Tm Molecules Research Articles

Development of conditionally active IL-10 INDUKINE TM molecules for the treatment of inflammatory bowel disease

Abstract Inflammatory bowel disease (IBD) encompasses two chronic inflammatory diseases of the gastrointestinal tract, Ulcerative Colitis (UC) and Crohn’s disease. Although great strides have been made in the treatment of IBD, subsets of patients do not respond or lose responsiveness to therapy, underlining a need for new treatments. Interleukin 10 (IL-10) is a immunoregulatory cytokine, known to suppress and inhibit inflammatory responses of a variety of immune cells including monocytes, macrophages, and T cells. Polymorphisms associated with reduced expression of IL-10 and IL-10R have been linked with IBD. Clinical trials using recombinant IL-10 in IBD have been unsuccessful, highlighting a need for further engineering to utilize IL-10 as a therapeutic agent. We have developed IL-10 INDUKINE molecules that are designed as prodrugs containing human IL-10, an inactivation domain and a half-life extension domain tethered together by protease-sensitive linkers. IL-10 INDUKINE molecules are peripherally inactive but due to dysregulation of the protease milieu in the inflamed colon, the linkers are cleaved, and IL-10 is released locally. We have identified proprietary linkers which are cleaved by UC and Crohn’s human colon samples. In a mouse model of colitis, IL-10 INDUKINE molecules prevent weight loss, intestinal damage and inhibit inflammatory cytokine production within the colon. Together these data support the potential of an IL-10 INDUKINE molecule for the treatment of IBD.

From Molecular Biology to Novel Immunotherapies and Nanomedicine in Uveal Melanoma.

Molecular biology studies of uveal melanoma have resulted in the development of novel immunotherapy approaches including tebentafusp-a T cell-redirecting bispecific fusion protein. More biomarkers are currently being studied. As a result, combined immunotherapy is being developed as well as immunotherapy with bifunctional checkpoint inhibitory T cell engagers and natural killer cells. Current trials cover tumor-infiltrating lymphocytes (TIL), vaccination with IKKb-matured dendritic cells, or autologous dendritic cells loaded with autologous tumor RNA. Another potential approach to treat UM could be based on T cell receptor engineering rather than antibody modification. Immune-mobilizing monoclonal T cell receptors (TCR) against cancer, called ImmTAC TM molecules, represent such an approach. Moreover, nanomedicine, especially miRNA approaches, are promising for future trials. Finally, theranostic radiopharmaceuticals enabling diagnosis and therapy with the same molecule bring hope to this research.

The structure of the junction region of the native cardiac thin filament.

Cardiac contraction depends on an intricate rearrangement of molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca2+ levels. Cardiac thin filament (cTF) consists of two strands comprised of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes. Tn binds Ca2+ to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits: Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is composed of adjacent Tm molecules that overlap “head-to-tail” along the actin filament. The N-terminus of TnT (e.g. TnT1) binds to the Tm overlap region to form the cTF junction region. Numerous studies have shown the crucial role of TnT1 in the Ca2+-dependent regulation of the cTF. Moreover, TnT1 is a hot spot for pathogenic mutations leading to cardiomyopathies in humans. However, a detailed structural description of the cTF junction region and, particularly, TnT1 interactions with Tm and actin remain unknown. Here we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa=8) Ca2+ conditions. We show that TnT1 stabilizes the Tm overlap region via mostly ionic interactions with the Tm C- and N-termini. Our data shows that TnT1 works as a joint that anchors the Tm overlap region to actin which stabilizes the off (e.g. Ca2+-free) state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.

High-resolution cryo-EM structure of the junction region of the native cardiac thin filament in relaxed state.

