Recombinant Chicken Muscle, skeletal receptor tyrosine protein kinase (MUSK), partial

What is MUSK and what is its primary function in skeletal muscle?

MUSK (muscle-specific kinase) is a receptor tyrosine kinase (RTK) that is specifically expressed in skeletal muscle tissue. It plays a central role in the formation and maintenance of the neuromuscular junction (NMJ), which is the synapse between motor neurons and skeletal muscle fibers. MUSK is expressed at low levels in proliferating myoblasts and is upregulated during differentiation and fusion of muscle cells. After development, MUSK expression becomes dramatically downregulated in mature muscle, where it remains prominent primarily at the neuromuscular junction, making it the only known RTK that specifically localizes to this structure .

The primary function of MUSK involves mediating the agrin signaling pathway. When neural agrin (released by motor neurons) binds to LRP4 (low-density lipoprotein receptor-related protein 4), it induces the formation of an agrin-LRP4-MUSK complex. This interaction triggers MUSK phosphorylation and activation, initiating downstream signaling cascades that regulate acetylcholine receptor (AChR) clustering at the postsynaptic membrane of the NMJ . This process is fundamental for proper neuromuscular communication and muscle function.

How is recombinant MUSK protein typically produced for research applications?

Recombinant MUSK protein can be produced using various expression systems, with the choice dependent on research needs and downstream applications. Based on available data, common expression systems include:

• Yeast expression system: This is frequently used for producing human MUSK protein fragments, as seen in the production of recombinant human MUSK protein (24-495 aa) with an N-terminal His-tag . Yeast systems offer advantages in post-translational modifications compared to bacterial systems.

• Mammalian cell expression: For applications requiring mammalian-specific post-translational modifications and proper protein folding, mammalian cell lines (typically HEK293 or CHO cells) may be preferred, though this information is not explicitly stated in the search results.

For chicken MUSK specifically, researchers often need to design expression vectors containing the chicken MUSK gene sequence. The expression system selection depends on the specific research requirements, including the need for proper glycosylation, disulfide bond formation, and other post-translational modifications crucial for MUSK function.

The recombinant protein production typically involves several key steps:

• Cloning the MUSK gene or its specific domains into an expression vector

• Transformation/transfection into the chosen expression system

• Protein expression induction

• Cell lysis and protein extraction

• Purification using affinity chromatography (e.g., using His-tag)

• Quality control assessment including SDS-PAGE for purity evaluation (>90% purity is typically desired)

What are the structural characteristics of MUSK that researchers should be aware of?

MUSK contains several distinct structural domains that are important for its function:

• Extracellular domain: The ectodomain of MUSK consists of:

  • Three consecutive immunoglobulin-like (Ig-like) domains, with the first two (Ig1-2) being primarily responsible for agrin-induced AChR clustering

  • A cysteine-rich domain (CRD) homologous to the CRD of Wnt-interacting Frizzled proteins

• Transmembrane domain: A single transmembrane helix that anchors the protein to the cell membrane

• Intracellular/cytoplasmic domain: Contains:

  • A juxtamembrane region with an NPXY motif (including Tyr553)

  • A tyrosine kinase domain with an activation loop (A-loop) containing critical tyrosine residues (Tyr750, Tyr754, and Tyr755)

Notable structural features revealed by crystallography include:

• The first two Ig-like domains (Ig1-2) adopt a semi-rigid rod-like structure with no obvious linker region between them

• Ig1 contains an additional disulfide bond critical for correct protein folding

• Ig1-2 can dimerize, with the dimer interface formed by interaction between domains

For researchers working with recombinant partial MUSK proteins, it's crucial to consider which domains are included in the construct, as this directly impacts functional studies. The Ig1-2 domains are particularly important for agrin-induced AChR clustering, while the intracellular tyrosine kinase domain is essential for downstream signaling.

How does the activation mechanism of MUSK differ from other receptor tyrosine kinases?

