Recombinant Photobacterium profundum Macrodomain Ter protein (matP)

What is the Macrodomain Ter protein (MatP) and what is its biological function?

MatP (Macrodomain Ter protein) is a DNA-binding protein that specifically recognizes and binds to matS sequences found within the Terminus (Ter) macrodomain of bacterial chromosomes. It plays a crucial role in chromosome organization and condensation. The protein contains a tripartite fold with a four-helix bundle DNA-binding motif, ribbon-helix-helix domain, and C-terminal coiled-coil region . MatP's primary function is to organize and compact the Ter macrodomain by bridging distant matS sites through the formation of tetramer structures, effectively looping DNA and creating a condensed chromosomal region . This organization is essential for proper chromosome segregation during cell division.

What are the conserved domains in MatP proteins across bacterial species?

The MatP protein contains three main conserved structural domains:

• Four-helix bundle motif: Serves as the primary DNA-binding domain, with residues from α4 helix making base-specific contacts with matS sequences .

• Ribbon-helix-helix (RHH) domain: Functions as the main dimerization region, with residues 97-129 in E. coli (120-142 in Y. pestis) being essential for MatP dimerization .

• C-terminal coiled-coil domain: Enables tetramerization through flexible bridging between MatP dimers bound to separate matS sites, facilitating DNA looping .

These domains are highly conserved across enterobacteria, suggesting evolutionary preservation of MatP's chromosome organization mechanism.

What are the optimal expression systems for recombinant P. profundum MatP?

For recombinant expression of P. profundum MatP, an E. coli-based expression system is recommended, similar to what has been used successfully for other MatP proteins. The choice of expression strain should consider the following methodological considerations:

• Use BL21(DE3) or its derivatives for high-level expression

• Consider using strains optimized for expression of proteins with rare codons (like Rosetta)

• Express at lower temperatures (15-20°C) to enhance proper folding and solubility

• Include HEPES buffer (pH 7.5) in the growth media to maintain stable pH

Given P. profundum's adaptation to high pressure environments , expression under pressure conditions might improve proper folding, though specialized equipment would be required. The gene should be cloned into vectors with strong inducible promoters (T7, tac) with a His-tag or other affinity tag to facilitate purification.

What purification strategy yields the highest purity and functionality for recombinant P. profundum MatP?

A multi-step purification strategy is recommended:

• Initial capture using affinity chromatography:

  • Ni-NTA for His-tagged protein

  • Optimize imidazole concentration in wash and elution buffers

• Intermediate purification using ion exchange chromatography:

  • Based on calculated pI of P. profundum MatP

  • Use salt gradient elution

• Polishing step using size exclusion chromatography:

  • Separates monomers, dimers, and tetramers

  • Provides information about oligomeric state

• Buffer optimization:

  • Include 100-150 mM NaCl to maintain solubility

  • Consider including small amounts of glycerol (5-10%)

  • Buffer pH around 7.5 (HEPES or Tris)

For functional studies, it's critical to verify DNA-binding activity using electrophoretic mobility shift assays with synthesized matS sequence oligonucleotides.

How does pressure affect the expression and stability of recombinant P. profundum MatP?

Since P. profundum is a piezophilic organism that grows optimally at 28 MPa , the expression and stability of its proteins, including MatP, are likely pressure-adapted. When expressing recombinant P. profundum MatP, consider:

• Expression under pressure conditions may yield properly folded protein with native characteristics, though specialized equipment is required

• At atmospheric pressure, temperature reduction (to 15-17°C) during expression may compensate for the absence of pressure

• The stability of purified protein might be enhanced under pressure conditions

For stability studies, compare the thermal denaturation profiles and activity of the protein at different pressures. Differential scanning fluorimetry or circular dichroism spectroscopy can be employed to assess protein stability under varying pressure conditions.

What experimental approaches can be used to assess the DNA-binding properties of P. profundum MatP?

