Conformational Changes In Myosin Research Articles

Exploring the Super-Relaxed State of Human Cardiac β-Myosin by Molecular Dynamics Simulations.

Human β-cardiac myosin plays a critical role in generating the mechanical forces necessary for cardiac muscle contraction. This process relies on a delicate dynamic equilibrium between the disordered relaxed state (DRX) and the super-relaxed state (SRX) of myosin. Disruptions in this equilibrium due to mutations can lead to heart diseases. However, the structural characteristics of SRX and the molecular mechanisms underlying pathogenic mutations have remained elusive. To bridge this gap, we conducted molecular dynamics simulations and free energy calculations to explore the conformational changes in myosin. Our findings indicate that the size of the phosphate-binding pocket can serve as a valuable metric for characterizing the transition from the DRX to SRX state. Importantly, we established a global dynamic coupling network within the myosin motor head at the residue level, elucidating how the pathogenic mutation E483K impacts the equilibrium between SRX and DRX through allosteric effects. Our work illuminates molecular details of SRX and offers valuable insights into disease treatment through the regulation of SRX.

Geldanaycin-encapsulated magnetic nanoparticle for isolation of myosin in proteomics

The grafting of a drug molecule, i.e., geldanamycin (GA) onto polyethyleneimine (PEI)-coated magnetic nanoparticle produces a novel composite, GA@Fe3O4–NH2. The composite is confirmed by characterizations with FT-IR, Raman, SEM, EDS, VSM and TEM. Due to the high binding-affinity of GA with myosin heavy chain (MYH), GA@Fe3O4–NH2 exhibits excellent adsorption performance towards myosin. Consequently, a solid-phase extraction procedure is established for highly efficient and selective separation of myosin from pig heart extract. At pH 6.0, an adsorption efficiency of 97.1 % is achieved for treating 100 μg mL−1 myosin (0.1 mL) with 0.1 mg GA@Fe3O4–NH2 as adsorbent. The adsorption behavior of myosin onto GA@Fe3O4–NH2 fits Langmuir model, corresponding to a theoretical adsorption capacity of 518.1 mg g−1. The adsorbed myosin can be readily recycled by the SDS solution (1 %, m/m) with an elution efficiency of 91.8 %. According to circular dichroism spectroscopy, the conformational changes of myosin during adsorption and elution are reversible. For practical application, myosin is successfully isolated from the pig left ventricular protein extract with GA@Fe3O4–NH2, and SDS-PAGE and LC-MS/MS showed that myosin had high purity and a total of 716 proteins could be identified. Significantly, Geldamycin-encapsulated magnetic nanoparticle for the separation of myosin well exploits the potential of the nanomaterials modified by drug molecules in the separation and purification of target proteins.

Collagens for surimi gel fortification: Type-dependent effects and the difference between type I and type II

Surimi products have unsatisfactory gel properties. Hence, this study evaluates the effect of collagen-adding on surimi gel properties and provides the first observation results regarding collagen type influence. With higher water solubility and more charged amino acids than type II, collagen type I intertwines with surimi myofibrillar proteins better to induce higher exposure of protein functional domains, more sufficient conformational changes of myosin and greater formation of chemical forces among proteins. These enhancements accelerate the gelation rate, leading to a well-stabilized surimi gel. The collagen I-containing surimi gels show more compact structures with uniformly distributed smaller pores than those containing collagen II, thereby providing the final products with higher water holding capacity and better textural profiles. As such, the surimi gel fortification performance of collagen I and the well-elucidated collagen-myofibrillar protein interaction mechanism will guide the further exploitation of collagen as an effective additive in the food industry.

Ameliorative effects of L-arginine? On heat-induced phase separation of Aristichthys nobilis myosin are associated with the absence of ordered secondary structures of myosin