MUSK activation represents a unique variant of RTK activation mechanisms, distinguishing it from most other receptor tyrosine kinases. The key differences include:

• Indirect ligand binding: Unlike conventional RTKs where ligands directly bind to and activate the receptor, MUSK does not directly interact with its primary activating ligand, agrin. Instead, activation requires the obligate co-receptor LRP4, which physically links agrin to MUSK . This creates a tripartite signaling complex (agrin-LRP4-MUSK) rather than the more common ligand-receptor binary complex.

• Sequential autophosphorylation: Upon agrin-induced activation, MUSK undergoes a specific sequence of autophosphorylation events. First, Tyr754 and Tyr553 become phosphorylated, followed by the remaining tyrosines in the activation loop (Tyr750 and Tyr755). This sequential phosphorylation dramatically increases the kinase catalytic efficiency .

• Dual inhibitory mechanisms: MUSK kinase activity is controlled by two inhibitory mechanisms:

  • Inhibition by the activation loop (A-loop)

  • Inhibition by the juxtamembrane region
    This combined inhibition is crucial for preventing ligand-independent activation and maintaining MUSK in a basal inactive state until appropriate signaling occurs .

• Positive feedback amplification: Activated MUSK creates a positive feedback loop that induces further MUSK clustering, enhancing signaling. This explains why overexpression of ectopic MUSK can induce agrin-independent autophosphorylation and non-synaptic AChR clustering .

These unique activation characteristics make MUSK an interesting model for studying variant RTK signaling mechanisms and highlight why standard RTK experimental approaches may need modification when studying MUSK.

What experimental considerations are important when comparing MUSK function across different species?

When comparing MUSK function across different species (e.g., human, mouse, and chicken), researchers should consider several important factors:

• Sequence and structural conservation: While MUSK is evolutionarily conserved, there are species-specific variations in protein sequence and domain structure. Researchers should analyze sequence alignments to identify conserved and variable regions before making cross-species comparisons. For instance, the extracellular domain topology may differ subtly between species, affecting ligand interaction.

• Expression patterns during development: The temporal and spatial expression patterns of MUSK during embryonic development may vary between species. In chicken, muscle development studies show that embryonic days (E) 12, 16, 19, and 21 represent important timepoints for examining muscle-related gene expression patterns . These developmental timelines should be adjusted when comparing with mammalian models.

• Co-receptor and downstream effector variations: The interaction between MUSK and its co-receptors or downstream signaling molecules may exhibit species-specific characteristics. For example, the binding affinity between agrin, LRP4, and MUSK may differ between species, affecting the sensitivity of the signaling pathway.

• Experimental systems: When working with recombinant proteins from different species, expression systems should be carefully selected to ensure proper post-translational modifications relevant to each species. For chicken MUSK, researchers developing recombinant expression systems have explored various promoter combinations to optimize expression, as seen in studies with chicken oviduct-specific genes .

• Functional assays: Assays measuring MUSK activity (e.g., phosphorylation, AChR clustering) may require species-specific antibodies, reagents, or cell lines for accurate assessment.

A methodological approach for cross-species comparison might include parallel experiments using recombinant MUSK proteins from different species in the same experimental system, or using species-specific cell lines to test conserved functions in their native context.

What are the critical phosphorylation sites in MUSK and how do they regulate its activity?

MUSK activity is tightly regulated through phosphorylation events at specific tyrosine residues. The critical phosphorylation sites and their regulatory functions include:

• Juxtamembrane region:

  • Tyr553: Located within the NPXY motif, this residue is among the first to be autophosphorylated upon agrin-induced activation of MUSK. Phosphorylation at Tyr553 creates a binding site for proteins containing phosphotyrosine binding (PTB) domains, such as the adaptor protein Dok7 . This interaction is crucial for downstream signaling and AChR clustering.

• Activation loop (A-loop):

  • Tyr750: One of the later phosphorylation sites in the activation sequence, phosphorylation of Tyr750 contributes to full activation of MUSK kinase activity.

  • Tyr754: Among the initial autophosphorylation sites following agrin stimulation, this residue plays a key role in initiating MUSK activation.

  • Tyr755: Another A-loop tyrosine that becomes phosphorylated following Tyr754 and Tyr553, contributing to the dramatic increase in kinase catalytic efficiency .