Cardiac contraction depends on molecular interactions among sarcomeric proteins coordinated by the rising and falling intracellular Ca2+ levels. Cardiac thin filament (cTF) consists of two strands composed of actin, tropomyosin (Tm), and equally spaced troponin (Tn) complexes forming regulatory units. Tn binds Ca2+ to move Tm strand away from myosin-binding sites on actin to enable actomyosin cross-bridges required for force generation. The Tn complex has three subunits-Ca2+-binding TnC, inhibitory TnI, and Tm-binding TnT. Tm strand is comprised of adjacent Tm molecules that overlap "head-to-tail" along the actin filament. The N-terminus of TnT (e.g., TnT1) binds to the Tm overlap region to form the cTF junction region-the region that connects adjacent regulatory units and confers to cTF internal cooperativity. Numerous studies have predicted interactions among actin, Tm, and TnT1 within the junction region, although a direct structural description of the cTF junction region awaited completion. Here, we report a 3.8 Å resolution cryo-EM structure of the native cTF junction region at relaxing (pCa 8) Ca2+ conditions. We provide novel insights into the "head-to-tail" interactions between adjacent Tm molecules and interactions between the Tm junction with F-actin. We demonstrate how TnT1 stabilizes the Tm overlap region via its interactions with the Tm C- and N-termini and actin. Our data show that TnT1 works as a joint that anchors the Tm overlap region to actin, which stabilizes the relaxed state of the cTF. Our structure provides insight into the molecular basis of cardiac diseases caused by missense mutations in TnT1.

Modulation of spin thermoelectric properties in transition metal porphyrin single-molecule spin caloritronic devices by Fano resonance

Abstract Single-molecule spin caloritronic devices, which convert the heat into electricity by using spin degree of freedom of electron, are attracting attention with the inspiration of finally shrinking electronic components by utilizing molecules as elementary blocks. In this work, one kind of molecular-scale spin caloritronic devices is theoretically proposed, which consists of a transition metal porphyrin (TM(Pr), TM=V, Mn, Fe, Co) molecule sandwiched between single-walled carbon nanotube (SWCNT) electrodes via carbon atomic chains (CACs). The corresponding spin transport properties are calculated. The results show there is robust Fano resonance in the transmission spectra around the Fermi energy for one spin component in each device, which is attributed to the quantum interference between continuous spin states originating from delocalized states over the pristine SWCNT-porphyrin device and “quasi-discrete” state mainly localized on the TM(Pr) molecule. Utilizing Fano resonance, spin-dependent Seebeck coefficients of the devices for one spin component can be strengthened. Interestingly, it is revealed the energy, spatial distributions, and parity of molecular orbitals of TM(Pr) molecules play an important role in the generation of Fano resonance, which is dependent on the kind of TM atom. Therefore, this further provides the possibility to optimize spin-dependent Seebeck coefficient by TM atoms. This work is helpful for the future design of spin caloritronic devices.

Predicting Effects of Tropomyosin Stiffness on Cardiac Muscle Contraction Using Coarse-Grained Stochastic Modeling

During the cardiac thin filament activation process, Tropomyosin (Tm) oscillates over the surface of actin filament in the azimuthal direction to regulate the access of Myosin heads to actin binding sites. These dynamical modes of oscillation and the resultant cooperative activation effects depend critically on the stiffness characteristics and the mobility of Tm molecules. In this study, we developed a stochastic coarse-grained computational model to describe the Tm motions over the surface of actin filament using Langevin-Brownian dynamics. The model represents the structural arrangement of Tm molecules as a flexible chain with variable torsional stiffness using a crystal elastic network approach. The model accounts for the spatial interactions among nearest-neighbor regulatory units (RUs), which are thought to appear from the structural coupling of adjacent Tropomyosins. This elastic coupling between RUs is accomplished by assigning a multi-well potential energy for each RU. The model is then used to study the effects of Tm torsional stiffness variations on the cooperative activation of the thin filament. The results suggest that small perturbations in Tm torsional stiffness can lead to a significant effect on force-Ca2+ sensitivity, the rate of tension redevelopment, relaxation rate, and other contraction characteristics. The present stochastic computational model draws for the first time a more detailed molecular connection between Tm torsional stiffness, Tm modes of oscillations over actin surface, cooperativity among RUs, and dynamic muscle twitches. Thus, this coarse-graining approach may be useful in explaining many cardiomyopathies induced by structural remodeling and stiffening in Tm molecules as a result of point mutations in the human gene TPM1.

