Recombinant Myosin-A (PY01232), partial

What is Recombinant Myosin-A (PY01232) and what is its functional significance?

Myosin-A belongs to the myosin superfamily of motor proteins responsible for muscle contraction and cellular motility. Recombinant Myosin-A (PY01232), partial, refers to a laboratory-synthesized fragment of the Myosin-A protein produced through recombinant DNA technology. Myosins function as molecular motors that convert chemical energy from ATP hydrolysis into mechanical force. In research contexts, recombinant myosins provide valuable experimental tools for studying molecular mechanisms of muscle contraction, cellular transport, and disease pathologies. Myosins are highly conserved across species but exhibit isoform-specific variations that confer specialized functions in different tissues and developmental stages .

How do experimental approaches for Myosin-A (PY01232) differ from those used for other myosin isoforms?

Experimental approaches for studying Myosin-A should be tailored to its specific biochemical and mechanical properties. While methodologies may overlap with those used for other myosin isoforms, researchers should consider potential differences in enzymatic activity, actin-binding properties, and force generation. Common experimental approaches include actin-activated ATPase assays, in vitro motility assays, and single-molecule optical trapping. The recombinant myosin motor domains can be expressed in expression systems such as mouse myoblast C2C12 cells and purified for detailed functional characterization. For comprehensive analysis, researchers should measure key parameters including ATP cycle rate, step size, and load-dependent actin-bound time, as these collectively determine the power production properties of the molecular motor .

What are the key parameters to evaluate when characterizing Myosin-A (PY01232) function?

When characterizing Myosin-A function, researchers should evaluate several key parameters that collectively determine its molecular power generation capabilities:

• ATPase activity: Measure both kcat (maximum turnover rate) and Km (apparent affinity for actin) using NADH-coupled solution-based assays

• Motility: Assess velocity at different temperatures using in vitro motility assays to determine Q10 temperature coefficient

• Molecular mechanics: Determine step size (d), actin-detachment rate at zero force (k0), and load sensitivity parameter (δ) using single-molecule optical trapping

• Duty ratio: Calculate the proportion of time the motor spends strongly bound to actin during its ATPase cycle

These parameters provide a comprehensive picture of the motor's functional properties and can be organized in a data table similar to the one below, adapted from studies of other myosin isoforms :

How can molecular dynamics simulations complement experimental approaches in studying Myosin-A (PY01232)?

Molecular dynamics (MD) simulations provide valuable insights into the conformational dynamics of myosins that may not be accessible through experimental techniques alone. For Myosin-A research, MD simulations can reveal structural mechanisms underlying its functional properties, particularly regarding allosteric communication between functional domains. When properly implemented, these simulations can predict:

• Conformational changes during the ATP hydrolysis cycle

• Interactions between nucleotide-binding sites and the lever arm

• Structural effects of mutations or post-translational modifications

• Pre-powerstroke lever arm positioning and ADP pocket dynamics

Effective MD simulation strategies should include multiple replicate simulations launched from experimentally validated structures, with simulations conducted in the specific sequence context of Myosin-A rather than relying solely on homology modeling. This approach is crucial because, as demonstrated with other myosin isoforms, the effects of identical mutations can vary dramatically depending on sequence context. Researchers should analyze the distribution of conformations adopted by the motor to explain functional differences observed experimentally .

What strategies can be employed to investigate the load-dependent kinetics of Myosin-A (PY01232)?

Investigating load-dependent kinetics of Myosin-A requires sophisticated biophysical approaches that can measure force-velocity relationships at the single-molecule level. The recommended methodological approach includes:

• Optical tweezer-based single-molecule force spectroscopy to directly measure force-dependent step sizes and kinetics

• Implementation of feedback-controlled force-clamp experiments to maintain constant resistive loads

• Analysis of actin-bound time distributions under varying loads to determine:

  • Load-free actin-detachment rate (k₀)

  • Load sensitivity parameter (δ)

  • Characteristic distance parameter that reflects how sensitive the motor is to external forces

Data should be analyzed using the Bell model relationship: k = k₀exp(-Fδ/kBT), where k is the rate of detachment under force F, kB is Boltzmann's constant, and T is temperature. This approach has revealed that different myosin isoforms exhibit distinct load sensitivities, with some showing greater force-sensitivity than others. For precise interpretation, actin-detachment rates should be measured at physiologically relevant ATP concentrations (typically 1-2 mM) .

How can the effects of mutations in Myosin-A (PY01232) be systematically compared to mutations in homologous positions of other myosin isoforms?

