Bifunctional Rhodamine Research Articles

Modeling the troponin core domain on thin filaments using data from cryoelectron microscopy and fluorescence approaches.

Our understanding of thin filament structure has been greatly enhanced by the publication of a number of models derived from cryo-electron microscopy studies (Yamada et al. (2020) and then later confirmed by Risi et al. (2021)). These studies elucidated atomic level details of the calcium-dependent activation of the thin filament leading to force development in cardiac muscle. However, while cryo-EM reconstruction is best suited to capture the high-resolution organization of static structures, troponin and tropomyosin are dynamic components of the thin filament and corresponding disorder is not always easily recorded and classified by the method. Thus, in order to better model these structures, it may be necessary to generate small ensembles of structures to incorporate known sources of disorder as guided by information derived from other biophysical techniques. One example is for troponin subunit C, which binds calcium and the troponin subunit I switch peptide during thin filament activation. While the cryo-EM structure has offered a single computed conformation of troponin C, polarized fluorescence of the troponin core domain labeled with bifunctional rhodamine indicates that in cardiac muscle troponin C is very dynamic, and its N-terminal lobe takes up multiple distinct orientations (Sevrieva et al., 2014). In this study, we incorporate data derived both from structural methodologies to build a set of unique models that satisfy the cryo-EM and the fluorescence data. Analysis of these structures shows the details of how the dynamic nature of the N-lobe of troponin C is important for its function in thin filament regulation.

Regulation of troponin and myosin conformation in thin- and thick-filament domains in the sarcomere of skeletal muscle myofibrils.

The contractility of striated muscle is controlled by a dual-filament regulatory mechanism which couples calcium-dependent structural changes in thin filaments with those in thick filaments triggering the release of folded-helical myosin motors from the filament surface. The folded-helical conformation of motors in relaxed muscle may be stabilized in the region of the thick filament containing myosin-binding protein-C (C-zone). Moreover, in active muscle actin-bound myosin motors might locally alter the activation level of the thin filament. To test these hypotheses we developed a novel fluorescence-based approach to measure troponin and myosin orientation in subdomains of the sarcomere in isolated myofibrils. Bifunctional rhodamine probes labelling troponin-C (TnC) and the myosin regulatory light chain (RLC) were exchanged into myofibrils from rabbit psoas muscle, and the spatial distribution of probe orientations in the sarcomere was measured by fluorescence polarization microscopy. In the absence of ATP, the RLCs of actin-attached myosin motors were perpendicular to the filament axis and the orientation of the TnC probes in the region of overlap with thick filaments was markedly different from that in the rest of the sarcomere, indicating that myosin binding to actin alters the conformation of troponin in the thin filament. In relaxing conditions all TnC probes had the same conformation which was insensitive to temperature. In contrast, the RLC probes became more parallel to the filament axis as temperature was increased above 12°C, and at 30°C in the presence of 4% Dextran their orientation was more parallel in the C-zone, indicating enrichment of folded myosin motors in this filament domain. These results show directly that troponin and myosin have distinct conformations in different sarcomere subdomains in situ (supported by the Wellcome Trust and the Royal Society, UK).

Rhodamine-Installed Polynorbornenes: Molecular Design, Structure, and Stimuli-Responsive Properties.

The synthesis of a number of tailored architectures of rhodamine dye–norbornene conjugate monomers and corresponding homopolymers derived from them is described. The impact of the monomer architecture on the mechanochromic, photochromic, and thermochromic properties of rhodamine-modified polynorbornenes is reported. Color changes were caused by the reversible interconversion between the “open” and “closed” spirolactam form of the covalently attached dye. Monomers were synthesized in two principle architectures that varied on: (1) the number of polymerizable norbornene groups tethered to a bifunctional rhodamine dye; (2) the presence of flexible methylene spacers between the dye and the polymerizable norbornene groups. Introduction of norbornene groups on each of the two hydroxy groups of a bifunctional rhodamine resulted in a cross-linked polymer that exhibited better mechanochromic, photochromic, and thermochromic properties compared to the corresponding polymer without cross-links, derived from the derivatization of bifunctional rhodamine with only one norbornene. The introduction of flexible methylene spacers between the two polymerizable norbornenes and the dye molecule resulted in a polymeric framework with rapidly reversible color-changing properties upon mechanical or photostimulation. The ideal monomer molecular structure, whereby (1) attaching norbornene on both sides of the rhodamine dye and (2) methylene spacers between the dye and norbornenes on both sides afforded the nonpareil polymer structure that was capable of thermoreversible mechanochromic and photochromic features, and irreversible thermochromic features. These new materials may find utility as multi-stimuli-responsive soft materials.