The phosphorylation of these residues follows a sequential pattern:

• Initial phosphorylation of Tyr754 and Tyr553

• Subsequent phosphorylation of Tyr750 and Tyr755

This sequential process is critical because:

• It creates a switch-like activation mechanism rather than a graded response

• It enables integration of multiple upstream signals

• It provides multiple regulatory points for fine-tuning MUSK activity

• It establishes binding sites for different downstream effectors, allowing signal diversification

For researchers studying MUSK regulation, site-directed mutagenesis of these tyrosine residues (e.g., Y→F substitutions) can provide valuable insights into the specific contribution of each phosphorylation event to MUSK function. Phospho-specific antibodies against these sites are also important tools for monitoring MUSK activation status in experimental settings.

What are the optimal storage and handling conditions for recombinant MUSK protein?

Proper storage and handling of recombinant MUSK protein are critical for maintaining its structural integrity and biological activity. Based on established protocols for similar proteins, the following guidelines are recommended:

• Storage temperature:

  • Long-term storage: -80°C or -20°C is recommended for both liquid and lyophilized forms

  • Working aliquots: Store at 4°C for up to one week

• Storage buffer composition:

  • Typically stored in Tris-based buffer with 50% glycerol for stability

  • The glycerol acts as a cryoprotectant to prevent freeze-thaw damage

• Aliquoting strategy:

  • Divide into small single-use aliquots before freezing

  • Repeated freeze-thaw cycles should be avoided as they can significantly reduce protein activity

• Shelf life considerations:

  • Liquid form: Approximately 6 months at -20°C/-80°C

  • Lyophilized form: Up to 12 months at -20°C/-80°C

• Reconstitution of lyophilized protein:

  • Use appropriate buffer (typically the same as the original formulation buffer)

  • Allow the protein to fully dissolve by gentle mixing rather than vortexing

  • Filter sterilize if intended for cell culture applications

• Handling recommendations:

  • Work with protein on ice when possible

  • Avoid extended exposure to room temperature

  • Use low-protein binding tubes for dilutions

• Quality control before use:

  • Verify protein concentration using appropriate methods (Bradford, BCA assay)

  • Check activity using phosphorylation assays if critical for experiments

It's worth noting that specific recombinant MUSK preparations may have unique handling requirements based on the expression system used, protein domains included, and specific tags attached. Always refer to the certificate of analysis (COA) provided with the specific protein lot for detailed information .

What approaches can be used to assess the functional activity of recombinant MUSK protein?

Assessing the functional activity of recombinant MUSK protein is essential to confirm its biological relevance for downstream applications. Several complementary approaches can be employed:

• Autophosphorylation assays:

  • In vitro kinase assays: Measure the ability of recombinant MUSK to phosphorylate itself or exogenous substrates in the presence of ATP

  • Phospho-specific antibodies: Use antibodies that specifically recognize phosphorylated tyrosine residues (pTyr750, pTyr754, pTyr755, pTyr553) to detect active MUSK

  • Mass spectrometry: Identify and quantify phosphorylation sites with high precision

• Protein-protein interaction assays:

  • Co-immunoprecipitation: Assess MUSK's ability to interact with known binding partners like LRP4, Dok7, or other downstream signaling molecules

  • Surface plasmon resonance (SPR) or Bio-Layer Interferometry (BLI): Measure binding kinetics and affinities between MUSK and its interaction partners

  • Yeast two-hybrid or mammalian two-hybrid assays: Screen for novel interaction partners

• Cell-based functional assays:

  • AChR clustering assays: Transfect muscle cells with recombinant MUSK and measure acetylcholine receptor clustering using fluorescently labeled α-bungarotoxin

  • Agrin-responsiveness assays: Determine if the recombinant MUSK can respond to agrin stimulation when introduced into MUSK-deficient cells

  • Phosphorylation cascade analysis: Examine the activation of downstream signaling molecules like Abl1, Src family kinases, or PAK1

• Structural confirmation:

  • Circular dichroism (CD) spectroscopy: Verify proper protein folding

  • Thermal shift assays: Assess protein stability

  • Size exclusion chromatography: Confirm protein oligomeric state

• Receptor dimerization assays:

  • As MuSK activation involves dimerization, assays that can detect dimer formation (crosslinking followed by western blot, FRET-based approaches) are valuable for confirming functional capacity

The combination of these approaches provides a comprehensive assessment of recombinant MUSK functionality. For chicken MUSK specifically, it's important to use appropriate cell lines (ideally chicken-derived muscle cells) and species-specific interaction partners when possible to ensure physiologically relevant results.

