Dissociation Of Actomyosin Research Articles

Single-Molecule Investigation of Load-Dependent Actomyosin Dissociation Kinetics for Cardiac and Slow Skeletal Myosin.

Myosins are ATP-powered, force-generating motor proteins involved in cardiac and muscle contraction. The external load experienced by the myosins modulates and coordinates their function in vivo. Here, this study investigates the tension-sensing mechanisms of rabbit native β-cardiac myosin (βM-II) and slow skeletal myosins (SolM-II) that perform in different physiological settings. Using mobile optical tweezers with a square wave-scanning mode, a range of external assisting and resisting loads from 0 to 15 pN is exerted on single myosin molecules as they interact with the actin filament. Influenced of load on specific strongly-bound states in the cross-bridge cycle is examined by adjusting the [ATP]. The results implies that the detachment kinetics of actomyosin ADP.Pi strongly-bound force-generating state are load sensitive. Low assisting load accelerates, while the resisting load hinders the actomyosin detachment, presumably, by slowing both the Pi and ADP release. However, under both high assisting and resisting load, the rate of actomyosin dissociation decelerates. The transition from actomyosin ADP.Pi to ADP state appears to occur with a higher probability for βM-II than SolM-II. This study interpret that dissociation of at least three strongly-bound actomyosin states are load-sensitive and may contribute to functional diversity among different myosins.

Differentiated texture and lipid-lowering effect of cooked pork belly by different cooking regimes: insights from myofibrillar protein aggregation and dissociation, micromorphology

The effects of traditional flame stewing (TFS), commercial pressure cooking (PC), and sous-vide cooking (SVC) on the texture of pork belly and the underlying mechanism were investigated. SVC treatment at a low temperature (60 °C) achieved weaker oxidation of sulfhydryl groups, lower α-helical loss, higher surface hydrophobicity, and actomyosin dissociation, which reduced the shrinkage of myofibrils and loss of immobilized water, thus contributed to a tender meat. However, the protein aggregation predominated without actomyosin dissociation during PC treatment, causing the toughening of meat. TFS and PC intensified the atrophy of adipocytes and damage of cell membranes, which helped for the softening of adipose tissue and achieved 13.82% and 17.62 % lipid loss, respectively, and contributed to a higher ratio of polyunsaturated fatty acids to saturated fatty acids (0.45–0.46) than SVC (0.34). Comparisons showed that TFS was a more suitable method to prepare cooked pork belly with tender and juicy texture, low lipid content, and high ratio of polyunsaturated fatty acids to saturated fatty acids. This study offered new insights into how cooking regimes affect the texture and lipid-lowering effects of stewed pork.

Changes in tenderness of beef M. semitendinosus and modification of actomyosin mediated by Fe(III)-protoporphyrin IX, protoporphyrin IX and free iron

The objective of the present study was to explore the effect of Fe(III)-protoporphyrin IX (hemin), protoporphyrin IX (PPIX) and free iron on beef M. semitendinosus tenderness and its molecular mechanism. Hemin, PPIX and FeCl3 treatment of beef muscles under heating tremendously decreased shear force of beef meat by approximately 24%, 26%, and 30% respectively and weakened the association of actin-myosin. The protein carbonyl content increased and sulfhydryl content decreased significantly in FeCl3 and hemin treated groups, indicating the oxidative modification of actomyosin. The higher surface hydrophobicity and intrinsic fluorescence intensity demonstrated that hemin, PPIX and FeCl3 increased the unfolding of actomyosin, and the crosslinking was formed by hydrophobic interaction and disulfide bonds. Hemin treatment increased the α-helices/random coil and initiated more structural changes in actomyosin as compared to that of PPIX and FeCl3. These biochemical changes might contribute to the dissociation of actomyosin. The obtained results established a link between meat tenderness and porphyrins/free iron content, and gave new insights into the mechanism of how hemin and released PPIX and free iron affect the physicochemical properties of actomyosin.