A systematic comparative approach to studying mutations in homologous positions across myosin isoforms provides insight into both disease mechanisms and protein allostery. The recommended methodological framework includes:

• Identification of conserved residues across myosin isoforms through sequence alignment

• Generation of equivalent mutations in recombinant constructs of multiple myosin isoforms

• Parallel characterization using identical experimental conditions to measure:

  • ATPase activity (kcat and Km)

  • In vitro motility velocities at different temperatures

  • Single-molecule properties (step size, load sensitivity, actin-detachment rate)

• Calculation of duty ratios to understand the proportion of the ATPase cycle spent in the strong actin-bound state

• Complementary MD simulations to predict structural mechanisms underlying functional differences

This approach has revealed that mutations at homologous positions can have dramatically different, or even opposite, effects depending on sequence context. For example, studies comparing mutations in β-cardiac, embryonic, and perinatal myosins showed that mutations causing hypertrophic cardiomyopathy, Freeman–Sheldon syndrome, and trismus-pseudocamptodactyly syndrome had divergent effects despite occurring at a conserved arginine residue. These findings underscore that predictions based solely on homology modeling may be insufficient for understanding mutation effects in different myosin isoforms .

What approaches can be used to characterize the mechanochemical coupling in Myosin-A (PY01232)?

Characterizing mechanochemical coupling in Myosin-A requires integrated approaches that connect biochemical states to mechanical outputs. A comprehensive methodology should include:

• Coupled ATPase and mechanical measurements to establish relationships between:

  • ATP hydrolysis rates

  • Force generation

  • Displacement parameters

  • Dwell times

• Analysis of how nucleotide states correlate with mechanical transitions through:

  • Transient kinetic studies with stopped-flow and quenched-flow techniques

  • Direct observation of structural changes using FRET-based sensors

  • Correlation of biochemical transitions with mechanical events using optical trapping

• Investigation of how mutations or small molecule effectors alter coupling by measuring:

  • Changes in step size

  • Effects on ATPase cycle rates

  • Alterations in load-dependent actin-detachment kinetics

These measurements should be performed under varying conditions of temperature, ionic strength, and ATP concentration to construct a comprehensive model of the mechanochemical cycle. For accurate interpretation, researchers should consider that predicted velocities based on step size and bound times should match those measured in motility assays, according to the relationship: V = d × k, where V is velocity, d is step size, and k is the actin-detachment rate .

What expression systems are most suitable for producing functional Recombinant Myosin-A (PY01232) for research?

The choice of expression system significantly impacts the yield, purity, and functionality of recombinant myosin proteins. For Myosin-A (PY01232), expression systems should be selected based on the following methodological considerations:

• Mammalian expression systems (e.g., mouse myoblast C2C12 cells) provide proper folding and post-translational modifications essential for motor function. These systems can co-express essential light chains and incorporate endogenous regulatory light chains, resulting in properly assembled myosin constructs. The approach involves transfection with vectors encoding the myosin motor domain with a C-terminal purification tag (typically 8-amino acid tags for surface attachment purposes) .

• Alternative systems like baculovirus-infected insect cells have also proven successful for myosin expression but may require co-expression of molecular chaperones to ensure proper folding.

• Critical quality control steps include:

  • Verification of proper folding through intrinsic tryptophan fluorescence measurements

  • Assessment of ATPase activity compared to native protein

  • Evaluation of actin-binding properties through co-sedimentation assays

  • Confirmation of motile activity in functional assays

Researchers should note that bacterial expression systems typically yield non-functional myosin and should be avoided for studies requiring enzymatically active protein .

What are the critical controls and validation steps for assessing the functional integrity of purified Myosin-A (PY01232)?

Ensuring the functional integrity of purified Myosin-A is essential for reliable experimental outcomes. A comprehensive validation protocol should include:

• Biochemical characterization:

  • SDS-PAGE and Western blotting to confirm molecular weight and immunoreactivity

  • Verification of light chain association (essential and regulatory light chains)

  • Assessment of aggregation state by size-exclusion chromatography or dynamic light scattering

  • Thermal stability analysis to confirm proper protein folding

• Functional validation:

  • Actin-activated ATPase assays with Michaelis-Menten analysis to determine kcat and Km

  • In vitro motility assays at multiple temperatures to calculate Q10 values

  • Single-molecule optical trapping to measure mechanical parameters

  • Comparison of measured and predicted velocities using the relationship V = d × k

• Structure-based validation:

  • Intrinsic tryptophan fluorescence to monitor conformational changes during ATP hydrolysis

  • Circular dichroism spectroscopy to verify secondary structure content

  • Limited proteolysis to assess domain organization and stability

These validation steps should be performed on each new preparation to ensure consistency across experiments and to detect any batch-to-batch variations that might affect experimental outcomes .

How can researchers effectively design experiments to compare the functional properties of Myosin-A (PY01232) with other myosin isoforms?

Designing experiments to compare Myosin-A with other myosin isoforms requires careful consideration of methodology to ensure meaningful comparisons. An effective experimental design framework includes:

• Standardization of constructs:

  • Generate comparable constructs with identical tags and fusion partners

  • Ensure equivalent domain boundaries across all isoforms

  • Use consistent light chain compositions or explicitly account for differences

• Parallel characterization under identical conditions:

  • Perform experiments at the same temperature, ionic strength, and ATP concentration

  • Use the same actin preparation across all experiments

  • Employ identical analysis methods and models for data fitting

• Comprehensive parameter measurement:

  • Determine full ATPase parameters (kcat and Km) to understand both maximum turnover and actin affinity

  • Measure motility at multiple temperatures to calculate temperature dependence (Q10)

  • Characterize single-molecule mechanics including step size and load sensitivity

  • Calculate duty ratios to understand the fraction of time spent in strong binding states

• Correlation of parameters:

  • Compare predicted velocities (from step size and detachment rate) with measured velocities

  • Analyze relationships between biochemical and mechanical parameters across isoforms

  • Consider evolutionary conservation and divergence in structure-function relationships

This systematic approach enables meaningful comparison of functional properties across myosin isoforms and provides insight into how sequence variations translate to functional differences .

