Recombinant Ningaui ridei Sperm protamine P1 (PRM1)

What is Ningaui ridei Sperm Protamine P1 and its biological function?

Ningaui ridei Sperm Protamine P1 (PRM1) is a small, arginine-rich basic protein that plays a primary role in packaging sperm DNA. Like protamines across mammalian species, it replaces histones during spermiogenesis to achieve a highly condensed, compact chromatin structure. The protein is characterized by numerous arginine residues that facilitate binding to DNA phosphate groups, enabling tight packaging of the sperm genome .

The sequence of recombinant Ningaui ridei PRM1 is: ARYRRHSRSRSRSRYRRRRRRRRSRHHNRRTYRRSRRHSRRRRGRRRGY SRRRYSRRGRRRY . This sequence reveals the characteristic arginine-richness (R) and serine content (S) typical of protamines, with arginine constituting approximately 48% of the amino acid composition.

The biological function of PRM1 is to facilitate the dramatic nuclear condensation required for sperm maturation, which is essential for:

• Protecting paternal DNA during transport through the female reproductive tract

• Enabling proper fertilization and subsequent protamine-to-histone exchange

• Contributing to paternal genome reprogramming following fertilization

How does PRM1 structure differ between species, and why is Ningaui ridei PRM1 of scientific interest?

PRM1 is highly conserved across mammalian species in terms of its basic function, but exhibits species-specific variations in sequence and post-translational modifications. In mammals, species vary in whether they utilize only PRM1 (as in some rodents) or both PRM1 and PRM2 (as in humans and mice) . The P1:P2 ratio is highly variable but maintaining a species-specific ratio is critical for normal fertility .

Ningaui ridei (Wongai ningaui) PRM1 is of scientific interest because:

• It represents a marsupial protamine variant, offering evolutionary insights into protamine diversification

• Comparative studies with eutherian mammal protamines can illuminate functional conservation and divergence

• Understanding species-specific differences in protamine structure provides insights into reproductive adaptations

The distinctive features of Ningaui ridei PRM1 include its specific pattern of arginine clusters and serine residues potentially involved in phosphorylation-mediated regulation, which influence DNA binding dynamics and chromatin compaction capabilities .

What post-translational modifications (PTMs) regulate PRM1 function, and how might they apply to Ningaui ridei PRM1?

Protamine P1 undergoes several critical post-translational modifications that regulate its function throughout spermiogenesis and early embryogenesis:

• Phosphorylation: Serine residues in PRM1 are phosphorylated during early embryogenesis, which is required to weaken protamine-DNA interactions and permit male pronuclear remodeling and protamine-to-histone exchange . SRPK1 (serine/arginine protein-specific kinase) catalyzes site-specific phosphorylation of protamine, triggering protamine-to-histone exchange in fertilized oocytes .

• Acetylation: Loss of acetylation at specific lysine residues (e.g., K49 in mouse P1) drastically alters sperm chromatin composition and results in subfertility, premature dismissal of P1 from paternal chromatin in the zygote, and altered DNA compaction and decompaction rates .

For Ningaui ridei PRM1 specifically, the sequence contains multiple serine residues (e.g., positions 7, 9, 11, 13), which likely serve as phosphorylation sites regulated by kinases similar to those in other mammals . These phosphorylation events would modulate:

• Protamine-DNA binding strength during spermiogenesis

• Chromatin condensation efficiency

• Protamine removal during fertilization

The specific kinases involved in Ningaui ridei PRM1 phosphorylation have not been directly characterized, but based on conservation of this regulatory mechanism, SRPK1 is a strong candidate for mediating these modifications .

How should researchers properly store and reconstitute recombinant Ningaui ridei PRM1 for optimal activity?

For optimal activity of recombinant Ningaui ridei PRM1, researchers should follow these storage and reconstitution protocols:

Storage recommendations:

• Store lyophilized protein at -20°C/-80°C for up to 12 months

• Store liquid form at -20°C/-80°C for up to 6 months

• Avoid repeated freeze-thaw cycles

• Working aliquots can be stored at 4°C for up to one week

Reconstitution protocol:

• Briefly centrifuge the vial prior to opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (recommended 50%) for long-term storage

• Prepare small working aliquots to minimize freeze-thaw cycles

The stability of recombinant protamines is influenced by multiple factors including buffer composition, storage temperature, and the intrinsic properties of the protein itself. Researchers should conduct activity assays after reconstitution to verify protein functionality before experimental use.