Correlation analysis of phosphorylation of myofibrillar protein and muscle quality of tilapia during storage in ice

In this study, the correlation between protein phosphorylation and deterioration in the quality of tilapia during storage in ice was examined by assessing changes in texture, water-holding capacity (WHC), and biochemical characteristics of myofibrillar protein throughout 7 days of storage. The hardness significantly decreased from 471.50 to 252.17 g, whereas cooking and drip losses significantly increased from 26.5% to 32.6% and 2.9% to 9.1%, respectively (P < 0.05). Myofibril fragmentation increased, while myofibrillar protein sulfhydryl content and Ca2+-ATPase activity decreased from 119.33 to 89.29 μmol/g prot and 0.85 to 0.46 μmolPi/mg prot/h, respectively (P < 0.05). Correlation analysis revealed that the myofibrillar protein phosphorylation level was positively correlated with hardness and Ca2+-ATPase activity but negatively correlated with WHC. Myofibrillar protein phosphorylation affects muscle contraction by influencing the dissociation of actomyosin, thereby regulating hardness and WHC. This study provides novel insights for the establishment of quality control strategies for tilapia storage based on protein phosphorylation.

L-arginine and l-lysine supplementation to NaCl tenderizes porcine meat by promoting myosin extraction and actomyosin dissociation

This study investigated the individual and combined effects of l-arginine, l-lysine, and NaCl on the ultrastructure of porcine myofibrils to uncover the mechanism underlying meat tenderization. Arg or Lys alone shortened A-bands and damaged M-lines, while NaCl alone destroyed M- and Z-lines. Overall, Arg and Lys cooperated with NaCl to destroy the myofibrillar ultrastructure. Moreover, these two amino acids conjoined with NaCl to increase myosin solubility, actin band intensity, and the protein concentration of the actomyosin supernatant. However, they decreased the turbidity and particle size of both myosin and actomyosin solutions, and the remaining activities of Ca2+- and Mg2+-ATPase. The current results revealed that Arg/Lys combined with NaCl to extract myosin and dissociate actomyosin, thereby aggravating the destruction of the myofibrillar ultrastructure. The present results provide a good explanation for the previous phenomenon that Arg and Lys cooperated with NaCl to improve meat tenderness.

Improved tenderness and water retention of pork pieces and its underlying molecular mechanism through the combination of low-temperature preheating and traditional cooking.

The effects of two-stage heating with different preheating combinations on the shear force and water status of pork-pieces were explored. The results showed that the combined preheating at 50 ℃ for 35min or at 60 ℃ for 5 or 20min with traditional high temperature heating reduced the shear force and improved the water retention of meat, which was attributed to uniformly separation of myofibers and smaller myofiber space. Visible dissociation of actomyosin in heating groups of 50 ℃-35min, and 60 ℃-5, 20min was related to the tenderization of meat. The higher surface hydrophobicity, tryptophan fluorescence intensity, and lower α-helices of actomyosin at 60 ℃ contributed to the liberation of actin. However, severe oxidation of sulfhydryl groups at 70 ℃ and 80 ℃ promoted the aggregation of actomyosin. This study presents the advantage of two-stage heating in improving meat tenderness and juiciness and its underlying mechanisms.

Single-molecule mechanics and kinetics of cardiac myosin interacting with regulated thin filaments

The cardiac cycle is a tightly regulated process wherein the heart generates force to pump blood to the body during systole and then relaxes during diastole. Disruption of this finely tuned cycle can lead to a range of diseases including cardiomyopathies and heart failure. Cardiac contraction is driven by the molecular motor myosin, which pulls regulated thin filaments in a calcium-dependent manner. In some muscle and nonmuscle myosins, regulatory proteins on actin tune the kinetics, mechanics, and load dependence of the myosin working stroke; however, it is not well understood whether or how thin-filament regulatory proteins tune the mechanics of the cardiac myosin motor. To address this critical gap in knowledge, we used single-molecule techniques to measure the kinetics and mechanics of the substeps of the cardiac myosin working stroke in the presence and absence of thin filament regulatory proteins. We found that regulatory proteins gate the calcium-dependent interactions between myosin and the thin filament. At physiologically relevant ATP concentrations, cardiac myosin’s mechanics and unloaded kinetics are not affected by thin-filament regulatory proteins. We also measured the load-dependent kinetics of cardiac myosin at physiologically relevant ATP concentrations using an isometric optical clamp, and we found that thin-filament regulatory proteins do not affect either the identity or magnitude of myosin’s primary load-dependent transition. Interestingly, at low ATP concentrations at both saturating and physiologically relevant subsaturating calcium concentrations, thin-filament regulatory proteins have a small effect on actomyosin dissociation kinetics, suggesting a mechanism beyond simple steric blocking. These results have important implications for the modeling of cardiac physiology and diseases.

