Rigor State Research Articles

Vertebrate Myosin VIIb Is a High Duty Ratio Motor Adapted for Generating and Maintaining Tension

Kinetic adaptation of muscle and non-muscle myosins plays a central role in defining the unique cellular functions of these molecular motor enzymes. The unconventional vertebrate class VII myosin, myosin VIIb, is highly expressed in polarized cells and localizes to highly ordered actin filament bundles such as those found in the microvilli of the intestinal brush border and kidney. We have cloned mouse myosin VIIb from a cDNA library, expressed and purified the catalytic motor domain, and characterized its actin-activated ATPase cycle using quantitative equilibrium and kinetic methods. The myosin VIIb steady-state ATPase activity is slow (approximately 1 s(-1)), activated by very low actin filament concentrations (K(ATPase) approximately 0.7 microm), and limited by ADP release from actomyosin. The slow ADP dissociation rate constant generates a long lifetime of the strong binding actomyosin.ADP states. ADP and actin binding is uncoupled, which enables myosin VIIb to remain strongly bound to actin and ADP at very low actin concentrations. In the presence of 2 mm ATP and 2 microm actin, the duty ratio of myosin VIIb is approximately 0.8. The enzymatic properties of actomyosin VIIb are suited for generating and maintaining tension and favor a role for myosin VIIb in anchoring membrane surface receptors to the actin cytoskeleton. Given the high conservation of vertebrate class VII myosins, deafness phenotypes arising from disruption of normal myosin VIIa function are likely to reflect a loss of tension in the stereocilia of inner ear hair cells.

Paramyosin phosphorylation site disruption affects indirect flight muscle stiffness and power generation in Drosophila melanogaster

The phosphoprotein paramyosin is a major structural component of invertebrate muscle thick filaments. To investigate the importance of paramyosin phosphorylation, we produced transgenic Drosophila melanogaster in which one, three, or four phosphorylatable serine residues in the N-terminal nonhelical domain were replaced by alanines. Depending on the residues mutated, transgenic lines were either unaffected or severely flight impaired. Flight-impaired strains had decreases in the most acidic paramyosin isoforms, with a corresponding increase in more basic isoforms. Surprisingly, ultrastructure of indirect flight muscle myofibrils was normal, indicating N-terminal phosphorylation is not important for myofibril assembly. However, mechanical studies of active indirect flight muscle fibers revealed that phosphorylation site mutations reduced elastic and viscous moduli by 21-59% and maximum power output by up to 42%. Significant reductions also occurred under relaxed and rigor conditions, indicating that the phosphorylation-dependent changes are independent of strong crossbridge attachment and likely arise from alterations in thick filament backbone properties. Further, normal crossbridge kinetics were observed, demonstrating that myosin motor function is unaffected in the mutants. We conclude that N-terminal phosphorylation of Drosophila paramyosin is essential for optimal force and oscillatory power transduction within the muscle fiber and is key to the high passive stiffness of asynchronous insect flight muscles. Phosphorylation may reinforce interactions between myosin rod domains, enhance thick filament connections to the central M-line of the sarcomere and/or stabilize thick filament interactions with proteins that contribute to fiber stiffness.

Should electrical stimulation be applied when cold shortening is not a risk?

This experiment was designed to show whether delayed high voltage stimulation (ES) is more beneficial than no stimulation (NS) to secure tenderness under circumstances where rigor conditions are difficult to control due to variations in carcass size, fatness and/or chilling capacity. Ten Charolais carcasses were split during slaughter, the left sides were stimulated at 45 min post-mortem for 45 s, and the right sides were left unstimulated. The carcass sides were then chilled at a medium chilling rate. Sarcomere length measurements confirm that there was neither cold nor heat shortening in the M. longissimus (LD). LD from ES sides aged for 2 days was more tender than non-stimulated LD (NES), although prolonged ageing eroded the advantage of ES to a non-significant advantage after 14 days. Initial tenderness differences coincided with lower 24 h calpain activity, suggesting an early onset of proteolysis and ageing (tenderisation). In contrast to conventional early ES, delayed ES, appears to be beneficial for the early development of tenderness without too much interference with enzyme. Myofibril fragment length (MFL) was a good indicator of the development of tenderness during prolonged ageing but not for the early post-mortem variation in tenderness. No colour ( L*, a*, b*) differences, occurred due to stimulation treatment, while drip loss was slightly higher at 24 h post-mortem for ES meat.