This investigation aimed to study the potential mechanism of L-arginine (L-Arg) on the heat-induced phase separation phenomenon of myosin from the perspective of conformational changes of myosin. L-Arg ameliorated the phase separation of myosin after a two-step heating procedure via suppression of heat-induced aggregation of myosin. The effect of L-Arg on the heating of myosin at high temperatures (75–85 °C) was more pronounced than that in the setting stage (35–45 °C), suggesting that the ameliorative effects of L-Arg on the heat-induced phase separation of myosin are mainly attributed to the inhibition of rod-rod cross-linking between denatured myosin molecules. Additionally, L-Arg without pH modification exhibited an increased ability to suppress the gelation of myosin compared with pH modification, indicating that both pH effects and the particular structure of L-Arg play noticeable roles in the suppression of myosin gelation. Far-UV circular dichroism, intrinsic fluorescence spectroscopy and differential scanning calorimetry demonstrated that L-Arg induced the absence of ordered secondary structures of myosin molecules, especially β-sheets, and thus generated a looser protein structure, which may represent the dominant suppression mechanisms of L-Arg on the heat-induced aggregation of myosin. This work provided support for the use of L-Arg as a food additive, and the results of this study will be attractive to the meat and beverage products.

Molecular Mechanisms of Muscle Weakness Associated with E173A Mutation in Tpm3.12. Troponin Ca2+ Sensitivity Inhibitor W7 Can Reduce the Damaging Effect of This Mutation.

Substitution of Ala for Glu residue in position 173 of γ-tropomyosin (Tpm3.12) is associated with muscle weakness. Here we observe that this mutation increases myofilament Ca2+-sensitivity and inhibits in vitro actin-activated ATPase activity of myosin subfragment-1 at high Ca2+. In order to determine the critical conformational changes in myosin, actin and tropomyosin caused by the mutation, we used the technique of polarized fluorimetry. It was found that this mutation changes the spatial arrangement of actin monomers and myosin heads, and the position of the mutant tropomyosin on the thin filaments in muscle fibres at various mimicked stages of the ATPase cycle. At low Ca2+ the E173A mutant tropomyosin shifts towards the inner domains of actin at all stages of the cycle, and this is accompanied by an increase in the number of switched-on actin monomers and myosin heads strongly bound to F-actin even at relaxation. Contrarily, at high Ca2+ the amount of the strongly bound myosin heads slightly decreases. These changes in the balance of the strongly bound myosin heads in the ATPase cycle may underlie the occurrence of muscle weakness. W7, an inhibitor of troponin Ca2+-sensitivity, restores the increase in the number of myosin heads strongly bound to F-actin at high Ca2+ and stops their strong binding at relaxation, suggesting the possibility of using Ca2+-desensitizers to reduce the damaging effect of the E173A mutation on muscle fibre contractility.

Protein Conformational Dynamics Probed Correlation Spectroscopy of Multiply Scattered Light

To assess the dynamics of proteins due to their external and internal degrees of freedom, we used a optical correlation spectroscopy method, that provides the spectrum of optical fluctuations due to protein dynamics in solution. In the case of acto-myosin forced out-of-equilibrium by its chemically driven motor activity, the difference spectrum between thermal and driven fluctuations reveals the frequency-amplitude properties of the conformational dynamics of myosin. This optical method is the so-called DWS (diffusive wave spectroscopy), first proposed by Maret and Wolf in 1987 (Z. Phys.). Practically, when illuminated by a highly monochromatic and coherent light beam in the strong scattering regime, the sample produces a diffusive light flux inside the sample. Outside of the sample, one obtains an intensity speckle field that is determined by the scattering properties and the spatial distribution of the scatterers, with fixed speckles if scatterers are immobile. By measuring the speckle intensity autocorrelation function, one obtains the fluctuation spectrum of the scaterrers. Using that method, we found that conformational changes of myosin occur at times-scales that are typically 2 to 3 orders of magnitude faster that the shortest time of the enzymatic activity cycle. To improve the sensitivity of that method, we introduce a new concept called Cavity Amplified Speckle Decorrelation Spectroscopy. The phase fluctuations of the light scattered by a given sample are amplified by placing it inside a closed Lambertian cavity with highly diffusively reflecting walls, which ergodically forces the light multiple times back into the sample. This new concept will be illustrated with a few biological and non-biological samples, showing that previously undetected fluctuations can be measured over 8-10 decades in the frequency domain, and resolved with unprecedented sensitivity.