How can researchers optimize expression and purification of recombinant chicken MUSK protein?

Optimizing the expression and purification of recombinant chicken MUSK protein requires careful consideration of multiple factors throughout the production process:

• Expression vector design:

  • Promoter selection: For chicken proteins, consider using recombinant promoters that combine regulatory elements from chicken genes. Studies have developed compact recombinant chicken promoters by linking regulatory regions of ovalbumin and other oviduct-specific genes .

  • Codon optimization: Adjust codons to match the preferred codon usage of the expression host to enhance translation efficiency.

  • Fusion tags: Include affinity tags (His-tag, GST, or Fc) for purification; His-tags are commonly used for MUSK purification .

  • Protease cleavage sites: Include sites for tag removal if the tag might interfere with functional studies.

• Expression system selection:

  • Yeast expression: Offers a balance between prokaryotic simplicity and eukaryotic post-translational modifications .

  • Insect cell system: Baculovirus-infected insect cells often provide good yields for complex proteins like receptor tyrosine kinases.

  • Mammalian expression: Consider for applications requiring mammalian-specific post-translational modifications.

• Expression conditions optimization:

  • Temperature: Lower temperatures (16-25°C) often improve protein folding for complex proteins.

  • Induction parameters: Optimize inducer concentration and induction time.

  • Media supplementation: Consider additives that enhance disulfide bond formation if expressing domains with disulfide bridges, such as MUSK's Ig domains .

• Extraction and solubilization:

  • Lysis buffers: Optimize buffer composition (pH, salt concentration, detergents for membrane-associated constructs).

  • Protease inhibitors: Include a comprehensive protease inhibitor cocktail to prevent degradation.

• Purification strategy:

  • Multi-step purification: Typically starts with affinity chromatography (e.g., Ni-NTA for His-tagged proteins) .

  • Secondary purification: Size exclusion chromatography or ion exchange chromatography to increase purity.

  • Quality control: SDS-PAGE analysis to confirm purity (aim for >90%) .

• Refolding considerations (if needed):

  • For inclusion body production, develop a careful refolding protocol that allows proper disulfide bond formation, particularly important for the Ig domains of MUSK .

• Stability enhancement:

  • Buffer optimization: Identify buffer conditions that maximize stability.

  • Addition of stabilizers: Consider glycerol (50%) or other stabilizers in final formulation .

When working specifically with chicken MUSK, researchers should also consider species-specific sequence characteristics that might affect expression. Analysis of chicken embryonic muscle transcriptome data may provide insights into expression patterns and potential regulatory elements that could be harnessed for optimal expression .

How can recombinant MUSK be used to study neuromuscular junction disorders?

Recombinant MUSK protein serves as a valuable tool for investigating neuromuscular junction (NMJ) disorders through multiple experimental approaches:

• Autoimmune neuromuscular disorders:

  • Recombinant MUSK can be used to develop ELISA or immunoprecipitation assays for detecting anti-MUSK antibodies in myasthenia gravis patients

  • The protein can serve as a target for screening therapeutic antibodies or small molecules that might block pathogenic autoantibody binding

  • Structure-function studies using various MUSK domains can identify specific epitopes targeted in autoimmune conditions

• Congenital myasthenic syndromes (CMS):

  • Recombinant wild-type MUSK can be compared with mutant versions harboring CMS-associated mutations to assess:

    • Autophosphorylation capacity

    • Interaction with LRP4 and agrin

    • Ability to induce AChR clustering

  • Such studies help establish pathogenic mechanisms and potential compensatory strategies