Modeling Predicts Non-Monotonic Dependence of Myofilament Ca2+ Sensitivity on Tropomyosin Stiffness

The cooperative Ca2+ activation of the thin filament in cardiac muscle plays a vital role in the contraction and relaxation of the heart. Nearest-neighbor interactions between thin filament regulatory units (RUs) are widely considered the principal mechanism of cooperativity, but descriptions of these interactions vary. Previous models have assumed that interactions can be partitioned into distinct categories, such as RU-RU, RU-crossbridge (XB), and XB-XB cooperativity. Under these schemes, the strength of each type of cooperativity is described by independently adjusted parameters. While such models have produced useful insight and predictions, we sought an approach that would relate the different types of nearest-neighbor interactions, thereby constraining the model's parameters and improving its predictive power. We first assumed that cooperative interactions are transmitted along the thin filament solely through tropomyosin (Tm) and its linkages with neighboring Tm molecules. We adopted published values from structural studies for the azimuthal angle displacements of Tm as it transitions between blocked, closed, and open regulatory states. We then postulated a mechanical model by which nearest-neighbor Tms alter the activation energy of tropomyosin state transitions according to structurally motivated strain energy functions. Making simple assumptions about the functional form of the strain energy then enables kinetic rates to depend explicitly on the characteristic stiffness of the Tm chain. Our formulation naturally results in multiple forms of cooperativity that are interrelated in a compact, structurally motivated manner while reducing the number of free parameters. The model successfully fits measured steady state and dynamic Ca2+-force relations, and preliminary simulations indicate that varying Tm stiffness has complex effects on thin filament activation. This behavior suggests that Tm-Tm coupling provides both inhibiting and activating cooperation between RUs, and that these effects are non-monotonically dependent on the magnitude of Tm stiffness.

α-Tropomyosin with a D175N or E180G Mutation in Only One Chain Differs from Tropomyosin with Mutations in Both Chains

α-Tropomyosin (Tm) carrying hypertrophic cardiomyopathy mutation D175N or E180G was expressed in Escherichia coli. We have assembled dimers of two polypeptide chains in vitro that carry one (αα*) or two (α*α*) copies of the mutation. We found that the presence of the mutation has little effect on dimer assembly, thereby predicting that individuals heterozygous for the Tm mutations are likely to express both αα* and α*α* Tm. Depending on the expression level, the heterodimer may be the predominant form in individuals carrying the mutation. Thus, it is important to define differences in the properties of Tm molecules carrying one or two copies of the mutation. We examined the Tm homo- and heterodimer properties: actin affinity, thermal stability, calcium regulation of myosin subfragment 1 binding, and calcium regulation of myofibril force. We report that the properties of the heterodimer may be similar to those of the wild-type homodimer (actin affinity, thermal stability, D175N αα*), similar to those of the mutant homodimer (calcium sensitivity, D175N αα*), intermediate between the two (actin affinity, E180G αα*), or different from both (thermal stability, E180G αα*). Thus, the properties of the homodimer are not a completely reliable guide to the properties of the heterodimer.

Coupling of Adjacent Tropomyosins Enhances Cross-Bridge-Mediated Cooperative Activation in a Markov Model of the Cardiac Thin Filament

We developed a Markov model of cardiac thin filament activation that accounts for interactions among nearest-neighbor regulatory units (RUs) in a spatially explicit manner. Interactions were assumed to arise from structural coupling of adjacent tropomyosins (Tms), such that Tm shifting within each RU was influenced by the Tm status of its neighbors. Simulations using the model demonstrate that this coupling is sufficient to produce observed cooperativity in both steady-state and dynamic force-Ca2+ relationships. The model was further validated by comparison with reported responses under various conditions including inhibition of myosin binding and the addition of strong-binding, non-force-producing myosin fragments. The model also reproduced the effects of 2.5 mM added Pi on Ca2+-activated force and the rate of force redevelopment measured in skinned rat myocardial preparations. Model analysis suggests that Tm-Tm coupling potentiates the activating effects of strongly-bound cross-bridges and contributes to force-Ca2+ dynamics of intact cardiac muscle. The model further predicts that activation at low Ca2+ concentrations is cooperatively inhibited by nearest neighbors, requiring Ca2+ binding to >25% of RUs to produce appreciable levels of force. Without excluding other putative cooperative mechanisms, these findings suggest that structural coupling of adjacent Tm molecules contributes to several properties of cardiac myofilament activation.