Effect of pH shifting on conformation and gelation properties of myosin from skeletal muscle of blue round scads(Decapterus maruadsi)

To clarify the gel formation mechanism of isolated proteins recovered from blue round scads by isoelectric solubilization/precipitation, the physiochemical, conformational and gelation properties of the major protein myosin treated by acidic-(AM) or alkaline-(KM) pH-shifting were investigated and compared with native myosin (NM). The solubility in all myosin samples dropped significantly at 40 °C, with remarkable increases of particle size, turbidity and surface hydrophobicity. Among the samples, AM exhibited the lowest solubility and highest turbidity than NM and KM as the temperature was higher than 40 °C, indicating that AM is easily to be denatured and aggregated. Circular dichroism (CD) analysis showed that both acidic and alkaline pH treatments led to conformational changes of myosin, together with α-helix content variations. When pH was readjusted to neutral, KM recovered its structure similar to NM, while AM failed to refold to its native state. NM exhibited the highest storage modulus (G′) at 90 °C, while AM revealed the lowest G′ value. Furthermore, AM did not change obviously during heating, especially at the gelling temperature range (60–90 °C). These results strongly suggested that acidic treatment led to an irreversible conformational change in myosin, consequently induced a fierce denaturation and aggregation at the initial stage of heating, and eventually led to a complete loss of gel-forming ability. Alkaline pH-shifting, on the other hand is thus more feasible for protein recovery from blue round scads.

Molecular Dynamics Studies of Myosin Structure with 2-Deoxy-ADP

Myosin uses ATP catalysis to drive muscle contraction by facilitating cross-bridge cycling. During the cross-bridge powerstroke, coordinated release of the byproducts of nucleotide catalysis (Pi, ADP) triggers a series of conformational changes in myosin that alter its interactions with actin and ultimately generate force. In addition to ATP, myosins can utilize other nucleotides, such as the naturally occurring 2-deoxy-ATP (dATP), to catalyze the powerstroke. We have reported that dATP enhances cross-bridge binding and cycling dynamics, resulting in stronger, faster contraction especially in cardiac muscle. In a recent study (Nowakowski 2017, Protein Sci 26:749-62) our simulations of myosin in the pre-powerstroke state showed that dADP induces conformational changes that expose polar residues and enhance its affinity for actin. In a current study using x-ray structural analysis, we found that dATP increases the rate of myosin detachment following tetanic contraction. To understand the molecular basis for the enhanced rate of detachment in the presence of elevated dATP, we performed molecular dynamics simulations of the myosin motor domain modeled in the post-powerstroke conformation in the presence of Mg2+ and either ADP or dADP. We found that dADP was more flexible than ADP, more mobile within the nucleotide binding pocket and had different interactions with myosin relative to ADP. Furthermore, changes in the interactions between dADP and myosin were transmitted to the actin binding surface. Additionally, we observed partial release of the nucleotides from the binding pocket and have delineated the molecular mechanisms by which regulatory loops in myosin control nucleotide release. Our results have revealed molecular mechanisms underlying dATP-enhanced cross-bridge cycling dynamics. More generally they have yielded insights for the development of novel myosin-targeted therapies for cardiomyopathies based the structural advantages produced by dATP in resting and contracting muscle.

Effect of Mild Ozone Oxidation on Structural Changes of Silver Carp (Hypophthalmichthys molitrix) Myosin

To produce high-quality surimi, we investigated the effect of ozone-induced mild oxidation on structural changes of silver carp myosin by analyzing circular dichroism (CD), intrinsic fluorescence, the sulfhydryl group, surface hydrophobicity, SDS-PAGE, and dynamic light scattering (DLS) of myosin. Mild ozone oxidation of myosin was performed by applying gaseous ozone for a short time. Results showed that the content of sulfhydryl group decreased, and carbonyl content and surface hydrophobicity increased with an increase in ozone treatment time. A red shift in the maximum wavelength of intrinsic fluorescence indicated the unfolding of oxidized myosin. Further conformational change of myosin was illustrated by CD; α-helical content decreased significantly and β-turn, β-sheet, and random coil content increased with increased ozone treatment time, which demonstrated the rearrangement of β-structure in myosin. The results from DLS and SDS-PAGE indicated that soluble oligomers or aggregates were formed by disulfide bonds under mild ozone treatment. Therefore, ozone-induced mild oxidation did not strongly alter myosin structure but promoted myosin partial unfolding, which might result in exposing more cross-linking sites for the formation of surimi gel.