Cardiac myosin's mechanics and load dependent kinetics are not altered by the thin-filament proteins.

The force necessary to power human heart contraction is generated by beta cardiac myosin pulling on regulated thin filaments. Myosin's force generation is a tightly regulated process, and its disruption causes diseases including genetic cardiomyopathies. Deciphering the details of force generation at the molecular scale is critical for our understanding of both cardiac physiology and disease. While excellent studies have elucidated the mechanics and kinetics underlying the interactions between cardiac myosin and actin at the single molecule scale, the majority of these studies have been conducted in the absence of regulatory proteins. In the case of several other myosin isoforms, regulatory proteins alter the mechanics and load-dependent kinetics of the myosin working stroke, and it is not clear what role, if any they play in tuning the cardiac myosin working stroke and load-dependent kinetics. Here, we used a single molecule optical trapping assay to measure myosin's mechanics in the presence and absence of reconstituted thin-filaments (RTFs). We observed that regulatory proteins do not affect the myosin working stroke or its substeps. Interestingly, at low ATP, we observed that regulatory proteins slow the rate of actomyosin dissociation, and we showed using stopped flow kinetics that this is due to tuning of the rate of ATP-induced dissociation. The difference in the dissociation rate disappears at physiologically relevant ATP concentrations. We also used an isometric force clamp to investigate the load dependence of the myosin working stroke at physiologically relevant ATP concentrations, and this was also unchanged with RTFs. Taken together, these results show that cardiac myosin's mechanics are not altered by regulatory proteins at physiologically relevant ATP concentrations, and they provide important insights into the molecular mechanisms underlying heart contraction.

Effects of actomyosin dissociation on the physicochemical and gelling properties of silver carp myofibrillar protein sol during freeze–thaw cycles

Myofibrillar protein (MP) system with different dissociation degrees of actomyosin was constructed by addition of ATP and the effects of actomyosin dissociation on the physicochemical and gelling properties of MP sol during freeze–thaw cycles were investigated. The results showed that the salt soluble protein content of the dissociated sample with 5 mM ATP (5-pH 6.2 group) was lower than that of other groups under unfrozen state, while the fluorescence intensity and hardness of all dissociated groups were significantly lower (P＜0.05) than that of the control group. After five freeze–thaw cycles, the low-field nuclear magnetic resonance (LF NMR) revealed that the immobile water (T22b) in MP sol with 5 mM ATP (pH 6.9) has lower fluidity compared with the control group. In addition, rheological studies revealed that the G′ (storage modulus) value at 90℃ of MP sol with 5 mM ATP (pH 6.9) showed less decrease than that of the other groups after five freeze–thaw cycles. Meanwhile, after five freeze–thaw cycles, scanning electron microscope (SEM) showed that the gel of the control group has large holes and rough structure, while the microstructure of sample with 5 mM ATP (pH 6.9) was more compact and uniform. The heat-induced gel of 5-pH 6.9 group had highest hardness and lowest cooking loss among the groups after five freeze–thaw cycles. Briefly, the dissociation of actomyosin before freezing could slow down the rate of MP denaturation and improve the gelling properties after freeze–thaw cycle.

The Local Environment of Loop Switch 1 Modulates the Rate of ATP-Induced Dissociation of Human Cardiac Actomyosin.