• Denervation models and NMJ remodeling:

  • MUSK is dramatically upregulated after denervation, electrical activity blockade, or physical immobilization

  • Recombinant MUSK can be used to study the molecular mechanisms underlying this upregulation and its functional consequences

  • Understanding these processes has implications for conditions involving denervation, such as amyotrophic lateral sclerosis (ALS) or peripheral nerve injuries

• High-throughput drug screening:

  • Recombinant MUSK protein can be used in biochemical assays to screen for compounds that:

    • Enhance MUSK kinase activity (potential therapeutics for MUSK-deficient CMS)

    • Stabilize MUSK-LRP4 interaction

    • Modulate MUSK-dependent signaling pathways

• Gene therapy development:

  • Recombinant MUSK can be used to validate gene therapy approaches before in vivo testing

  • Structure-function studies with recombinant protein can inform the design of gene therapy constructs targeting specific MUSK functions

• Cross-species comparisons:

  • Comparing recombinant MUSK from different species (human, mouse, chicken) can reveal evolutionary conservation of pathogenic mechanisms

  • This approach is particularly valuable for validating animal models of human NMJ disorders

Notably, the chicken model offers unique advantages for studying certain aspects of NMJ development due to accessibility of embryos and the well-characterized embryonic muscle development timeline . Recombinant chicken MUSK therefore enables comparative studies that might reveal conserved or divergent mechanisms relevant to human disease.

What role does MUSK play in muscle development, and how can recombinant MUSK advance developmental biology research?

MUSK plays critical roles in multiple aspects of muscle development, and recombinant MUSK protein offers valuable tools for investigating these developmental processes:

• Temporal expression pattern during myogenesis:

  • MUSK is expressed at low levels in proliferating myoblasts

  • Its expression increases upon differentiation and fusion of myoblasts

  • In embryonic development, MUSK is specifically expressed in early myotomes and developing muscle

  • After development, MUSK is dramatically downregulated in mature muscle, remaining prominent only at the neuromuscular junction

• Functional roles in muscle development:

  • Prepatterning of AChR clusters: MUSK is involved in the initial clustering of acetylcholine receptors before nerve contact

  • NMJ formation: Central role in the agrin-LRP4-MUSK signaling pathway that induces postsynaptic differentiation

  • Cytoskeletal reorganization: MUSK activation regulates reorganization of the actin cytoskeleton through effectors like PAK1 and Rho family GTPases

  • Synapse-specific gene expression: MUSK signaling influences the expression of genes in subsynaptic nuclei

Recombinant MUSK can advance developmental biology research through:

• In vitro developmental models:

  • Recombinant MUSK can be applied to muscle cell cultures at different developmental stages to assess stage-specific responses

  • When combined with time-lapse imaging, this approach can reveal dynamic processes in AChR clustering and cytoskeletal remodeling

• Transcriptional regulation studies:

  • Chicken embryonic muscle shows dynamic transcriptome changes during development (E12-E21)

  • Recombinant MUSK can be used to identify MUSK-dependent gene expression changes by applying it to cultured muscle cells and performing RNA-seq analysis

  • This approach helps map the MUSK-dependent transcriptional networks during myogenesis

• Developmental pathway interactions:

  • MUSK's CRD domain is homologous to the Wnt-interacting domain of Frizzled proteins

  • Recombinant MUSK can be used to investigate potential crosstalk between MUSK and Wnt signaling during muscle development

  • Such studies illuminate how developmental pathways are integrated during myogenesis

• Species-specific developmental differences:

  • Comparing the activity of recombinant MUSK from different species in standardized assays can reveal evolutionary adaptations in developmental mechanisms

  • The chicken model offers advantages for certain developmental studies, and recombinant chicken MUSK enables direct comparison with mammalian systems

• Functional rescue experiments:

  • In MUSK knockout or knockdown models, applying recombinant MUSK at different developmental timepoints can reveal critical windows for MUSK function

  • These experiments help establish the causality between MUSK activity and specific developmental outcomes

By leveraging recombinant MUSK protein in these research approaches, developmental biologists can gain deeper insights into the molecular mechanisms governing muscle development and neuromuscular junction formation.