Cryo-EM structure of a human cytoplasmic actomyosin complex at near-atomic resolution

The interaction of myosin with actin filaments is the central feature of muscle contraction and cargo movement along actin filaments of the cytoskeleton. The energy for these movements is generated during a complex mechanochemical reaction cycle. Crystal structures of myosin in different states have provided important structural insights into the myosin motor cycle when myosin is detached from F-actin. The difficulty of obtaining diffracting crystals, however, has prevented structure determination by crystallography of actomyosin complexes. Thus, although structural models exist of F-actin in complex with various myosins, a high-resolution structure of the F-actin–myosin complex is missing. Here, using electron cryomicroscopy, we present the structure of a human rigor actomyosin complex at an average resolution of 3.9 Å. The structure reveals details of the actomyosin interface, which is mainly stabilized by hydrophobic interactions. The negatively charged amino (N) terminus of actin interacts with a conserved basic motif in loop 2 of myosin, promoting cleft closure in myosin. Surprisingly, the overall structure of myosin is similar to rigor-like myosin structures in the absence of F-actin, indicating that F-actin binding induces only minimal conformational changes in myosin. A comparison with pre-powerstroke and intermediate (Pi-release) states of myosin allows us to discuss the general mechanism of myosin binding to F-actin. Our results serve as a strong foundation for the molecular understanding of cytoskeletal diseases, such as autosomal dominant hearing loss and diseases affecting skeletal and cardiac muscles, in particular nemaline myopathy and hypertrophic cardiomyopathy.

Ca2+-Induced Conformational Changes of Myosin from Silver Carp (Hypophthalmichthys molitrix) in Gelation

The role of Ca2+ in gelation of myosin extracted from silver carp (Hypophthalmichthys molitrix) was investigated. Compared with the control, myosin with CaCl2 showed higher turbidity and larger particle size, indicating that CaCl2 contributed to myosin aggregation. Addition of CaCl2 decreased the myosin thermostability and promoted the unfolding of myosin α-helical structure during heating. Moreover, CaCl2 could increase surface hydrophobicity at low temperatures (20–40 °C) and promoted hydrophobic interactions. Meanwhile, it decreased the temperature of disulfide bond formation by about 10 °C. Addition of 10–40 mM CaCl2 favored activation of the Ca2+-ATPase activity at 20 to 30 °C, but not obvious with elevated temperatures. Ca2+-ATPase activity of myosin was undetectable when heated above 60 °C because of severe structural damage of myosin heads. G’ at both the first and second peaks increased with increasing CaCl2 concentration. Therefore, CaCl2 was beneficial for gelation of myosin by inducing the unfolding of myosin and enhancing disulfide bonds along with hydrophobic interactions between neighboring myosin molecules.

Fast monitoring of in-vivo conformational changes in myosin using single scan polarization-SHG microscopy

Fast imaging of molecular changes under high-resolution and label-free conditions are essential for understanding in-vivo processes, however, current techniques are not able to monitor such changes in real time. Polarization sensitive second harmonic generation (PSHG) imaging is a minimally invasive optical microscopy technique capable of quantifying molecular conformational changes occurring below the diffraction limit. Up to now, such information is generally retrieved by exciting the sample with different linear polarizations. This procedure requires the sample to remain static during measurements (from a few second to minutes), preventing the use of PSHG microscopy from studying moving samples or molecular dynamics in living organisms. Here we demonstrate an imaging method that is one order of magnitude faster than conventional PSHG. Based on circular polarization excitation and instantaneous polarimetry analysis of the second harmonic signal generated in the tissue, the method is able to instantaneously obtain molecular information within a pixel dwell time. As a consequence, a single scan is only required to retrieve all the information. This allowed us to perform PSHG imaging in moving C. elegans, monitoring myosin's dynamics during the muscular contraction and relaxation. Since the method provides images of the molecular state, an unprecedented global understanding of the muscles dynamics is possible by correlating changes in different regions of the sample.

