Recombinant Histone H4-1

What is recombinant histone H4-1 and how is it typically produced?

Recombinant human histone H4-1 (UniProt: P62805, Entrez Gene: NM_003538.3) is a fundamental chromosomal protein typically produced in E. coli expression systems for research applications. The recombinant production process involves bacterial expression followed by purification using Fast Protein Liquid Chromatography (FPLC) . This approach allows researchers to obtain highly pure histone H4-1 protein for various experimental applications.

For proper experimental use, researchers should verify protein identity and purity through analytical techniques such as HPLC and mass spectrometry. When conducting structural or functional studies, it's essential to consider that recombinant H4.1 lacks the post-translational modifications present in native histones isolated from mammalian cells, which may affect certain experimental outcomes depending on research objectives.

What are the primary research applications of recombinant histone H4-1?

Recombinant histone H4-1 serves multiple critical functions in chromatin biology research. The primary applications include:

• Enzyme activity assays - particularly for studying histone-modifying enzymes such as acetyltransferases, methyltransferases, and their inhibitors .

• Western blotting - as standards and controls when investigating histone modifications in experimental samples .

• Structural characterization studies - using techniques like ion mobility spectrometry and mass spectrometry to understand conformational dynamics .

• Nucleosome reconstitution - combining with other core histones and DNA to form nucleosomes for chromatin structure/function studies .

• Drug discovery platforms - serving as targets for screening potential inhibitors of histone-modifying enzymes .

Additionally, recombinant H4-1 is valuable in investigating epigenetic mechanisms, as it provides a controlled substrate for studying how different modifications affect subsequent enzymatic reactions.

How can researchers accurately quantify recombinant histone H4-1 concentrations for experiments?

Accurate quantification of recombinant histone H4-1 is crucial for experimental reproducibility. While conventional protein quantification methods like Bradford or BCA assays provide estimates, nuclear magnetic resonance (NMR) spectroscopy offers superior precision for histone quantification.

The recommended procedure involves:

• Preparing a D₂O solution containing your histone H4-1 sample (approximately 4.5 mM based on weight) with 1 mM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS) as an external standard .

• Collecting ¹H NMR spectra at room temperature.

• Calculating the actual concentration by comparing the integration ratio between the DSS proton peak (0 ppm) and the two proton peaks from the imidazole group of His-18 (δ = 7–9 ppm) .

This method ensures significantly greater accuracy than colorimetric assays, particularly important when conducting kinetic studies or calibrating enzyme activity assays where precise substrate concentrations are essential.

What structural conformations does histone H4-1 adopt and how can researchers investigate them?

Histone H4-1 adopts diverse conformational states ranging from compact (C) to partially folded (P) to elongated (E) structures, reflecting its dynamic nature in different cellular contexts . These conformational variations significantly impact biological function and interactions with modifying enzymes.

Advanced research techniques to investigate these conformations include:

• FAIMS-TIMS-MS (Field Asymmetric Ion Mobility Spectrometry-Trapped Ion Mobility Spectrometry-Mass Spectrometry) - This tandem approach allows detailed characterization of different H4.1 conformational states in the gas phase .

• Collision-Induced Unfolding (CIU) - Reveals structural stability and unfolding pathways, with H4.1 showing intriguing "refolding after unfolding" phenomena for certain charge states .

• Molecular Dynamics (MD) simulations - Provides candidate structures and unfolding trajectories that complement experimental observations .

Research findings indicate that H4.1 gas-phase structures depend significantly on starting solution conditions, evidenced by differences in charge state distributions, mobility distributions, and unfolding pathways . The conformational diversity appears particularly important when studying interactions with other chromatin components.

How do solution conditions influence histone H4-1 structure and what methodological approaches reveal these relationships?

Solution conditions critically affect histone H4-1 conformation, with significant implications for experimental design. Research shows that:

The nature and concentration of buffer components directly influence H4.1 structural profiles. In native conditions (10-65 mM ammonium acetate), H4.1 maintains more compact conformations compared to denaturing conditions (methanol/water/formic acid, 50:49:1), which promote extended structures .