How can researchers address inconsistencies between predicted and measured velocities in Myosin-A (PY01232) studies?

Discrepancies between predicted velocities (based on single-molecule parameters) and measured velocities (from in vitro motility assays) represent a common challenge in myosin research. To address these inconsistencies:

• Verify experimental conditions:

  • Ensure temperature consistency across all measurements

  • Check buffer compositions, especially ionic strength which affects actin-myosin interactions

  • Confirm ATP concentrations are consistently at saturating levels

• Apply temperature corrections:

  • Use Q10 values to adjust for temperature differences between single-molecule (typically 23°C) and motility experiments (often 30°C)

  • Apply the correction factor to single-molecule detachment rates using the Q10 relationship: k30°C = k23°C × Q10^((30-23)/10)

• Account for ensemble effects:

  • Consider that multiple motors working together may exhibit different kinetics than single molecules

  • Measure motility at varying motor densities to assess cooperative effects

  • Determine if the duty ratio influences the relationship between single-molecule and ensemble measurements

• Validate measurement methods:

  • Perform calibration controls for both optical trapping and motility assays

  • Use well-characterized myosin isoforms as internal standards to validate experimental systems

  • Consider the effects of surface attachments and fluorescent labels on motor function

By systematically addressing these factors, researchers can reconcile discrepancies and develop more accurate models of how molecular properties translate to ensemble behavior .

What strategies can be employed to investigate potential allosteric mechanisms in Myosin-A (PY01232)?

Investigating allosteric mechanisms in Myosin-A requires a multifaceted approach that integrates experimental and computational methodologies. Effective strategies include:

These approaches have revealed that myosins are highly allosteric proteins, with mutations at communication hotspots (e.g., between the nucleotide-binding site and lever arm) having long-range effects on motor function. Importantly, the same mutation in different isoforms can produce dramatically different functional outcomes, highlighting the context-dependent nature of allosteric mechanisms .

How might advanced biophysical techniques enhance our understanding of Myosin-A (PY01232) function?

Emerging biophysical techniques offer opportunities to advance our understanding of Myosin-A function beyond current methodological limitations. Promising approaches include:

• High-resolution structural techniques:

  • Cryo-electron microscopy to capture myosin in different states of the ATPase cycle

  • Time-resolved X-ray crystallography to visualize structural transitions

  • Single-particle tracking to monitor conformational dynamics in real-time

• Advanced force spectroscopy:

  • Improved optical trapping with higher spatial and temporal resolution

  • Magnetic tweezers for long-duration measurements under constant force

  • AFM-based force measurements to probe mechanical stability of protein domains

• Integrative methodologies:

  • Combined fluorescence and force measurements to correlate chemical and mechanical events

  • High-throughput single-molecule assays for comprehensive characterization of mutant libraries

  • Microfluidic platforms for rapid assessment of motor properties under varying conditions

These advanced techniques would enable researchers to address fundamental questions about the relationship between structural dynamics and mechanical function in Myosin-A, potentially revealing novel therapeutic targets for myosin-related diseases .

What implications does research on Myosin-A (PY01232) have for understanding disease mechanisms and developing therapeutic strategies?

Research on Myosin-A has significant implications for understanding disease mechanisms and developing targeted therapeutics. Key considerations include:

• Disease mechanism insights:

  • Identification of how structural perturbations lead to functional defects in myosin-related diseases

  • Understanding of tissue-specific effects of mutations in widely expressed myosin isoforms

  • Correlation between molecular defects and disease severity to guide prognostic assessments

• Therapeutic development opportunities:

  • Design of small molecule effectors that specifically target defective parameters of myosin function

  • Development of precision therapies tailored to specific molecular defects rather than downstream symptoms

  • Screening of compounds that could reverse specific mechanical or enzymatic defects

• Translational research approaches:

  • Development of in vitro diagnostic assays to characterize patient-specific myosin defects

  • Creation of humanized animal models expressing disease-associated myosin variants

  • Implementation of high-throughput screening platforms to identify modulators of myosin function

Research has shown that prolonged actin-bound times may be a common defect in several myosin-related diseases. Compounds like 2-deoxy-ATP, which has been shown to increase actin-detachment rates of myosin molecules by approximately 70%, represent potential therapeutic strategies. Other myosin effectors have been identified that can alter step size, change ATP cycle rates, inhibit actin binding, or promote specific conformational states, providing a diverse toolkit for targeted therapeutic intervention .