Structural Transitions within the Actomyosin Complex in the Presence of Saturating ATP, Detected by Time-Resolved FRET

Myosin generates force by cycling between actin-attached (strong binding) and actin-detached (weak binding) states during the actomyosin ATPase cycle. However, it remains unclear what specific conformational changes in myosin take place upon binding to actin, and how these structural changes lead to product release and force. To characterize these changes, we have used site-directed FRET to determine distance between specific sites in actin and myosin subfragment 1 (S1) in the presence and absence of ATP. Rabbit skeletal muscle myosin has two essential light chain (ELC) isoforms, A1 and A2.The ELC isoform has significant effects on the interaction of S1 with actin, resulting in higher catalytic efficiency for the S1A1-isoenzyme but higher in vitro motility and muscle shortening velocity for S1A2.We have focused on S1A1, since it binds to actin in the presence of ATP with higher affinity. Actin was labeled at C374 with a donor, AlexaFluor 568. A1 light chain, was expressed,purified and labeled at C177 with an acceptor, AlexaFluor 647. The labeled A1 was exchanged into myosin S1, and S1A1 was isolated using a Talon affinity resin. The efficiency of energy transfer between C374 of actin and C177 of A1 was measured by time-resolved fluorescence, allowing us to detect directly the FRET within the actin-S1A1 complex, even in the presence of saturating ATP. Addition of ATP to the strongly bound complex resulted in a significant decrease in FRET within the weakly bound complex, corresponding to a significant increase in the donor-acceptor distance. We conclude that the transition between strongly and weakly bound states of acto-S1 is associated with a structural transition in the acto-S1 complex that affects the distance from actin to the light-chain domain of myosin.

Conformational changes at the nucleotide site in the presence of bound ADP do not set the velocity of fast Drosophila myosins

The conformational changes in myosin associated with ADP release and their influence on actin sliding velocity are not understood. Following actin binding, the myosin active site is in equilibrium between a closed and open ADP bound state, with the open state previously thought to favor ADP release and thus expected to be favored in faster myosins. However, our recent work with a variety of myosins suggests the opposite, that the open conformation is dominant in slower myosins, which have higher ADP affinities. To test if this correlation holds for fast myosin isoforms, we determined the relationships between conformational pocket dynamics, ADP affinity and velocity of four Drosophila myosins: indirect flight muscle (IFM) myosin (IFI), embryonic muscle myosin (EMB) and two IFI/EMB chimeras. Electron paramagnetic resonance spectra of nucleotide-analog spin probes (SLADP) bound to IFI subfragment-1 in the absence of actin showed a high degree of immobilization, indicating a predominately closed nucleotide pocket. The A·M·SLADP spectra of all four myosins in fibers (actin bound) also indicated an equilibrium favoring the closed conformation with the closed state closing even further. However, the energetics of pocket closure did not correlate with Drosophila myosin actin velocity suggesting our previous model relating pocket dynamics to velocity does not hold for fast myosin isoforms. We conclude that for these fast myosins, and possibly other fast myosins, velocity is controlled by factors other than the ratio of open to closed nucleotide pocket conformation.

2PS017 Contribution of the conformational change of myosin II in the coupled sliding and binding process of the force-generation(The 50th Annual Meeting of the Biophysical Society of Japan)

2PS017 Contribution of the conformational change of myosin II in the coupled sliding and binding process of the force-generation(The 50th Annual Meeting of the Biophysical Society of Japan)

Conformational Changes in Myosin V Monitored by Hydrogen-Deuterium Exchange Mass Spectrometry

Conformational changes between the apo and the BeF.ADP states of Myosin V were determined by hydrogen/deuterium exchange monitored by FT-ICR mass spectrometry.In the BeF.ADP state we have observed opening of the 50K domain cleft (actin binding cleft) (aa 566-578) as expected from the x-ray structure (Coureux et al., Nature 2003). The opening of the actin binding cleft was coupled to the closure (decreased solvent accessibility) of the active site - a stretch of aa 197-231 that includes switch 1, loop1 and B6 β-strand and an additional peptide at the entrance to the active site (aa 233-237).Unexpected changes were observed in the in the SH1 helix (aa 678-692), which was more exposed and the upper 50 kDa domain (aa 331-339 and 350-381) which was less exposed. More protection was observed in the loop coupling B3 β-strand to the SH1 helix (aa 662-676).Figure: Conformational changes in Myosin V mapped on the crystal structure. Increased solvent accessibility in BeF.ADP in blue wide cylinders. Decreased accessibility in narrow red cylinders.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Biochemical and Conformational Changes of Myosin Purified from Pacific Sardine at Various pHs