Methodological approaches to investigate these solution-dependent conformations include:

• Comparative analysis using different buffer systems (native vs. denaturing) coupled with ion mobility-mass spectrometry

• Observation of charge state distributions - higher charge states typically indicate more denatured conformations

• Molecular dynamics simulations in different solvent environments to predict structural changes

When designing histone modification experiments, researchers should carefully consider buffer composition, pH, and salt concentration, as these parameters significantly influence histone structure and consequently enzyme accessibility to modification sites.

How do lysine acetylation patterns on histone H4-1 affect subsequent arginine methylation?

The interplay between histone modifications represents a critical regulatory mechanism in chromatin biology. Research demonstrates that lysine acetylation on histone H4-1 differentially modulates arginine methylation in a site-specific manner. This relationship is not uniform but depends on both the acetylation site and the type of methylation .

Lysine acetylation at positions K5, K8, K12, and K16 of the H4 tail creates distinct modification patterns that either promote or inhibit subsequent arginine methylation by specific protein arginine methyltransferases (PRMTs) . The molecular basis for this regulation involves:

• Altered substrate recognition by methyltransferases due to changes in charge distribution

• Conformational changes in the H4 tail induced by acetylation

• Potential disruption of enzyme binding surfaces

Methodologically, researchers investigating these relationships should employ:

• Synthetic peptide substrates with specific modification patterns

• In vitro methyltransferase assays with purified enzymes

• MS/MS analysis to quantify modification levels

• Complementary in vivo validation using histone mutants

What is the role of histone H4-1 N-terminal domain in H3K79 methylation and how can researchers study this interaction?

The N-terminal domain of histone H4-1 plays an essential and direct role in facilitating H3K79 methylation by the Dot1 methyltransferase. Unlike other histone tails, the H4 N-terminal region specifically interacts with Dot1 and is absolutely required for H3K79 methylation to occur .

Key research findings demonstrate:

• Deletion of the H4 N-terminal tail completely eliminates bulk methylation of H3K79 in vivo

• Point mutations of known modification sites (including all four conserved lysines) do not reproduce this effect, suggesting a structural rather than modification-dependent mechanism

• In vitro histone methyltransferase (HMT) assays confirm that Dot1 cannot methylate nucleosomal substrates lacking the H4 tail

Researchers investigating this cross-talk should consider:

• Using recombinant nucleosome core particles with and without the H4 tail in HMT assays

• Employing both gel-based and liquid scintillation-based methylation assays to quantify activity

• Generating comprehensive mutation series across the H4 tail to identify critical residues

• Validating findings through complementary in vivo and in vitro approaches

This interaction represents a key example of inter-histone crosstalk that regulates chromatin structure and function.

What mass spectrometry approaches are most effective for characterizing histone H4-1 and its modifications?

Mass spectrometry (MS) techniques have revolutionized histone H4-1 characterization, enabling detailed analysis of modifications and conformational states. The most effective approaches include:

• Tandem nonlinear and linear ion mobility spectrometry (FAIMS-TIMS) coupled to mass spectrometry - This powerful combination provides exceptional resolution of different H4.1 conformational states and charge variants, revealing structural dynamics impossible to detect with traditional methods .

• Top-down MS approaches with two-dimensional liquid chromatography - These methods enable identification of numerous H4 isoforms with different modification patterns, with studies identifying up to 74 distinct H4 isoforms in differentiating human embryonic stem cells .

• MALDI-MS - Useful for confirming peptide identity and purity after synthesis, particularly for studies employing synthetic H4 tail peptides with specific modification patterns .

When implementing these techniques, researchers should consider:

• Sample preparation methods that preserve native structures when desired

• Appropriate buffer exchange procedures to remove interfering components

• The complementary nature of different MS approaches for comprehensive characterization

What are the methodological considerations for studying histone H4-1 in nucleosome contexts?