The effect of various types of poultry pre- and post-rigor meats on emulsification capacity, water-holding capacity and cooking loss

In this study, the relationship between various kinds of poultry meat (quail, partridge, chicken and turkey) on pH, emulsification capacity, water-holding capacity and cooking loss was investigated. The effect of rigor state on pH, emulsification capacity, water-holding capacity and cooking loss was also determined. To investigate these parameters, immediately after slaughter and deboning, meat parts were submitted to both pre- and post-rigor analyses. The results indicated that the emulsification capacity of quail and chicken meat was higher than the values for partridge and turkey meat. Quail meat showed the highest water-holding capacity value in the post-rigor stage. The lowest cooking loss value was found in partridge meat, in both pre- and post-rigor stages. The state of rigor had a significant (P<0.05; P≤0.01) effect on pH and cooking loss values, respectively.

Kinetic Mechanism of Blebbistatin Inhibition of Nonmuscle Myosin IIB

We examined the effect of blebbistatin on the kinetic properties of nonmuscle myosin IIB subfragment 1 (NMIIB S1). Blebbistatin is a small molecule that affects cell blebbing during the process of cell division, which has been shown to decrease the myosin ATPase activity of a number of myosins [Straight et al. (2003) Science 299, 1743-1747]. The steady-state actin-activated ATPase activity of NMIIB S1 was decreased approximately 90% at 40 microM actin in the presence of blebbistatin. Stopped-flow techniques were employed to elucidate the effect of blebbistatin on the various steps of the NMIIB S1 cross-bridge cycle. Blebbistatin did not affect ATP binding and hydrolysis. Binding to actin in the presence of ADP (0.57 +/-0.08 microM(-1) s(-1)) was reduced slightly in the presence of blebbistatin (0.38 +/- 0.03 microM(-1) s(-1)), while mantADP dissociation from acto-NMIIB S1 was reduced (approximately 30%). P(i) release was blocked in the presence of blebbistatin. Accordingly, the apparent affinity of NMIIB S1 for actin in the presence of ATP was greatly reduced. Based on the above data, we surmise that blebbistatin inhibits the ATPase activity of NMIIB S1 primarily by blocking entry into the strong binding state; secondarily, it reduces the rate of ADP release. These effects are likely mediated by binding of blebbistatin within the myosin cleft that progressively closes in forming the acto-myosin rigor state.

Repriming the actomyosin crossbridge cycle.

The central features of the mechanical cycle that drives the contraction of muscle are two translational steps: the working stroke, whereby an attached myosin crossbridge moves relative to the actin filament, and the repriming step, in which the crossbridge returns to its original orientation. Although the mechanism of the first of these is understood in some detail, that of the second has received less attention. Here, we show that repriming occurs after detachment of the crossbridge from the actin, rather than intervening between two actomyosin states with ATP bound [Eisenberg, E. & Greene, L. E. (1980) Annu. Rev. Physiol. 42, 293-309]. To discriminate between these two models we investigated the single-molecule mechanics of the myosin-actin interaction in the presence of ATP analogues such as GTP, for which the hydrolytic step itself limits the actomyosin GTPase rate to a much lower rate than for ATP. The lifetimes of bound states was proportional to 1/[GTP], indicating that during the bound period myosin was in the actomyosin rigor configuration. Moreover, despite the very low actomyosin GTPase, the rate of actin binding and formation of the rigor state was higher than with ATP; it follows that most interactions with actin result in the release of GTP and not of the products, GDP and phosphate. There was no significant movement of the actin during this interaction, so repriming must occur while myosin is dissociated, as in the original Lymn-Taylor scheme [Lymn, R. W. & Taylor, E. W. (1971) Biochemistry 10, 4617-4624].

Physical and quality attributes of salted cod ( Gadus morhua L.) as affected by the state of rigor and freezing prior to salting

The effects of the rigor state and freezing of cod prior to salting on the mass transfer during production and the quality of heavily cured cod have been investigated. Pre-rigor salting lead to a larger reduction in weight, a higher water loss and a lower uptake of NaCl than in fish salted post-rigor and in fish salted after frozen storage. The cause of this is believed to be the simultaneous influx of NaCl and rigor contractions in the fish muscle. In order to reduce the loss of proteins from the raw material and to increase the instrumental lightness values ( L *) of the salt ripened product, the fish should be salted in pre-rigor state. However, if the primary focus is on the weight yield, the fish should be salted post-rigor after chilling or freezing pre- or post-rigor. Freezing and thawing of cod prior to salting post-rigor increased the firmness of the salt cured product compared to chilled fish salted post-rigor.

Myo1c is designed for the adaptation response in the inner ear.