Preparation of traceable heterobifunctional cross-linking probes from an unsymmetrical bifunctional rhodamine

We describe the preparation and utility of fluorescent heterobifunctional probes derived from an easily prepared bifunctional rhodamine core possessing carboxylic and sulfonic acid functional groups. Trichloroacetonitrile/PPh3 was employed as a mild chlorinating reagent for this complex system to allow functionalization of the sulfonic acid. This technique is described as an alternative to harsh chlorinating conditions. The synthesized probes allow for the introduction of functionality (ie. biotin) to biomolecules and direct quantification of incorporation through absorbance or fluorescence measurements.

A bifunctional rhodamine derivative as chemosensor for recognizing Cu2+ and Hg2+ ions via different spectra

A new simple bifunctional chemosensor 1 based on rhodamine was synthesized by hydrazide and formylformic acid, which could detect Cu2+ and Hg2+via different detecting methods in CH3CN-HEPES buffer solution (20 mmol/L, pH 7.4) (1:9, v/v) respectively. When sensor 1 bound with Cu2+, it showed a colorimetric change, while a selective enhancement in fluorescence occurred upon 1 binding with Hg2+, resulting from the spirolatam-ring opening process. The binding modes of 1 with Cu2+ and Hg2+ were investigated based on UV, fluorescence change, ESI-Mass and Job’s Plot data. Moreover, sensor 1 could selectively detect target ion in a mixed solution of Cu2+ and Hg2+, and the two metal ions do not interfere with each other in the process of detecting Cu2+ or Hg2+ with 1.

Resolution of ångström-scale protein conformational changes by analyzing fluorescence anisotropy.

SummaryConformational changes within typical protein molecules are rapid and small, making their quantitative resolution challenging. These changes generally involve rotational motions, and may thus be resolved by determining changes in the orientation of a fluorescent label that assumes a unique orientation in each conformation. Here, by analyzing fluorescence intensities collected using a polarization microscope at a rate of 50 frames per second, we can follow the changes of 10 to 16° in the orientation of a single bifunctional rhodamine molecule attached to an RCK domain of the MthK channel, and thus the transitions among its three conformational states, with effective standard deviation (σ) of 2 to 5°. Based on available crystal structures, the position of the fluorophore's center differ by 3.4 to 8.1 Å among the states. Thus, the present approach allows the resolution of protein-conformational changes involving angstrom-scale displacements.

Structural Kinetics of the RLC Domain of Myosin during Activation of Skeletal Muscle Fibers by Photolysis of Caged-Calcium

Recent fluorescence polarization studies in skeletal muscle fibers using bifunctional rhodamine (BSR) probes on the C-lobe of the regulatory light chain (RLC) of myosin showed that the steady-state calcium-sensitivity and co-operativity of the orientation change of the RLC C-lobe are higher than those of force (Fusi et al., Nat Commun 2016,doi:10.1038/ncomms13281). We have now investigated the in situ kinetics of the conformational change of the RLC during isometric contraction of a muscle fiber triggered by a rapid (>104 s−1) increase in [Ca2+] following UV-photolysis of caged-calcium (NP-EGTA). We monitored by polarized fluorescence changes in the orientation of the E-helix of a BSR-labeled RLC exchanged into demembranated fibers from rabbit psoas muscle during activation by photolysis of NP-EGTA, under conditions which preserve the physiological resting structure of the thick filament (T=25°C, 5% Dextran, 2.45 µm sarcomere length). After photolysis force increased with a sigmoidal time course similar to that of an isometric tetanus in an intact muscle fiber. The change in the order parameter P2 of the RLC probe, associated with it becoming more perpendicular to the fiber axis, also had a sigmoidal time course. The fractional change in P2 was faster than that of force. The P2 change was 25% complete 20ms after photolysis when activation of the thin filament is already maximal (T=12°C, Fusi et al., PNAS 2014, 111:4626-31) but force had developed to only 10% of its plateau value (T0). The P2 change was essentially complete by ca 70ms after photolysis when force was only 60% T0. These results show that the change in RLC orientation associated with the activation of the myosin motors is slower than thin-filament activation but faster than force generation. Supported by MRC, UK.