Two isoforms of human cardiac myosin, alpha and beta, share significant sequence similarities but show different kinetics. The alpha isoform is a faster motor; it spends less time being strongly bound to actin during the actomyosin cycle. With alpha isoform, actomyosin dissociates faster upon ATP binding, and the affinity of ADP to actomyosin is weaker. One can suggest that the isoform-specific actomyosin kinetics is regulated at the nucleotide binding site of human cardiac myosin. Myosin is a P-loop ATPase; the nucleotide-binding site consists of P-loop and loops switch 1 and 2. All three loops position MgATP for successful hydrolysis. Loops sequence is conserved in both myosin isoforms, and we hypothesize that the isoform-specific structural element near the active site regulates the rate of nucleotide binding and release. Previously we ran molecular dynamics simulations and found that loop S291-E317 near loop switch 1 is more compact and exhibits larger fluctuations of the position of amino acid residues in beta isoform than in alpha. In alpha isoform, the loop forms a salt bridge with loop switch 1, the bridge is not present in beta isoform. Two isoleucines I303 and I313 of loop S291-E317 are replaced with valines in alpha isoform. We introduced a double mutation I303V:I313V in beta isoform background and studied how the mutation affects the rate of ATP binding and ADP dissociation from actomyosin. We found that ATP-induced actomyosin dissociation occurs faster in the mutant, but the rate of ADP release remains the same as in the wild-type beta isoform. Due to the proximity of loop S291-E317 and loop switch 1, a faster rate of ATP-induced actomyosin dissociation indicates that loop S291-E317 affects structural dynamics of loop switch 1, and that loop switch 1 controls ATP binding to the active site. A similar rate of ADP dissociation from actomyosin in the mutant and wild-type myosin constructs indicates that loop switch 1 does not control ADP release from actomyosin.

Electrostatic interaction of loop 1 and backbone of human cardiac myosin regulates the rate of ATP induced actomyosin dissociation.

Double mutation D208Q:K450L was introduced in the beta isoform of human cardiac myosin to remove the salt bridge D208:K450 connecting loop 1 and the seven-stranded beta sheet within the myosin head. Beta isoform-specific salt bridge D208:K450, restricting the flexibility of loop 1, was previously discovered in molecular dynamics simulations. Earlier it was proposed that loop 1 modulates nucleotide affinity to actomyosin and we hypothesized that the electrostatic interactions between loop 1 and myosin head backbone regulate ATP binding to and ADP dissociation from actomyosin, and therefore, the time of the strong actomyosin binding. To examine the hypothesis we expressed the wild type and mutant of the myosin head construct (1-843 amino acid residues) in differentiated C2C12 cells, and the kinetics of ATP-induced actomyosin dissociation and ADP release were characterized using stopped-flow spectrofluorometry. Both constructs exhibit a fast rate of ATP binding to actomyosin and a slow rate of ADP dissociation, showing that ADP release limits the time of the strongly bound state of actomyosin. We observed a faster rate of ATP-induced actomyosin dissociation with the mutant, compared to the wild type actomyosin. The rate of ADP release from actomyosin remains the same for the mutant and the wild type actomyosin. We conclude that the flexibility of loop 1 is a factor affecting the rate of ATP binding to actomyosin and actomyosin dissociation. The flexibility of loop 1 does not affect the rate of ADP release from human cardiac actomyosin.

Effects of chilling rate on progression of rigor mortis in postmortem lamb meat

This work investigated the effects of chilling rate on the progression of rigor mortis and explored possible mechanisms. Silverside from 18 lamb carcasses was assigned to control group (1.94 °C/h), very fast chilling-I group (VFC-I, 12.19 °C/h) and VFC-II group (15.10 °C/h). The shear force, myofibril fragmentation index (MFI), actomyosin ATPase activity, protein degradation and actomyosin dissociation were determined. There was no increase in the shear force in VFC-II group. The activation of actomyosin ATPase at 2–4 h postmortem in VFC-II group resulted in super-contracted sarcomeres and an increase in MFI. The degradation of μ-calpain, troponin T and desmin in VFC-II group was higher than that in control group from 6 to 24 h postmortem. These results suggested that rigor mortis was influenced which resulted in decreased shear force at a chilling rate of 15.10 °C/h by activating actomyosin ATPase and μ-calpain at early postmortem and promoted actomyosin dissociation.

Effects of acetylation on dissociation and phosphorylation of actomyosin in postmortem ovine muscle during incubation at 4 °C in vitro

This study aimed to assess the effects of acetylation levels on actomyosin disassociation and phosphorylation of lamb during incubation at 4 °C. Samples of whole proteins from lamb longissimus thoracis muscles were prepared and assigned into three treatments (high, middle and low acetylation groups). The results showed that deacetylation of myosin heavy chain and actin was inhibited by lysine deacetylase inhibitor trichostatin A and nicotinamide in this study. Phosphorylation levels of myosin heavy chain and actin were inhibited by their acetylation during incubation in vitro. Actomyosin disassociation degree in high acetylation group was significantly lower than that in middle and low acetylation groups (P < 0.05). The ATPase activity in high acetylation group was significantly higher than that in middle and low acetylation groups (P < 0.05). In conclusion, acetylation of myosin heavy chain and actin inhibited actomyosin dissociation by inhibiting their phosphorylation at 4 °C in vitro.