Biochemical and conformational changes of purified sardine myosin were investigated at various pHs. The purity of myosin, as determined by SDS-PAGE, was approximately 94.6%. One major band at 205 kDa, corresponding to myosin heavy chain, and 3 light chains at 31, 24, and 23 kDa were observed on the SDS-PAGE gel. The greatest myosin protein solubility was observed at pH 7 and remained constant up to pH 11. Sardine myosin showed no solubility at pHs 2.5 to 5.0. Three endothermic peaks were obtained for samples prepared at pHs 7 and 10, while no peaks were shown for pH 2 samples, indicating chemical denaturation of myosin occurred before thermal treatment. The greatest Ca(2+)-ATPase activity was observed at pH 7, while no activity was observed between pHs 2 and 5 and at pH 11. Total sulfhydryl content was not measured at pHs 2.5 to 4 while the greatest measure was obtained for samples at pH 5.5. Surface hydrophobicity was not detected from pHs 2.5 to 5.0; thereafter the content remained consistent through pH 11. Storage modulus, indicating the elastic element of myosin gels, was minimally affected at pH 2, indicating myosin was chemically denatured before the temperature sweep treatment. However, at pH 10, the thermal exposure of myosin, as evidenced by dynamic thermograms with deeper valleys at 40 to 60 degrees C, was noted, indicating myosin was not damaged by adjustment to pH 10 and therefore was still able to undergo thermal gelation.

Intermediate states of myosin head during atp hydrolysis cycle in psoas muscle fibres by EPR and DSC

Force generation in muscle during contraction arises from direct interaction of the two main protein components of the muscle, myosin and actin. The process is driven by the energy liberated from the hydrolysis of ATP. In the presence of CaATP the energy released from hydrolysis produces conformational changes in myosin and actin, which can be manifested as an internal motion of myosin head while bound to actin. It is suggested that myosin heads attached to actin produce conformational changes during the hydrolysis process of ATP, which results in a strain in the head portion of myosin in an ATP-dependent manner. These structural changes lead to a large rotation of myosin neck region relieving the strain.

Biochemical and physicochemical properties of actomyosin from pre- and post-spawned flounder ( Paralichtys patagonicus) stored on ice

The biochemical and physicochemical properties of actomyosin from pre- and post-spawned flounder stored on ice were studied. Irrespective of the gonadal condition of the fish, a gradual decrease in reduced viscosity, Mg 2+ATPase and Mg 2+Ca 2+ATPase activities of actomyosin was observed. A similar decrease in the Mg 2+ EGTA ATPase activity of actomyosin from post-spawned flounder was also observed. The decrease in enzymatic activities was accompanied by an increase in surface hydrophobicity of the protein. No signs of proteolysis of the major components of the actomyosin complex were detected. The relative percentage of myosin decreased and that of actin increased in actomyosin from pre- and post-spawned flounder stored on ice. These changes occur earlier in actomyosin from prespawned flounder. The results of this paper indicate that actomyosin from flounder denatured during fish storage on ice and suggest that this denaturation is due to structural and conformational changes in myosin which led to aggregation of this protein.

Exploration of the conformational space of myosin recovery stroke via molecular dynamics

Muscle contractions are driven by cyclic conformational changes of myosin, whose molecular mechanisms of operation are being elucidated by recent advances in crystallographic studies and single molecule experiments. To complement such structural studies and consider the energetics of the conformational changes of myosin head, umbrella sampling molecular dynamics (MD) simulations were performed with the all-atom model of the scallop myosin sub-fragment 1 (S1) with a bound ATP in solution in explicit water using the crystallographic near-rigor and transition state conformations as two references. The constraints on RMSD reaction coordinates used for the umbrella sampling were found to steer the conformational changes efficiently, and relatively close correlations have been observed between the set of characteristic structural changes including the lever arm rotation and the closing of the nucleotide binding pocket. The lever arm angle and key residue interaction distances in the nucleotide binding pocket and the relay helix show gradual changes along the recovery stroke reaction coordinate, consistent with previous crystallographic and computational minimum energy studies. Thermal fluctuations, however, appear to make the switch-2 coordination of ATP more flexible than suggested by crystal structures. The local solvation environment of the fluorescence probe, Trp 507 (scallop numbering), also appears highly mobile in the presence of thermal fluctuations.