What are the recommended methods for studying protamine-DNA interactions using recombinant PRM1?

Researchers can employ several methodologies to study protamine-DNA interactions using recombinant PRM1:

In vitro binding assays:

• Electrophoretic Mobility Shift Assay (EMSA): Mix varying concentrations of recombinant PRM1 with labeled DNA fragments to observe mobility shifts indicating complex formation

• DNA condensation assay: Monitor changes in light scattering or fluorescence as PRM1 condenses DNA

• Atomic Force Microscopy (AFM): Visualize PRM1-induced DNA condensation and toroidal structure formation

Chromatin packaging analysis:

• DNA protection assay: Assess protection of DNA from nuclease digestion when bound to PRM1

• Chromatin compaction assay: Measure changes in DNA accessibility using intercalating dyes or antibody binding

Analytical techniques for studying PRM1-DNA dynamics:

• Circular dichroism spectroscopy to monitor changes in DNA structure upon PRM1 binding

• Isothermal titration calorimetry for thermodynamic parameters of binding

• Fluorescence anisotropy to measure binding kinetics

Recent research has shown that protamine undergoes a DNA-dependent phase transition to gel-like condensates. Researchers can study this phenomenon using recombinant PRM1 by employing fluorescence microscopy with labeled protamine to visualize condensate formation in the presence of DNA .

How can researchers effectively analyze PRM1 phosphorylation states and their functional consequences?

To analyze PRM1 phosphorylation states and their functional consequences, researchers should employ a combination of biochemical, cellular, and functional approaches:

Analytical methods for phosphorylation detection:

• Mass spectrometry: Identify exact phosphorylation sites and their occupancy

• Phospho-specific antibodies: Detect specific phosphorylated residues in PRM1

• Acid-urea polyacrylamide gel electrophoresis: Separate and quantify different phosphorylated forms of PRM1

• Radioisotope incorporation assays: Measure phosphate incorporation using γ-32P-ATP and recombinant kinases like SRPK1

Functional analysis of phosphorylation:

• Mutagenesis studies: Generate phosphomimetic (S→D/E) or phospho-deficient (S→A) PRM1 mutants

• DNA binding assays: Compare DNA binding properties of wild-type and mutant PRM1

• Chromatin condensation assays: Assess how phosphorylation affects chromatin packaging efficiency

In vivo approaches:

• Transgenic models: Generate animals expressing mutant PRM1 at critical phosphorylation sites

• Microinjection studies: Inject wild-type or mutant PRM1 into oocytes or zygotes to study effects on chromatin remodeling

• Chromatin accessibility assays: Perform ATAC-seq to determine how phosphorylation impacts chromatin organization

A specific experimental approach based on recent research would include:

• Express and purify wild-type and phosphorylation-site mutant Ningaui ridei PRM1

• Perform in vitro kinase assays with SRPK1 to verify phosphorylation sites

• Measure DNA binding affinity of phosphorylated versus non-phosphorylated PRM1

• Assess chromatin condensation capability using fluorescence microscopy

• Test interaction with nucleoplasmin (NPM2) and HIRA to understand the mechanism of protamine-to-histone exchange

How does Ningaui ridei PRM1 compare to other mammalian protamines in genome condensation efficiency?

This advanced research question requires comparative analysis of protamine function across species. Researchers should design experiments that directly compare the DNA condensation properties of Ningaui ridei PRM1 with those from other mammals:

Experimental design:

• Express and purify recombinant protamines from multiple species (Ningaui ridei, mouse, human, etc.) using identical expression systems

• Perform DNA condensation assays under identical conditions:

  • Measure DNA compaction using intercalating dyes (decreased fluorescence indicates condensation)

  • Analyze toroid formation using electron microscopy or atomic force microscopy

  • Quantify DNA protection from nuclease digestion

Data collection and analysis:

• Compare condensation kinetics across species

• Measure DNA binding affinity using fluorescence anisotropy

• Analyze protamine-DNA complex stability under increasing salt concentrations

• Correlate differences to specific sequence features or post-translational modifications

Expected results table:

This comparative approach would reveal whether the unique sequence features of Ningaui ridei PRM1, such as its specific arginine distribution pattern, confer different DNA packaging properties compared to other mammals .