What are the challenges in designing MUSK-targeted therapeutic strategies, and how can recombinant MUSK help address them?

Designing MUSK-targeted therapeutic strategies faces several significant challenges, with recombinant MUSK protein serving as a valuable tool to address these obstacles:

• Challenge: Receptor specificity and off-target effects

  • Problem: MUSK belongs to the RTK family, which includes numerous members with structural similarities, raising the risk of off-target effects for MUSK-directed therapeutics.

  • Solution using recombinant MUSK:

    • Recombinant MUSK domains (particularly the extracellular and kinase domains) can be used in competitive binding assays to screen therapeutic candidates for specificity

    • Comparative binding studies with other RTK family members help identify MUSK-specific binders

    • Structure-guided design of therapeutics targeting unique MUSK features becomes possible

• Challenge: Maintaining physiological signaling balance

  • Problem: MUSK signaling requires precise regulation; excessive activation or inhibition could disrupt neuromuscular function.

  • Solution using recombinant MUSK:

    • Dose-response studies with recombinant MUSK can define the therapeutic window for potential modulators

    • Phosphorylation assays using recombinant MUSK can assess whether candidates achieve partial vs. complete activation/inhibition

    • Activity assays comparing wild-type and mutant forms help understand how disease mutations affect this balance

• Challenge: Blood-nerve barrier penetration

  • Problem: Therapeutic agents targeting MUSK at the NMJ must cross the blood-nerve barrier.

  • Solution using recombinant MUSK:

    • Recombinant MUSK extracellular domain can be used to screen for small molecules or peptides with high affinity but favorable pharmacokinetic properties

    • In vitro models incorporating barrier components can be developed to test passage of MUSK-targeted therapeutics

• Challenge: Validation of animal models for human diseases

  • Problem: Species differences in MUSK structure and function may limit the translational value of animal models.

  • Solution using recombinant MUSK:

    • Comparative studies with recombinant human, mouse, and chicken MUSK can identify conserved and divergent features

    • Binding and activation assays can determine whether therapeutic candidates interact similarly with MUSK across species

    • Understanding these differences helps in selecting appropriate animal models and interpreting preclinical results

• Challenge: Developmentally appropriate interventions

  • Problem: MUSK plays different roles during development versus maintenance of mature NMJs.

  • Solution using recombinant MUSK:

    • Studies comparing recombinant MUSK effects on developing versus mature muscle cultures can identify stage-specific responses

    • This information helps design therapeutics appropriate for developmental versus degenerative conditions

• Challenge: Complex activation mechanism

  • Problem: MUSK activation requires LRP4 co-receptor and agrin, creating a complex regulatory system .

  • Solution using recombinant MUSK:

    • Reconstitution systems with recombinant MUSK, LRP4, and agrin enable screening for agents that modulate specific components of this tripartite complex

    • Structure-function studies help identify potential binding sites for therapeutics that could stabilize or enhance complex formation

These approaches using recombinant MUSK not only advance basic understanding of MUSK biology but also provide practical platforms for therapeutic discovery and validation.

What are emerging areas of MUSK research beyond the neuromuscular junction?

While MUSK has been primarily studied in the context of neuromuscular junction formation and maintenance, several emerging research areas explore its functions beyond this traditional role:

• Central nervous system functions:

  • Recent evidence suggests MUSK may play roles within the central nervous system by mediating cholinergic responses, synaptic plasticity, and memory formation

  • Recombinant MUSK can be used to investigate these potential roles through binding studies with CNS-expressed proteins and functional assays in neuronal cultures

  • This represents a significant expansion of our understanding of MUSK biology beyond the peripheral nervous system

• Potential roles in non-muscle tissues:

  • While MUSK is traditionally considered muscle-specific, sensitive detection methods may reveal low-level expression in other tissues

  • Investigating potential non-canonical functions using tissue-specific transcriptomic and proteomic approaches may reveal unexpected roles