Tropomyosin Flexibility Evaluated by Electron Microscopy Image Analysis

Movement of tropomyosin (Tm) on thin filaments in response to Ca2+-binding to troponin and myosin binding to actin is an inherent feature of muscle regulation. As part of this process, the cable-like mechanical properties of the Tm coiled-coil are thought to underlie cooperative on and off switching of contraction. In principle, movement of Tm over the flat surface of actin may not require significant molecular flexibility. However, local perturbations caused, for example, by myosin binding on actin may necessitate some plasticity of the Tm molecule. In contrast, any large-scale Tm flexibility might dampen its cooperative movement. In the current study, we directly assessed the flexibility of Tm by examining EM images of both rotary shadowed and negatively stained molecules. Single Tm molecules in both image sets showed no obvious signs of sharp bending or kinks, and displayed contours close to those predicted from a high-resolution 3D model of Tm (Lorenz et al., 1995). Short multimeric strings of end-to-end bonded Tm were commonly observed in the rotary shadowed images. These showed no pronounced bending or joints at the intermolecular junctions. The persistence length of Tm was calculated to be over twice the length of the molecule based on these EM images. The data as a whole imply that Tm molecules exhibit an intrinsic stiffness sufficient to contribute to cooperativity on thin filaments and thus are consistent with models of muscle regulation (e.g. Lehrer & Geeves, 1998; Lehman et al., 2000). A tropomyosin mutant in which the coiled-coil interface was destabilized by introduction of Ala clusters in the 2nd and 3rd periodic repeats (Y60A-L64A-L106A; Singh & Hitchcock-DeGregori, unpublished) was also examined by EM. No obvious extra bending was observed, although the calculated persistence length was significantly shortened, showing the sensitivity of the technique.

Tropomyosin Phosphorylation Has Filament-Level And Crossbridge-Level Effects On Actin-Myosin Interactions

The α-isoform of tropomyosin (Tm) can be phosphorylated near the C-terminus, and dephosphorylation of Tm has been shown to decrease actin-myosin ATPase rates (Heeley et al., 1989). Additionally, Tm polymerizes with adjacent Tm molecules in a head-to-tail manner. Phosphorylation of Tm might therefore influence the strength of these interactions, and thus regulate the degree of cooperative activation by myosin binding. We tested Tm phosphorylation effects by measuring the force and velocity of actin-myosin interactions at the level of single actin filaments using a combination of actin-Tm binding assays, in vitro motility assays, and a novel, high throughput laser trap assay to measure isometric force. In these assays we used purified heavy meromyosin (HMM) and actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. Dephosphorylation of Tm did not result in significant changes in actin-Tm binding or unloaded sliding velocities. Isometric force measurements showed that thin filaments were cooperatively activated by myosin, but only when Tm was phosphorylated. When Tm was dephosphorylated, the reconstituted filaments behaved like bare actin filaments. These results suggest transmission of cooperative activation beyond one thin filament regulatory unit when Tm is phosphorylated. Moreover, Tm phosphorylation increased isometric force production ∼50% compared to bare actin filaments at intermediate HMM surface densities. In combination with sliding velocity data, this result suggests that Tm phosphorylation may have effects at the level of a single crossbridge that cannot be explained by steric hindrance. We hypothesize that Tm phosphorylation accelerates the rate of crossbridge attachment. Single molecule kinetic and force spectroscopy experiments are underway to gain insight on this possibility.

Phosphorylation of tropomyosin extends cooperative binding of myosin beyond a single regulatory unit

Tropomyosin (Tm) is one of the major phosphoproteins comprising the thin filament of muscle. However, the specific role of Tm phosphorylation in modulating the mechanics of actomyosin interaction has not been determined. Here we show that Tm phosphorylation is necessary for long-range cooperative activation of myosin binding. We used a novel optical trapping assay to measure the isometric stall force of an ensemble of myosin molecules moving actin filaments reconstituted with either natively phosphorylated or dephosphorylated Tm. The data show that the thin filament is cooperatively activated by myosin across regulatory units when Tm is phosphorylated. When Tm is dephosphorylated, this "long-range" cooperative activation is lost and the filament behaves identically to bare actin filaments. However, these effects are not due to dissociation of dephosphorylated Tm from the reconstituted thin filament. The data suggest that end-to-end interactions of adjacent Tm molecules are strengthened when Tm is phosphorylated, and that phosphorylation is thus essential for long range cooperative activation along the thin filament.