The molecular motor, Myo1c, a member of the myosin family, is widely expressed in vertebrate tissues. Its presence at strategic places in the stereocilia of the hair cells in the inner ear and studies using transgenic mice expressing a mutant Myo1c that can be selectively inhibited implicate it as the mediator of slow adaptation of mechanoelectrical transduction, which is required for balance. Here, we have studied the structural, mechanical and biochemical properties of Myo1c to gain an insight into how this molecular motor works. Our results support a model in which Myo1c possesses a strain-sensing ADP-release mechanism, which allows it to adapt to mechanical load.

Rigor-Force Producing Cross-Bridges in Skeletal Muscle Fibers Activated by a Substoichiometric Amount of ATP

Isometric skinned muscle fibers were activated by the photogeneration of a substoichiometric amount of ATP and their cross-bridge configurations examined during the development of the rigor force by x-ray diffraction and electron microscopy. By the photogeneration of ∼100μM ATP, ∼2/3 of the concentration of the myosin heads in a muscle fiber, muscle fibers originally in the rigor state showed a transient drop of the force and then produced a long-lasting rigor force (∼50% of the maximal active force), which gradually recovered to the original force level with a time constant of ∼4s. Associated with the photoactivation, muscle fibers revealed small but distinct changes in the equatorial x-ray diffraction that run ahead of the development of force. After reaching a plateau of force, long-lasting intensity changes in the x-ray diffraction pattern developed in parallel with the force decline. Two-dimensional x-ray diffraction patterns and electron micrographs of the sectioned muscle fibers taken during the period of 1–1.9s after the photoactivation were basically similar to those from rigor preparations but also contained features characteristic of fully activated fibers. In photoactivated muscle fibers, some cross-bridges bound photogenerated ATP and underwent an ATP hydrolysis cycle whereas a significant population of the cross-bridges remained attached to the thin actin filaments with no available ATP to bind. Analysis of the results obtained indicates that, during the ATP hydrolysis reaction, the cross-bridges detached from actin filaments and reattached either to the same original actin monomers or to neighboring actin monomers. The latter cross-bridges contribute to produce the rigor force by interacting with the actin filaments, first producing the active force and then being locked in a noncycling state(s), transforming their configuration on the actin filaments to stably sustain the produced force as a passive rigor force.

Modelling muscle motor conformations using low-angle X-ray diffraction

New results on myosin head organization using analysis of low-angle X-ray diffraction patterns from relaxed insect flight muscle (IFM) from a giant waterbug, building on previous studies of myosin filaments in bony fish skeletal muscle (BFM), show that the information content of such low-angle diffraction patterns is very high despite the 'crystallographically low' resolution limit (65 A) of the spacings of the Bragg diffraction peaks being used. This high information content and high structural sensitivity arises because: (i) the atomic structures of the domains of the myosin head are known from protein crystallography; and (ii) myosin head action appears to consist mainly of pivoting between domains which themselves stay rather constant in structure, thus (iii) the intensity distribution among diffraction peaks in even the low resolution diffraction pattern is highly determined by the high-resolution distribution of atomically modelled domain mass. A single model was selected among 5000+ computer-generated variations as giving the best fit for the 65 reflections recorded within the selected resolution limit of 65 A. Clear evidence for a change in shape of the insect flight muscle myosin motor between the resting (probably like the pre-powerstroke) state and the rigor state (considered to mimic the end-of-powerstroke conformation) has been obtained. This illustrates the power of the low-angle X-ray diffraction method. The implications of these new results about myosin motor action during muscle contraction are discussed.

Effect of Muscular Contraction on Magnetization Transfer Detected at 1.5T

The effect of rigor (formation of actomyosin complexes) on magnetization transfer was observed with a 1.5 T clinical magnetic resonance (MR) imaging system. The magnetization transfer ratio of chemically skinned (calcium-sensitive) muscle fiber preparations increased much more in a rigor state than in a relaxed state, while that of calcium-insensitive fiber preparations and solutions showed no difference. These results suggest that the formation of actomyosin complexes increased the magnetization transfer ratio. A clinical MR system is not only effective for medical imaging, but also has the potential to demonstrate physiological characteristics.

Double freezing of cod fillets: influence on sensory, physical and chemical attributes of battered and breaded fillet portions.