Bifunctional Rhodamine Linker Simplifies Colocalization Studies in Single-Molecule Imaging

Xanthene dyes (e.g., fluoresceins, rhodamines and rhodols) containing linkers for bio-conjugation are used extensively as fluorescent labels and probes to study biological systems. We have synthesized unsymmetrical, single isomer, bright, photostable rhodamine dyes. The asymmetric nature of the dye allowed us to introduce two independent functional groups into the rhodamine dye system. This, in turn, enabled us to generate a biotinylated and fluorescently-labeled protein, ready to be immobilized onto a streptavidin-coated surface for imaging.We demonstrated the utility of this bifunctional dye in a simple colocalization experiment. We showed that that by using anti-NGAL antibody attached to the biotin-linked rhodamine dye (bt-Rh-mAb), we can easily determine the surface density of this capture antibody on a glass slide. We reacted several different concentrations of bt-Rh-mAb onto a streptavidin surface, washed, and took single-molecule images. Simply counting the fluorophores generated a good estimate of the surface concentration of capture antibody. Without the fluorescent label, the surface concentration of the capture antibody must be either assumed or determined through a more complex and less direct process. We also conducted binding experiments using Cy5-labeled NGAL under equilibrium conditions. This colocalization measurement provided the fraction bound and thus a quick estimate of the dissociation constant.Lastly, we performed a sandwich immunoassay utilizing the bt-Rh-mAb capture antibody and a Cy5-labeled detection antibody. Colocalization analysis provided the analyte concentration and simultaneously reduced the contribution of nonspecific surface binding since “Cy5-only signals” were excluded. The bifunctional rhodamine linker is therefore a useful reagent to simplify a wide range of single-molecule experiments.

The Conformation of Myosin Heads in Relaxed Skeletal Muscle: Implications for Myosin-Based Regulation

In isolated thick filaments from many types of muscle, the two head domains of each myosin molecule are folded back against the filament backbone in a conformation called the interacting heads motif (IHM) in which actin interaction is inhibited. This conformation is present in resting skeletal muscle, but it is not known how exit from the IHM state is achieved during muscle activation. Here, we investigated this by measuring the in situ conformation of the light chain domain of the myosin heads in relaxed demembranated fibers from rabbit psoas muscle using fluorescence polarization from bifunctional rhodamine probes at four sites on the C-terminal lobe of the myosin regulatory light chain (RLC). The order parameter 〈P2〉 describing probe orientation with respect to the filament axis had a roughly sigmoidal dependence on temperature in relaxing conditions, with a half-maximal change at ∼19°C. Either lattice compression by 5% dextran T500 or addition of 25 μM blebbistatin decreased the transition temperature to ∼14°C. Maximum entropy analysis revealed three preferred orientations of the myosin RLC region at 25°C and above, two with its long axis roughly parallel to the filament axis and one roughly perpendicular. The parallel orientations are similar to those of the so-called blocked and free heads in the IHM and are stabilized by either lattice compression or blebbistatin. In relaxed skeletal muscle at near-physiological temperature and myofilament lattice spacing, the majority of the myosin heads have their light chain domains in IHM-like conformations, with a minority in a distinct conformation with their RLC regions roughly perpendicular to the filament axis. None of these three orientation populations were present during active contraction. These results are consistent with a regulatory transition of the thick filament in skeletal muscle associated with a conformational equilibrium of the myosin heads.