Tropomyosin in the groove? Molecular insights into an inherited myopathy

Tropomyosin in the groove? Molecular insights into an inherited myopathy

The effect of single residue substitutions of serine-283 on the strength of head-to-tail interaction and actin binding properties of rabbit skeletal muscle alpha-tropomyosin.

Vertebrate skeletal muscle alpha-tropomyosin polymerizes in a head-to-tail manner and binds cooperatively to actin. It has been postulated that the cooperative actin binding is governed by the strength of the head-to-tail interaction. In order to know the relationship between the head-to-tail affinity and actin binding, we studied the properties of tropomyosin variants with single residue substitutions at serine-283, the penultimate residue at the carboxyl terminus that is involved in the head-to-tail interaction. It has been shown that the phosphorylation of serine-283 strengthens the head-to-tail interaction. Viscometry was employed to compare the head-to-tail affinity of tropomyosin variants. Variant S283E showed higher viscosity whereas variant S283K showed lower viscosity compared with the wild type non-phosphorylated alpha-tropomyosin. The results confirm the idea that the interaction is sensitive to the ionic properties of residue 283. The strength of the head-to-tail interaction was assessed directly by sedimentation equilibrium using two pairs of tropomyosin variants designed so that only dimeric interactions were allowed within each pair. From one pair of variants with serine-283, the association constant was determined to be 2.6 x 10(4) M(-1) (SD =1.0 x 10(4)), whereas for the second pair with glutamate-283, the affinity was 3.9 x 10(4) M(-1) (SD =1.6 x 10(4)), slightly stronger than the former, consistent with the results of viscometry. The results indicate that the head-to-tail association is weak as previously implicated from light scattering measurements. Cosedimentation was employed to measure the cooperative actin binding of tropomyosin variants. Although previous results indicated the phosphorylation has no significant influence on the actin affinity, variant S283E shows a lower affinity compared with the control. Variants S283K and S283A show even lower affinities to actin, although these species bind to actin more cooperatively than does variant S283E. The results indicate that the affinity of the head-to-tail interaction between adjacent tropomyosin molecules is weak, and is substantially influenced by an extra charge at residue 283. On the other hand, the interaction with actin, the affinity and the cooperativity in actin binding, is dependent on amino acid residues at 283 and is not simply correlated with the strength of the head-to-tail interaction between Tm molecules in solution.

Electron Microscopic Study of Tropomyosin Binding to Actin Filaments Reveals Tethered Molecules Representing Weak Binding

The binding of tropomyosin (Tm) to actin filaments was studied using the rotary shadowing technique. Electron micrographs showed, in addition to free Tm molecules, some Tm molecules bound to actin filaments at only one end creating “tethered molecules.” The ratio of free and bound Tm was determined by comparing densities of free Tm molecules in micrographs with those in Tm alone specimens. At a 1 to 7 ratio of 17 nMchicken gizzard Tm (gTm) and 120 nMactin filaments, the sum of the tethered (7%) and the free was only 29% of total Tm. The missing Tm molecules are likely to be those bound to actin filaments lengthwise and not visualized by the technique. At a lower ratio of Tm (3 nM) to actin (120 nM), the majority of bound Tm appeared to be tethered. At a lower actin concentration but a higher ratio of Tm (12 nM) to actin (24 nM), 57% of bound Tm appeared to be tethered. The populations of free and tethered molecules were also affected by different salt and glycerol concentrations or type of Tm used: more with rabbit skeletal (sTm) than with gTm. The tethering indicates that the interaction of Tm molecules to actin filaments is stronger at one end than at the rest of the molecule. The bundling of actin filaments was observed under certain conditions (e.g., ∼2 μMactin and ∼0.5 μMsTm), suggesting that both ends of Tm molecules can bind to actin filaments but one end binds much more weakly than the other. All of these observations support the fact that free and tethered molecules are under rapid equilibrium and that incidental encounter of the ends of two tethered Tm molecules which are at the right positions, or of one tethered and a free molecule, would strengthen the remaining actin binding site(s), resulting in tight binding, thus, the tethered molecules represent a weak form of binding.