Fillet samples were processed on board a research vessel from cod (Gadus morhua) in different states of rigor mortis. In addition, headed and gutted fish in different states of rigor were frozen and, after ten days of frozen storage, the fish were thawed, processed into fillets and refrozen. During subsequent frozen storage at -24 degrees C of breaded and battered portions processed from the different fillet batches several quality attributes were tested using sensory, physical, and chemical methods. The measurements indicated differences in the quality attributes depending on refreezing and rigor states. Especially the sensory texture attributes gumminess and, with some restrictions, firmness were found to be suitable for distinguishing single frozen (SF) from double frozen (DF) samples. This finding was supported by the outcome of the measurement of penetration force where generally a significantly (p < 0.05) higher force was necessary to penetrate the DF compared to SF samples. DF samples were generally lighter (higher L*) than SF samples. The colour difference delta E between both sample types was at least very clear and therefore visible. However, no clear differences between SF and DF samples were found when evaluating the flavour attributes and using instrumental texture profile analysis for texture characterisation. Also, all chemical parameters measured did not allow to clearly distinguish between single and double frozen samples. Therefore, it can be stated that, at least when using cod as raw material for processing battered and breaded portions, the different methods for preparing the fillet blocks will, if at all, only affect the textural quality of the final products.

Conformational change of the actomyosin complex drives the multiple stepping movement.

Actin-myosin (actomyosin) generates mechanical force by consuming ATP molecules. We apply the energy landscape perspective to address a controversial issue as to whether the myosin head moves with multiple steps after a single ATP hydrolysis or only a single mechanical event of the lever-arm swinging follows a single ATP hydrolysis. Here we propose a theoretical model in which the refolding of the partially unfolded actomyosin complex and the movement of the myosin head along the actin filament are coupled. A single ATP hydrolysis is followed by the formation of a high free-energy partially unfolded actomyosin complex, which then gradually refolds with a concomitant multiple stepping movement on the way to the lowest free-energy rigor state. The model quantitatively explains the single-molecular observation of the multiple stepping movement and is consistent with structural observations of the disorder in the actomyosin-binding process. The model also explains the observed variety in dwell time before each step, which is not accounted for by previous models, such as the lever-arm or ratchet models.

Myosin II from rabbit skeletal muscle and Dictyostelium discoideum and its interaction with F-actin studied by 1H NMR spectroscopy

Mg-F-actin occurs in two conformational states, I and M, where the N-terminal amino acids are either immobile or highly mobile. In the rigor or ADP complex of rabbit myosin S1 with Mg-F-actin the N-terminal acetyl group of actin stays in its highly mobile state. The same is true for the complexes with the myosin motor domain from Dictyostelium discoideum. This excludes a direct strong interaction of the N-terminal amino acids with myosin in the rigor state as suggested. An interaction of the N-terminus of F-actin with myosin is also not promoted by occupying its low-affinity binding site(s) with divalent ions. The N-terminal high-mobility region may be part of a structural system which has evolved for releasing inadequate stress applied to the actin filaments

Mutation of the myosin converter domain alters cross-bridge elasticity.

Elastic distortion of a structural element of the actomyosin complex is fundamental to the ability of myosin to generate motile forces. An elastic element allows strain to develop within the actomyosin complex (cross-bridge) before movement. Relief of this strain then drives filament sliding, or more generally, movement of a cargo. Even with the known crystal structure of the myosin head, however, the structural element of the actomyosin complex in which elastic distortion occurs remained unclear. To assign functional relevance to various structural elements of the myosin head, e.g., to identify the elastic element within the cross-bridge, we studied mechanical properties of muscle fibers from patients with familial hypertrophic cardiomyopathy with point mutations in the head domain of the beta-myosin heavy chain. We found that the Arg-719 --> Trp (Arg719Trp) mutation, which is located in the converter domain of the myosin head fragment, causes an increase in force generation and fiber stiffness under isometric conditions by 48-59%. Under rigor and relaxing conditions, fiber stiffness was 45-47% higher than in control fibers. Yet, kinetics of active cross-bridge cycling were unchanged. These findings, especially the increase in fiber stiffness under rigor conditions, indicate that cross-bridges with the Arg719Trp mutation are more resistant to elastic distortion. The data presented here strongly suggest that the converter domain that forms the junction between the catalytic and the light-chain-binding domain of the myosin head is not only essential for elastic distortion of the cross-bridge, but that the main elastic distortion may even occur within the converter domain itself.

The alanine-scanning mutagenesis of Dictyostelium myosin II at the ionic interface with actin.