What role does PRM1 phosphorylation play in the protamine-to-histone exchange during early embryogenesis?

Understanding the role of PRM1 phosphorylation in early embryogenesis requires sophisticated experimental approaches that connect biochemical mechanisms to developmental outcomes:

Experimental approaches:

• Zygote microinjection studies:

  • Inject wildtype versus phosphorylation-deficient mutant PRM1 into fertilized eggs

  • Track chromatin decondensation, protamine removal, and histone deposition using fluorescence microscopy

  • Monitor embryonic development to assess functional consequences

• Biochemical interaction analysis:

  • Investigate how phosphorylation affects PRM1 interaction with key factors:

    • Nucleoplasmin (NPM2): mediates protamine removal

    • HIRA: deposits histone H3.3

  • Use co-immunoprecipitation, pull-down assays, and surface plasmon resonance

• Genomic approaches:

  • Perform ATAC-seq to analyze how PRM1 phosphorylation impacts chromatin accessibility in early pronuclei

  • Compare wild-type versus embryos with phosphorylation-deficient PRM1

Recent research has demonstrated that SRPK1-mediated phosphorylation of protamine is essential for initiating the protamine-to-histone exchange. Specifically, SRPK1 phosphorylates serine residues in P1, weakening protamine-DNA interactions and facilitating interactions with NPM2 for removal and HIRA for H3.3 deposition .

This process is critical for proper embryonic development, as embryos with SRPK1 depletion or expression of phosphorylation-deficient protamine exhibit developmental arrest at the 1-cell stage due to failure in paternal genome decondensation .

How do bacterial infections affect protamine structure and function in sperm, and can recombinant PRM1 studies provide insights?

This question explores the intersection between reproductive biology and infectious disease, requiring integrated experimental approaches:

Experimental design:

• In vitro protamine-bacteria interaction studies:

  • Expose recombinant Ningaui ridei PRM1 to bacterial products from common urogenital pathogens

  • Analyze changes in PRM1 structure, post-translational modifications, and DNA binding capacity

  • Compare effects between different bacterial species (Staphylococcus, Escherichia, etc.)

• Functional analysis of bacterially-modified PRM1:

  • Assess DNA condensation efficiency of PRM1 exposed to bacterial products

  • Measure changes in P1/P2 ratios in mixed protamine systems

  • Evaluate protamine-to-histone exchange capacity using in vitro fertilization models

• Comparative analysis with human samples:

  • Compare findings with human sperm data from infected versus non-infected samples

  • Correlate bacterial effects on recombinant PRM1 with observed clinical findings

Research has shown that bacterial infections significantly impact human sperm parameters and protamine function. Specifically, bacterial infection is associated with abnormal P1/P2 ratios, decreased sperm chromatin condensation, reduced motility, and increased DNA fragmentation .

Comparative data from human studies:

By studying how bacterial products affect recombinant Ningaui ridei PRM1, researchers can develop controlled models to understand the molecular mechanisms underlying these clinical observations and potentially develop interventions to protect sperm chromatin integrity .

What are common challenges in expressing and purifying functional recombinant protamines, and how can they be addressed?

Recombinant protamine expression and purification present several technical challenges due to their unique biochemical properties:

Common challenges and solutions:

• Poor expression yields:

  • Challenge: Protamines' high arginine content can be toxic to expression hosts

  • Solutions:

    • Use specialized E. coli strains optimized for toxic protein expression

    • Employ inducible expression systems with tight regulation

    • Express as fusion proteins with solubility tags (e.g., GST, MBP)

    • Optimize codon usage for expression host

• Protein aggregation:

  • Challenge: Protamines readily bind nucleic acids and self-aggregate

  • Solutions:

    • Include high salt (0.5-1.0 M NaCl) in purification buffers

    • Add polyethyleneimine during lysis to remove nucleic acids

    • Perform purification under denaturing conditions followed by refolding

    • Use size exclusion chromatography as a final purification step

• Protease degradation:

  • Challenge: Small basic proteins can be targets for proteolysis

  • Solutions:

    • Include protease inhibitors in all purification buffers

    • Perform purification at lower temperatures (4°C)

    • Minimize purification time by optimizing protocols

• Functional verification:

  • Challenge: Confirming that recombinant protamine retains native function

  • Solutions:

    • Perform DNA binding assays to confirm functionality

    • Verify correct folding using circular dichroism

    • Compare activity to native protamine isolated from sperm

The recombinant Ningaui ridei PRM1 described in the product datasheet demonstrates >85% purity by SDS-PAGE, suggesting successful purification strategies have been employed .

How can researchers distinguish between normal and abnormal protamine function in experimental settings?

Distinguishing normal from abnormal protamine function requires multiple complementary approaches:

Functional assays for protamine activity:

• DNA binding and condensation:

  • Normal: Efficient, uniform DNA condensation at physiological protamine:DNA ratios

  • Abnormal: Incomplete condensation, irregular structures, or aggregation

  • Methods:

    • Fluorescence assays using DNA intercalating dyes

    • Electron or atomic force microscopy to visualize condensed structures

    • Protection of DNA from nuclease digestion

• Protamine ratios and modifications:

  • Normal: Species-specific P1:P2 ratios and appropriate post-translational modifications

  • Abnormal: Altered ratios or modification patterns

  • Methods:

    • Acid-urea gel electrophoresis to quantify P1 and P2 levels

    • Mass spectrometry to characterize post-translational modifications

• Chromatin accessibility:

  • Normal: Appropriately condensed chromatin with specific regions remaining accessible

  • Abnormal: Globally altered accessibility patterns

  • Methods: ATAC-seq to map accessible chromatin regions

• DNA integrity:

  • Normal: Protected DNA with minimal fragmentation

  • Abnormal: Increased DNA fragmentation

  • Methods: Comet assay, TUNEL assay, or sperm chromatin structure assay (SCSA)

Research has shown that abnormal P1:P2 ratios in humans and mice correlate with increased sperm DNA fragmentation, diminished fertilization rates, and defects in sperm morphology and motility . Bacterial infections significantly alter the P1:P2 ratio and increase DNA fragmentation .

What are the latest methodological advances for studying protamine-DNA interactions at the genome-wide level?

Recent methodological advances have expanded our ability to study protamine-DNA interactions at the genome-wide level, though significant challenges remain due to the highly condensed nature of protamine-bound chromatin:

Cutting-edge methodologies:

• Modified ChIP-seq approaches:

  • Challenge: Traditional ChIP protocols are ineffective due to the tight protamine-DNA interactions

  • Advances:

    • Specialized crosslinking methods optimized for arginine-rich proteins

    • Enhanced sonication and fragmentation protocols for condensed chromatin

    • Antibody development against specific protamine epitopes or modifications

• Accessibility mapping:

  • ATAC-seq adapted for sperm to identify regions with differential protamine occupancy

  • DNase-seq with modified nuclease concentrations

  • Micrococcal nuclease (MNase) titration approaches to distinguish protamine-bound versus histone-bound regions

• Structural biology approaches:

  • Cryo-electron microscopy of protamine-DNA complexes

  • Integrative modeling combining data from multiple structural techniques

  • Next-generation DNA footprinting with chemical probes

• Novel crosslinking strategies:

  • Development of arginine-specific crosslinkers to capture protamine-DNA interactions

  • Optimized formaldehyde crosslinking conditions for basic proteins

  • Photo-activatable nucleotide analogs for precise interaction mapping

The field still faces significant challenges in determining whether protamine protein placement varies along the sperm genome. Current models suggest uniform binding throughout the genome, but definitive data remains limited. The scarcity of lysine residues in protamines makes traditional crosslinking approaches difficult, and the super-condensed state of protamine-packaged chromatin presents technical barriers to mechanistic investigations .

Researchers are developing novel chromatin remodeling systems that hold promise for identifying candidate remodelers and uncovering molecular details of histone-to-protamine exchange, which would provide valuable insights into both packaging and unpackaging mechanisms .