Phosphorylation of myosin regulatory light chain at Ser17 regulates actomyosin dissociation

Phosphorylation of myosin regulatory light chain (MRLC) can regulate muscle contraction and thus affect actomyosin dissociation and meat quality. The objective of this study was to explore the mechanism by how MRLC phosphorylation regulates actomyosin dissociation and thus develop strategies for improving meat quality. Here, the phosphorylation status of MRLC was modulated by myosin light chain kinase and myosin light chain kinase inhibitor. MRLC phosphorylation at Ser17 decreased the kinetic energy and total energy of actomyosin, thus stabilized the structure, facilitating the interaction between myosin and actin; this was one possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation. Moreover, MRLC phosphorylation at Ser17 was beneficial to the formation of ionic bonds, hydrogen bonds, and hydrophobic interaction between myosin and actin, and was the second possible way that MRLC phosphorylation at Ser17 negatively affects actomyosin dissociation.

Duck breast muscle proteins, free fatty acids and volatile compounds as affected by curing methods

The aim of this study was to investigate the effects of different curing methods on protein structure, protein and lipid oxidation, lypolysis and volatile compounds in duck breast meat. The results showed that compared to static brining and pulsed pressure salting, the vacuum tumbling curing significantly decreased the oxidation of proteins and lipids, and the surface hydrophobicity of proteins, increased α-helix structure but decreased the proportion of β-sheet, and increased actomyosin dissociation, liplysis and the free fatty acid content in meat. Meanwhile, vacuum tumbling curing decreased the amount of volatile flavor compounds, hexanal, 2,3-octanone, and off-flavor compounds 1-octen-3-ol and 1-hexanol. This study suggests that concerns on healthiness and the sensory quality of processed meat products should be paid in the selection of curing methods and vacuum tumbling curing is superior in terms of both aspects.

Quantitative phosphoproteomics analysis of actomyosin dissociation affected by specific site phosphorylation of myofibrillar protein

Phosphorylation of myofibrillar proteins can regulate muscle contraction and thus affect actomyosin dissociation. To explore the mechanism by which myofibrillar protein phosphorylation and phosphorylation site affects actomyosin dissociation, mass spectrometry-based quantitative phosphoproteomics was used to analyze ovine longissimus thoracis muscles with different actomyosin dissociation. A total of 2073 phosphorylated peptides from 826 phosphoproteins were identified among the myofibrillar proteins of muscles with different actomyosin dissociation, and 176 phosphorylated peptides from 114 phosphoproteins were significantly different (P < 0.05) between the high and low group of actomyosin dissociation. Phosphorylation of myosin heavy chain 1 at Ser1896, myosin heavy chain 7 at Thr1019 and Tyr1307, and titin at Ser265, Ser6085, Ser21930, and Ser26576 can inhibit actomyosin dissociation, whereas phosphorylation of tropomyosin 2 at Ser86, Thr252, and Ser279 contribute to actomyosin dissociation. The phosphorylation sites associated with actomyosin dissociation were mostly serine residues, and the phosphorylation status of myosin regulatory light chain at Ser15 was negatively correlated with actomyosin dissociation. In conclusion, the phosphorylation of some myofibrillar proteins and myosin at specific serine residues may be crucial for actomyosin dissociation.

L-arginine and L-lysine degrade troponin-T, and L-arginine dissociates actomyosin: Their roles in improving the tenderness of chicken breast

This work investigated the effects of L-arginine (Arg) and L-lysine (Lys) on the tenderness of chicken breast and explored the possible mechanisms underlying this effect for the first time. The results showed that both Arg and Lys decreased the shear force and increased the pH value, sarcomere length and myofibrillar fragmentation index as well as degraded the troponin-T by keeping calpain activity in chicken breast. In addition, Arg effectively reduced Ca2+/Mg2+-ATPase activities and promoted actomyosin dissociation. These results indicated that both Arg and Lys could enhance the tenderness of chicken breast, and it could also explain why Arg was more effective than Lys in improving the tenderness of chicken breast. These results will help facilitate the development of industrial-scale methods for improving the tenderness of meat products.

