorophore.

The synthesis and characterization of a bifunctional rhodamine dye bearing 2-(iodoacetamido)ethyl substituents on the 3'- and 6'-nitrogen atoms is described. Aspects of the conversion of chloroacetamides to iodoacetamides are discussed, including a remarkably mild dehalogenation of an aromatic haloacetamide in the presence of NaI and camphorsulfonic acid. The bifunctional rhodamine has been designed for two-site, 1:1 labeling of proteins that contain two suitably disposed cysteine residues and is intended to constrain the orientation of the rhodamine absorption and emission dipoles in a predictable relationship to the protein structure.

On the Treatment of Puerperal Convulsions

We simultaneously measure both the step size, via FIONA, and the 3-D orientation, via DOPI, of the light-chain domain of individual dimeric myosin VIs. This allows for the correlation of the change in orientation of the light chain domain to the stepping of the motor. Three different pairs of positions were tested using a rigid bifunctional rhodamine on the calmodulin of the IQ domain. The data for all three labeling positions support the model that the light chain domain undergoes a significant rotation of approximately 180°. Contrary to an earlier study [Sun, Y. et al. (2007) Mol Cell 28, 954–964], our data does not support a model of multiple angles of the lever arm of the lead head, nor “wiggly” walking on actin. Instead, we propose that for the two heads of myosin VI to coordinate their processive movement, the lever arm of the lead head must be uncoupled from the converter until the rear head detaches. More specifically, intramolecular strain causes the myosin VI lever arm of the lead head to uncouple from the motor domain, allowing the motor domain to go through its product-release (phosphate and ADP) steps at an unstrained rate. The lever arm of the lead head rebinds to the motor and attains a rigor conformation when the rear head detaches. By coupling the orientation and position information with previously described kinetics, this allows us to explain how myosin VI coordinates its heads processively while maintaining the ability to move under load with a (semi-) rigid lever arm.