Cyclic interaction of actin and myosin coupled with ATP hydrolysis is essential for generation of force by the actin-myosin system. The major force maintaining the actin-myosin contact during ATP hydrolysis, actin-myosin sliding and force generation seems to be ionic interactions, since these functions are highly sensitive to the ionic strength of the solvent. In the absence of ATP, i.e., under rigor conditions, however, hydrophobic interactions become dominant in maintaining the actin-myosin association. Based on the three-dimensional reconstruction of electron microscopic images of the rigor complex of actin and myosin subfragment 1 (S1), these ionic and hydrophobic interaction sites have been tentatively assigned to several locations of actin and myosin (Schroder et al. 1993; Milligan 1996).KeywordsActin FilamentMalachite GreenBasic ResidueHeavy Chain GeneDictyostelium CellThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structure and Nucleotide-Dependent Changes of Thick Filaments in Relaxed and Rigor Plaice Fin Muscle

The myosin crossbridge array, positions of non-crossbridge densities on the backbone, and the A-band “end filaments” have been compared in chemically skinned, unfixed, uncryoprotected relaxed, and rigor plaice fin muscles using the freeze-fracture, deep-etch, rotary-shadowing technique. The images provide a direct demonstration of the helical packing of the myosin heads in situ in relaxed muscle and show rearrangements of the myosin heads, and possibly of other myosin filament proteins, when the heads lose ATP on going into rigor. In the H-zone these changes are consistent with crossbridge changes previously shown by others using freeze-substitution. In addition, new evidence is presented of protein rearrangements in the M-region (bare zone), associated with the transition from the relaxed to the rigor state, including a 27-nm increase in the apparent width of the M-region. This is interpreted as being mostly due to loss or rearrangement of a nonmyosin (M9) protein component at the M-region edge. The structure and titin periodicity of the end-filaments are described, as are suggestions of titin structure on the myosin filament backbone.

Motor-driven dynamics in actin-myosin networks.

The effect of myosin motor protein activity on the filamentous actin (F-actin) rheological response is studied using diffusing wave spectroscopy. Under conditions of saturating motor activity, we find an enhancement of longitudinal filament fluctuations corresponding to a scaling of the viscoelastic shear modulus G(d)(omega) approximately omega(7/8). As the adenosine tri-phosphate reservoir sustaining motor activity is depleted, we find an abrupt transient to a passive, "rigor state" and a return to dissipation dominated by transverse filament modes. Single-filament measurements of the apparent persistence length support the notion that motor activity leads to an increase in the effective temperature for tangential motion.

Nucleotide-induced changes in muscle fibres studied by DSC and TMDSC

Differential scanning calorimetry (DSC) was used in conventional and temperature-modulated mode to study the energetics of myosin in skeletal muscle fibres in different states of the actomyosin ATPase cycle. Psoas muscle fibres from rabbit were used in the experiments with and without the presence of nucleotides (ATP, ADP, AMP–PNP) and ATP or ADP+orthovanadate. In the complex DSC pattern, the higher transition referred to the head region of myosin. The enthalpy of the thermal unfolding depended on the nucleotides, the conversion from a strongly attached state of myosin to actin to a weakly binding state was accompanied with an increase of the transition temperature which was due to the change of the affinity of nucleotide binding to myosin. This was more pronounced in TMDSC mode, indicating that the strong-binding state and rigor state differ energetically from each other. The different transition temperatures indicated alterations in the internal microstructure of myosin head region. The monoton decreasing TMDSC heat capacities show that the C p of biological samples should not be temperature independent.

Crossbridge and tropomyosin positions observed in native, interacting thick and thin filaments

Tropomyosin movements on thin filaments are thought to sterically regulate muscle contraction, but have not been visualized during active filament sliding. In addition, although 3-D visualization of myosin crossbridges has been possible in rigor, it has been difficult for thick filaments actively interacting with thin filaments. In the current study, using three-dimensional reconstruction of electron micrographs of interacting filaments, we have been able to resolve not only tropomyosin, but also the docking sites for weak and strongly bound crossbridges on thin filaments. In relaxing conditions, tropomyosin was observed on the outer domain of actin, and thin filament interactions with thick filaments were rare. In contracting conditions, tropomyosin had moved to the inner domain of actin, and extra density, reflecting weakly bound, cycling myosin heads, was also detected, on the extreme periphery of actin. In rigor conditions, tropomyosin had moved further on to the inner domain of actin, and strongly bound myosin heads were now observed over the junction of the inner and outer domains. We conclude (1) that tropomyosin movements consistent with the steric model of muscle contraction occur in interacting thick and thin filaments, (2) that myosin-induced movement of tropomyosin in activated filaments requires strongly bound crossbridges, and (3) that crossbridges are bound to the periphery of actin, at a site distinct from the strong myosin binding site, at an early stage of the crossbridge cycle.