Combined effect of ultrasound and sodium bicarbonate marination on chicken breast tenderness and its molecular mechanism

The objective of this study was to explore the combined effect of ultrasound (20 kHz ultrasound probe (12 mm diameter), an output power of 350 W for 5 min (on-time and off-time pulse durations of 2 s and 3 s, respectively)) with low concentration (0.2 M) sodium bicarbonate solution (USB) on chicken breast tenderness and its preliminary molecular mechanism. Therefore, myofibril fragmentation index (MFI), filtering residues, cooking loss, shear force, histology of meat as well as SDS-PAGE, circular dichroism (CD), synchronous fluorescence spectroscopy, differential scanning calorimetrv (DSC) of actomyosin were investigated and compared with untreated (Control), deionized water (DW), ultrasound in deionized water (UDW), 0.2 M sodium bicarbonate solution (SB), respectively. The results showed that USB can effectively increase MFI, and reduce filtering residues, cooking loss and shear force by compared with UDW or SB. Furthermore, myofibril was presented with the largest interfibrillary spaces and the highest degree of actomyosin dissociation in USB group. The increase in α-helix content and decrease in fluorescence intensity of tyrosine and tryptophan implied that USB caused the conformation change in actomyosin. Additionally, actomyosin in USB group became more sensitive to temperature. Therefore, the treatment of ultrasound combined with low concentration of sodium bicarbonate accelerated actomyosin degeneration was considered as a promising and efficient technique in meat processing, especially for the fitness enthusiasts.

Phosphorylation of myosin regulatory light chain affects actomyosin dissociation and myosin degradation

SummaryThis research aimed to investigate the influence of myosin regulatory light chain (MRLC) phosphorylation on actomyosin dissociation and myosin degradation in post‐mortem ovine longissimus thoracis (LT) muscle with a purpose for strategies to improve meat quality in practice. Data obtained show that the highest actomyosin dissociation occurred at 48 h post‐mortem. Based on the cluster analysis of the actomyosin dissociation degree at 48 h post‐mortem, samples were divided into high, middle and low‐actomyosin dissociation groups. During 0.5–72 h post‐mortem, the phosphorylation level of MRLC significantly decreased (P &lt; 0.05), while actomyosin dissociation, and degradation of MRLC and myosin heavy chains (MHC) significantly increased (P &lt; 0.05). The level of MRLC phosphorylation was negatively correlated with the degree of actomyosin dissociation (r = −0.794, P &lt; 0.05), and the degradation of MRLC (r = −0.764, P &lt; 0.05) and MHC (r = −0.826, P &lt; 0.05). In conclusion, phosphorylation of MRLC may regulate actomyosin dissociation, as well as degradation of MRLC and MHC.

Macromolecular Crowding Affects the Rate of ADP Release from Actomyosin

We have used macromolecular crowding agent Ficoll PM 70 to mimic the intracellular environment and study the effect of crowding on the kinetics of actomyosin dissociation and ADP release. Our previous studies of the heavily mutated D. discoideum myosin S1 construct showed that the effect of macromolecular crowding results in a more compact state of the myosin head. In the current study, we have used the wild-type rabbit skeletal myosin S1. Addition of 10% w/v Ficoll led to the faster rate of ATP induced actomyosin dissociation, in agreement with the excluded volume theory. The kinetics of ADP dissociation from actomyosin is best fitted to the two-exponential function. One component of the kinetics transient reflects ADP dissociation, which was slower than the rate of ADP dissociation without Ficoll in solution. Our data show that ADP binds actomyosin stronger in the presence of Ficoll. The rate of the second component of the transient does not depend on the concentration of nucleotides in solution. This component of the observed transient can be interpreted as the kinetics of myosin conformational change, leading to the opening of the active site for nucleotide to bind. We conclude that macromolecular crowding not only increases the chemical potential of solutes, but affects protein structural state due to change in the osmotic pressure.