Regulatory domain of troponin moves dynamically during activation of cardiac muscle

Heart muscle is activated by Ca2+ to generate force and shortening, and the signaling pathway involves allosteric mechanisms in the thin filament. Knowledge about the structure-function relationship among proteins in the thin filament is critical in understanding the physiology and pathology of the cardiac function, but remains obscure. We investigate the conformation of the cardiac troponin (Tn) on the thin filament and its response to Ca2+ activation and propose a molecular mechanism for the regulation of cardiac muscle contraction by Tn based uniquely on information from in situ protein domain orientation. Polarized fluorescence from bifunctional rhodamine is used to determine the orientation of the major component of Tn core domain on the thin filaments of cardiac muscle. We show that the C-terminal lobe of TnC (CTnC) does not move during activation, suggesting that CTnC, together with the coiled coil formed by the TnI and TnT chains (IT arm), acts as a scaffold that holds N-terminal lobe of TnC (NTnC) and the actin binding regions of troponin I. The NTnC, on the other hand, exhibits multiple orientations during both diastole and systole. By combining the in situ orientation data with published in vitro measurements of intermolecular distances, we construct a model for the in situ structure of the thin filament. The conformational dynamics of NTnC plays an important role in the regulation of cardiac muscle contraction by moving the C-terminal region of TnI from its actin-binding inhibitory location and enhancing the movement of tropomyosin away from its inhibitory position.

Molecular Mechanism for the Regulation of Cardiac Muscle Contraction by Troponin

Contraction of cardiac muscle is regulated by Ca2+ ions binding to troponin (Tn) in the actin-containing thin filament, leading to a movement of tropomyosin around the filament that allows myosin heads to bind to actin and generate force. However the molecular structural basis of this Tn-mediated signalling pathway has remained obscure. We investigated the conformation of the cardiac Tn on the thin filament and its response to binding of Ca2+ to elucidate the molecular mechanism of the regulation of contraction in cardiac muscle.Polarized fluorescence from bifunctional rhodamine was used to determine the orientation of the major component of Tn core domain on the thin filaments of cardiac muscle. We showed that the C-terminus of TnC (CTnC), together with the coiled coil formed by the TnI and TnT chains, did not move during activation and acted as a scaffold that holds N-terminus of TnC (NTnC) and the actin binding regions of troponin I. The NTnC, on the other hand, exhibited multiple orientations during both diastole and systole. By combining the in situ orientation data with published in vitro measurements of intermolecular distances, we constructed an atomic model for the in situ structure of the thin filament that suggests a plausible molecular mechanism for the regulation of heart muscle.

Tilting and Wobble of Myosin V by High-Speed Single-Molecule Polarized Fluorescence Microscopy

Myosin V is biomolecular motor with two actin-binding domains (heads) that take multiple steps along actin by a hand-over-hand mechanism. We used high-speed polarized total internal reflection fluorescence (polTIRF) microscopy to study the structural dynamics of single myosin V molecules that had been labeled with bifunctional rhodamine linked to one of the calmodulins along the lever arm. With the use of time-correlated single-photon counting technology, the temporal resolution of the polTIRF microscope was improved ∼50-fold relative to earlier studies, and a maximum-likelihood, multitrace change-point algorithm was used to objectively determine the times when structural changes occurred. Short-lived substeps that displayed an abrupt increase in rotational mobility were detected during stepping, likely corresponding to random thermal fluctuations of the stepping head while it searched for its next actin-binding site. Thus, myosin V harnesses its fluctuating environment to extend its reach. Additional, less frequent angle changes, probably not directly associated with steps, were detected in both leading and trailing heads. The high-speed polTIRF method and change-point analysis may be applicable to single-molecule studies of other biological systems.

Fluorescent Labeling of Calmodulin with Bifunctional Rhodamine: Figure 1.

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to label chicken calmodulin (CaM) with bifunctional rhodamine (BR) at two engineered cysteine (Cys) residues (P66C and A73C) so that it cross-links the two Cys sites. The resulting BR-CaM protein is then purified by high-performance liquid chromatography (HPLC) and concentrated by filter centrifugation. To confirm that the two Cys residues in the labeled CaM are actually cross-linked by BR, a sample of purified BR-CaM is digested by an endoproteinase and analyzed by mass spectrometry. The BR-CaM can then be used to label myosin V, which can in turn be used in a polTIRFM processive motility assay.