  • Recombinant MUSK can serve as a positive control and standard in such studies

• Interactions with Wnt signaling pathway:

  • MUSK contains a cysteine-rich domain (CRD) homologous to the Wnt-interacting Frizzled proteins

  • This structural feature suggests potential crosstalk between MUSK and Wnt signaling pathways

  • Recombinant MUSK can be used in binding assays with Wnt proteins and related signaling components to explore this hypothesis

• Genetic associations with unexplored conditions:

  • In humans, MUSK maps to chromosome 9q31.3-32, which overlaps with the region reported to contain the Fukuyama muscular dystrophy mutation

  • This genomic localization suggests potential involvement in conditions beyond classic MUSK-associated disorders

  • Recombinant MUSK carrying variants identified in these conditions can be functionally characterized

• Evolutionary biology perspectives:

  • Comparative studies of MUSK across species can provide insights into the evolution of neuromuscular systems

  • Functional conservation versus innovation can be assessed using recombinant MUSK proteins from diverse species

  • Such studies might reveal species-specific adaptations in neuromuscular development and function

These emerging research directions highlight the potential for discoveries beyond MUSK's established role at the neuromuscular junction and underscore the value of recombinant MUSK as a versatile tool for exploring these new frontiers.

How might advances in structural biology techniques enhance our understanding of MUSK function?

Recent and emerging advances in structural biology techniques offer unprecedented opportunities to deepen our understanding of MUSK structure-function relationships:

• Cryo-electron microscopy (Cryo-EM):

  • Application to MUSK research: Cryo-EM can potentially resolve the complete structure of the agrin-LRP4-MUSK signaling complex, which has been challenging to study using X-ray crystallography alone

  • Advantages: Allows visualization of large protein complexes and flexible regions without crystallization, potentially revealing dynamic interactions during complex formation

  • Research opportunities: Using recombinant MUSK in complex with LRP4 and agrin for single-particle cryo-EM analysis to understand the full assembly process

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Application to MUSK research: Can map conformational changes in MUSK upon activation, identifying regions that undergo structural rearrangements

  • Advantages: Provides dynamic information about protein structure in solution, complementing static crystal structures

  • Research opportunities: Comparing HDX patterns between inactive and activated recombinant MUSK to identify allosteric mechanisms of activation

• AlphaFold and other AI-based structure prediction:

  • Application to MUSK research: Can predict structures of MUSK domains or variants for which experimental structures are unavailable

  • Advantages: Rapidly generates structural models to guide hypothesis formation and experimental design

  • Research opportunities: Predicting structures of species-specific MUSK variants (e.g., chicken MUSK) to guide recombinant protein design and functional studies

• Integrative structural biology approaches:

  • Application to MUSK research: Combining multiple techniques (X-ray crystallography, NMR, SAXS, crosslinking-MS) to build comprehensive models of MUSK complexes

  • Advantages: Overcomes limitations of individual techniques to provide more complete structural information

  • Research opportunities: Using purified recombinant MUSK domains in integrative approaches to understand domain interactions and complex assembly

• Single-molecule techniques:

  • Application to MUSK research: Single-molecule FRET or force spectroscopy can reveal conformational dynamics of MUSK during activation

  • Advantages: Provides information about transient states and heterogeneity not accessible through ensemble measurements

  • Research opportunities: Studying the dimerization dynamics of recombinant MUSK ectodomain and how it relates to activation

• In-cell structural biology:

  • Application to MUSK research: Techniques like in-cell NMR or proximity labeling can study MUSK structure in its native environment

  • Advantages: Preserves cellular context, including membrane environment and full complement of interaction partners

  • Research opportunities: Comparing recombinant MUSK behavior in reconstituted systems versus cellular environments

Current structural information on MUSK is limited to the crystal structure of the first two Ig-like domains (Ig1-2) . Expanding structural knowledge to include the full ectodomain, transmembrane region, and intracellular domain would significantly advance our understanding of MUSK function. Recombinant protein production strategies will need to be optimized for each structural biology approach, potentially requiring different constructs and expression systems depending on the technique employed.