Studying histone H4-1 within nucleosome contexts requires specific methodological considerations to maintain structural integrity and functional relevance. When working with reconstituted nucleosomes:

• Nucleosome assembly approaches:

  • Commercial kits like EpiMark assembly systems provide standardized methods for reproducible reconstitution

  • Core histones should be carefully quantified and mixed in equimolar ratios

  • Salt gradient dialysis techniques help ensure proper nucleosome formation

• Experimental validation of nucleosome integrity:

  • Native gel electrophoresis to confirm proper assembly

  • Micrococcal nuclease digestion patterns to verify DNA wrapping

  • Analytical ultracentrifugation to assess homogeneity

• Modification studies with nucleosomal substrates:

  • Pre-modification of histone H4 prior to assembly versus post-assembly modification

  • Gel-based assays (15-17% SDS-PAGE) for nucleosome modification analysis

  • Storage phosphorimaging for visualizing methylated nucleosomal substrates

• Structural considerations:

  • The H4 tail extends outside the nucleosome core particle and can interact with adjacent nucleosomes

  • The basic patch of H4 (residues 16-20) is particularly important for internucleosomal contacts

These methodological considerations ensure that studies investigating H4-1 in nucleosomal contexts accurately reflect the protein's native environment and functional interactions.

How can researchers investigate the potential of histone H4-1 as a drug target for sepsis and related conditions?

Extracellular histone H4-1 has emerged as a promising drug target due to its role in organ failure during sepsis and other inflammatory conditions . Researchers investigating H4-1 as a therapeutic target should consider:

• Structural characterization approaches:

  • Detailed 3D structural coordinates are essential for in silico drug discovery

  • Conformational dynamics studies using FAIMS-TIMS-MS provide critical information about structural flexibility

  • Molecular dynamics simulations help identify potential binding pockets and their accessibility

• Functional screening methods:

  • Development of high-throughput assays to identify compounds that disrupt H4-1 interactions with inflammatory mediators

  • Cell-based assays to evaluate the impact of candidate compounds on inflammatory pathways

  • Binding affinity measurements with techniques like surface plasmon resonance or isothermal titration calorimetry

• Target validation considerations:

  • Confirmation that candidate compounds specifically bind extracellular rather than nuclear H4-1

  • Demonstration that binding alters pathological activity while minimizing impact on normal physiological functions

  • Investigation of structure-activity relationships to optimize lead compounds

This research direction requires integrating structural biology, medicinal chemistry, and inflammation research to develop effective therapeutic strategies targeting histone H4-1.

What technical challenges exist in analyzing dipole alignment of histone H4-1 structures and what solutions are available?

Analysis of dipole alignment in histone H4-1 structures presents significant technical challenges due to the protein's complex charge distribution and conformational heterogeneity. Research reveals that:

Charge distribution in elongated (E-like) H4-1 structures, where basic and acidic residues are exposed, significantly influences dipole alignment behaviors at high electric fields . This is particularly evident when CCS (collision cross-section) profiles exhibit narrow band distributions.

Technical challenges and solutions include:

• Distinguishing dipole effects from other factors:

  • Challenge: Separating dipole alignment effects from other phenomena affecting ion mobility

  • Solution: Comparative analysis of different charge states with similar CCS values but different charge distributions

• Quantitative analysis of dipole moments:

  • Challenge: Accurate calculation of dipole moments for flexible protein structures

  • Solution: Theoretical dipole calculations coupled with experimental FAIMS-TIMS data to validate models

• Conformational averaging:

  • Challenge: Accounting for dynamic averaging of dipole moments across conformational ensembles

  • Solution: Using molecular dynamics simulations to sample conformational space and calculate ensemble-averaged properties

These advanced analytical approaches provide deeper insights into histone H4-1 structural dynamics that are essential for understanding its biological functions and potential as a therapeutic target.