Orientation and Rotational Motions of Single Molecules by Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM)

In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin.

Fluorescent Labeling of Myosin V for Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM) Motility Assays

Polarized total internal reflection fluorescence microscopy (polTIRFM) can be used to detect the spatial orientation and rotational dynamics of single molecules. polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. This protocol describes how to exchange bifunctional rhodamine-calmodulin (BR-CaM) for wild-type calmodulin (WT-CaM) on the lever arm of myosin V. BR-CaM is exchanged at low stoichiometry (∼0.4 BR-CaM per double-headed myosin V) to obtain myosin V molecules with one BR-CaM and to limit the proportion of myosin V molecules with two or more probes. The stoichiometry is very sensitive to the concentration of calcium during the exchange reaction. The labeled myosin V can subsequently be used for investigating the motility of myosin V in vitro with a polTIRFM processive motility assay, which is performed on substrate-attached actin.

Orientation of the N-Terminal Lobe of the Myosin Regulatory Light Chain in Skeletal Muscle Fibers

The orientation of the N-terminal lobe of the myosin regulatory light chain (RLC) in demembranated fibers of rabbit psoas muscle was determined by polarized fluorescence. The native RLC was replaced by a smooth muscle RLC with a bifunctional rhodamine probe attached to its A, B, C, or D helix. Fiber fluorescence data were interpreted using the crystal structure of the head domain of chicken skeletal myosin in the nucleotide-free state. The peak angle between the lever axis of the myosin head and the fiber or actin filament axis was 100—110° in relaxation, isometric contraction, and rigor. In each state the hook helix was at an angle of ∼40° to the lever/filament plane. The in situ orientation of the RLC D and E helices, and by implication of its N- and C-lobes, was similar in smooth and skeletal RLC isoforms. The angle between these two RLC lobes in rigor fibers was different from that in the crystal structure. These results extend previous crystallographic evidence for bending between the two lobes of the RLC to actin-attached myosin heads in muscle fibers, and suggest that such bending may have functional significance in contraction and regulation of vertebrate striated muscle.

The azimuthal path of myosin V and its dependence on lever-arm length

Myosin V (myoV) is a two-headed myosin capable of taking many successive steps along actin per diffusional encounter, enabling it to transport vesicular and ribonucleoprotein cargos in the dense and complex environment within cells. To better understand how myoV navigates along actin, we used polarized total internal reflection fluorescence microscopy to examine angular changes of bifunctional rhodamine probes on the lever arms of single myoV molecules in vitro. With a newly developed analysis technique, the rotational motions of the lever arm and the local orientation of each probe relative to the lever arm were estimated from the probe’s measured orientation. This type of analysis could be applied to similar studies on other motor proteins, as well as other proteins with domains that undergo significant rotational motions. The experiments were performed on recombinant constructs of myoV that had either the native-length (six IQ motifs and calmodulins [CaMs]) or truncated (four IQ motifs and CaMs) lever arms. Native-length myoV-6IQ mainly took straight steps along actin, with occasional small azimuthal tilts around the actin filament. Truncated myoV-4IQ showed an increased frequency of azimuthal steps, but the magnitudes of these steps were nearly identical to those of myoV-6IQ. The results show that the azimuthal deflections of myoV on actin are more common for the truncated lever arm, but the range of these deflections is relatively independent of its lever-arm length.

Structural Kinetics of Troponin during Activation of Skeletal Muscle Fibers by Photolysis of Nitrophenyl-EGTA (NP-EGTA)