Deletion of the first 45 NH2-terminal residues of rabbit skeletal troponin T strengthens binding of troponin to immobilized tropomyosin

The binding of the NH2-terminal region of troponin T (TnT) to the COOH-terminal region of tropomyosin (Tm) and the head-to-tail overlap between Tm molecules is thought to provide a pivotal link between troponin (Tn) and Tm (White, S.P., Cohen, C., and Phillips, G.N., Jr. (1987) Nature 325, 826-828). To further explore the structure-function relationship of the NH2-terminal region of TnT, we studied the binding of a 26,000-dalton TnT fragment (26K-TnT, Ohtsuki, I., Shiraishi, F., Suenaga, N., Miyata, T., and Tanokura, M.J. (1984) J. Biochem. (Tokyo) 95, 1337-1342) which corresponds to residues 46-259 of TnT2f, the major isoform of TnT in rabbit fast twitch muscle, to immobilized alpha-Tm. Both 26K-TnT and TnT2f were retained by the alpha-Tm affinity column in the presence of 150 mM NaCl. However, upon increasing the NaCl concentration 26K-TnT was eluted from the column at a higher ionic strength than was TnT. When applied alone, the binary complex of TnI and TnC (TnC.TnI) was not retained by the alpha-Tm affinity column. When applied subsequently to prebound TnT2f or 26K-TnT, TnI.TnC was retained by the alpha-Tm affinity column and eluted together with TnT2f or 26K-TnT as ternary troponin complexes. Whether Ca2+ was present or not, Tn containing 26K-TnT was eluted at a higher ionic strength than was Tn containing TnT2f, indicating that removal of the first 45 residues of TnT2f strengthens the binding of Tn to Tm. In the presence of Tm, reconstituted Tn containing 26K-TnT conferred Ca2+ sensitivity on actomyosin-S1 MgATPase, and the steepness of the pCa-ATPase relation was unchanged with respect to the actoS1 ATPase regulated by TnT2f. It is concluded that the first 45 residues of TnT2f are not essential for anchoring the troponin complex to the thin filament and do not play a crucial role in the cooperative response of regulated actoS1 ATPase to Ca2+.

Cross-helix separation of tropomyosin molecules in acto-tropomyosin as determined by neutron scattering

The cross-helix separation of Tm molecules in acto-tropomyosin has been determined using neutron scattering. Deuterated Dictyostelium discoideum actin was density matched in a 93% D2O buffer so that effectively only the protonated tropomyosin was "visible" to neutrons. Analysis of the solution scattering pattern in the region of the first oscillation yielded a value for the cross-helix separation of 7.9 +/- 0.3 nm. The implications of this value for the mechanism of the regulation of muscle contraction are discussed in light of recent results by others.

Recent neutron scattering studies of muscle contraction and its control

We have presented two applications of the method of neutron scattering utilizing selective deuteration of actin. In these experiments the actin was rendered effectively invisible to neutrons by matching the scattering-length densities of deuterated actin and the solvent. The scattering of neutrons by myosin S1 and by Tm bound to this actin was studied. For free chymotrypsin-generated S1 it was found that Rg = 4.0 +/- 0.15 nm, while for papain-generated S1 it was found that Rg = 4.6 +/- 0.2 nm. Upon binding of papain-generated S1 to actin at low NS1/N actin ratios, the change in Rg in difference experiments was delta Rg = 0.05 +/- 0.15 nm. This lack of significant change in Rg in the very low-s domain confirms and extends our earlier neutron scattering work in the higher-s domain. The longest chords of S1, as well as shorter ones, are not significantly altered upon actin binding. These results indicate that muscle contraction does not occur as a result of large-scale changes in S1 structure. In actin-Tm complexes, a measurement of the mean cross-helix separation, d, of Tm molecules has been made using neutron scattering. With deuterated actin matched out in 93% D2O buffer, it was found that d = 7.9 +/- 0.3 nm. This value is in good agreement with a model based on Tm crystallography and also with recent electron microscopy results. These experiments demonstrate the feasibility and value of neutron diffraction and scattering techniques in the study of muscle contraction and its control. One can expect that the further employment of emerging cell biology techniques for generating deuterated proteins will aid our understanding of muscle in the future.