Mutants of troponin C (TnC) with bifunctional rhodamine (BR) cross-linking pairs of cysteine residues 56 and 63 or 96 and 103, along the C- and E-helices respectively, were exchanged into demembranated fibers from rabbit psoas muscle. Changes in the orientation of the two helices following a sudden increase in [Ca2+] elicited by photolysis of NP-EGTA were monitored by polarized fluorescence. The second- and fourth-rank order parameters, and respectively, that describe the orientation distribution of the BR dipoles with respect to the muscle fiber axis were calculated from the polarized fluorescence intensities. 5 ms after photolysis, when force was only 3% of maximum, the changes in and were 90% complete for the C-helix and 65% for the E-helix. The results reveal the kinetics of the first steps in the signaling pathway in the troponin complex: the [Ca2+] jump induces a fast structural change in the N-terminal lobe of TnC, containing the regulatory Ca2+ sites near the C helix, followed by a structural change in its C-lobe (containing the E helix) that is still faster than force generation. When active force development was inhibited by N-benzyl-p-toluene-sulphonamide (BTS), the kinetics and amplitude of the orientation change in the C-helix were not affected, whereas the orientation change in the E-helix was reduced. The effect of BTS on the kinetics also revealed a component of the E-helix orientation change that is synchronous with force development and accounts for the amplitude decrease, suggesting that myosin head binding to actin completes the structural change of the C-lobe initially triggered by calcium binding to the N-lobe.Supported by Medical Research Council and Wellcome Trust, UK.

Structural Dynamics of SERCA and Phospholamban by TR-FRET and TIRF

We used fluorescence to investigate the structure and dynamics of phospholamban (PLB) and its inhibited target, sarcoplasmic reticulum Ca-ATPase (SERCA). Time-resolved fluorescence energy transfer (TR-FRET) was used to study the oligomeric interaction between PLB and SERCA. Previous work on our lab has shown that PLB is primarily pentameric but SERCA binds preferentially to the monomeric form in lipid vesicles. Recent EM studies suggest that the PLB pentamer might also bind to SERCA. We tested this hypothesis by labeling SERCA at C674 with a fluorescent donor (TMRIA) and labeling PLB at K3 with a non-fluorescent acceptor (MGITC), then reconstituting the proteins into lipid vesicles and performing TR-FRET as a function of the fraction of acceptor-labeled PLB (xA), keeping the total PLB/SERCA molar ratio constant at 10. Simulations showed that if a PLB monomer binds to SERCA, the dependence of FRET on xA should be linear, but the binding of a PLB oligomer should produce distinct curvature in the plot. The observed plot was quite linear, and was indistinguishable from control experiments using a monomeric mutant of PLB. We conclude that PLB binds to SERCA only as a monomer. We used polarized TIRF to study the structure of PLB in the presence and absence of SERCA in a supported lipid bilayer. Polarized TIRF data shows that the cytoplasmic helix of PLB, labeled with bifunctional rhodamine, is close to be parallel to the membrane surface. SERCA binding lifts the cytoplasmic helix away from the membrane surface. These results have important implications for the design of PLB mutants to be used in gene therapy for heart failure.

Orientation of the Myosin Regulatory Light Chain in Cardiac Muscle Determined by Polarized Fluorescence

The myosin regulatory light chain (RLC) is a component of the myosin lever arm, and the cardiac RLC isoforms are common sites of mutations associated with familial hypertrophic cardiomyopathy. Phosphorylation of the RLC can modulate cardiac contractility independently of troponin/tropomyosin-mediated regulation, but the molecular mechanism of such modulation is poorly understood. We are investigating structure-function relationships in the cardiac myosin RLC in the native environment of cardiac muscle cells. Pairs of cysteine residues were genetically introduced at six different pairs of surface-accessible sites in the RLC, each of which are 10-15 A apart. Three pairs of cysteines were introduced in the N-terminal lobe of the RLC, and three in the C-terminal lobe. Each pair of cysteines was cross-linked with a bifunctional rhodamine: bis-[N-iodoacetyl(piperazyl)]-sulforhodamine (BSR; B-10621, Invitrogen) and the labelled protein purified to 95% homogeneity. The pure 1:1 BSR-RLC conjugates are exchanged into demembranated cardiac muscle cells and the orientation of the BSR fluorescence dipole determined by polarized fluorescence.With three probes on each lobe of the RLC, the orientation of the lobe with respect to the actin filament axis and the bend between the lobes can be determined. We are measuring these orientations under various physiological conditions including relaxation, active contraction, and rigor, and investigating the effects of RLC phosphorylation by myosin light chain kinase. These experiments should provide insight into RLC function in cardiac muscle and the mechanism of modulation of cardiac contractility by RLC phosphorylation.Supported by the British Heart Foundation.