Several complementary approaches are recommended for comprehensive characterization of P. profundum MatP's DNA-binding properties:

• Fluorescence polarization (FP) assays:

  • Can determine binding affinity (Kd) using fluorescently labeled matS oligonucleotides

  • Allows for quantitative comparison with E. coli MatP (which has Kd=2-5 nM)

  • Enables assessment of binding to mutated matS sequences

• Electrophoretic mobility shift assays (EMSA):

  • Visualizes protein-DNA complexes

  • Can distinguish between different oligomeric states

• Surface plasmon resonance (SPR):

  • Provides kinetic parameters (kon and koff)

  • Enables real-time monitoring of binding

• ChIP-seq analysis:

  • For genome-wide binding site identification

  • Validates the specificity for matS sequences in vivo

For all methods, compare binding to the consensus matS sequence (GTGACRNYGTCAC) versus control DNA sequences to establish specificity . Testing binding under different pressure conditions would provide insights into potential pressure-dependent functional adaptations.

How can I assess the DNA bridging activity of P. profundum MatP?

The DNA bridging activity of P. profundum MatP can be assessed using multiple complementary approaches:

• Atomic force microscopy (AFM):

  • Directly visualizes DNA looping and bridging

  • Can observe MatP-mediated DNA compaction at single-molecule level

  • Compare with E. coli MatP as a positive control

• Electron microscopy (EM):

  • Provides complementary structural evidence for DNA bridging

  • Negative staining EM can visualize protein-DNA complexes

• Single-molecule DNA stretching experiments:

  • Using optical or magnetic tweezers

  • Measures forces involved in DNA bridging

• Tethered particle motion (TPM) analysis:

  • Detects changes in DNA conformation upon protein binding

  • Measures effective length changes resulting from looping

For all these methods, DNA substrates containing two or more matS sites separated by various distances should be used to evaluate the distance dependence of bridging activity. Additionally, MatP coiled-coil domain mutants can be generated as negative controls, as this domain is crucial for bridging activity in E. coli MatP .

What mutations in P. profundum MatP affect its function and how can they be characterized?

Based on structural and functional studies of MatP from other species, several key regions can be targeted for mutational analysis in P. profundum MatP:

• DNA-binding domain mutations:

  • Target residues in the four-helix bundle, particularly in α4 (equivalent to Gln85, Arg88, Ala89, and Arg93 in Y. pestis MatP)

  • Characterize using fluorescence polarization to measure changes in DNA binding affinity

  • Verify with ChIP experiments to assess in vivo binding

• Dimerization interface mutations:

  • Target the RHH domain (equivalent to L104G, Q109G, and E127G in E. coli)

  • Assess using size exclusion chromatography and analytical ultracentrifugation

  • Use bacterial two-hybrid assays to verify disruption of dimerization

• Coiled-coil domain mutations:

  • Critical for tetramerization and DNA bridging

  • Characterize using atomic force microscopy and electron microscopy to assess DNA looping

  • Evaluate Ter condensation in vivo using fluorescence microscopy

For in vivo functional characterization, complementation assays can be performed, introducing mutant P. profundum MatP into MatP knockout strains and assessing chromosome organization phenotypes.

What structural methods are most suitable for studying P. profundum MatP-DNA complexes?

Multiple structural approaches provide complementary information:

• X-ray crystallography:

  • Provides high-resolution structures of MatP-matS complexes

  • Can reveal the specific base contacts and DNA recognition mechanism

  • Requires crystallization of protein-DNA complexes, which may be challenging

  • Has been successfully used for Y. pestis and E. coli MatP-matS structures

• Cryo-electron microscopy (cryo-EM):

  • Particularly useful for larger assemblies like MatP tetramers bridging DNA

  • Does not require crystallization

  • May reveal flexible conformations inaccessible to crystallography

• Small-angle X-ray scattering (SAXS):

  • Provides low-resolution structural information in solution

  • Useful for studying flexible conformations of MatP-DNA complexes

  • Can be performed under varying pressure conditions

• Nuclear magnetic resonance (NMR) spectroscopy:

  • For studying dynamics of specific domains

  • Useful for smaller fragments of MatP

For P. profundum MatP, structural studies under varying pressure conditions would be particularly valuable to understand potential pressure-dependent conformational changes. High-pressure crystallography or high-pressure NMR could provide insights into pressure adaptation mechanisms.

How does the structure of P. profundum MatP compare to MatP proteins from non-piezophilic bacteria?

While specific structural data for P. profundum MatP is not available in the provided search results, comparative analysis would likely reveal:

• Core structural conservation:

  • Preservation of the tripartite fold (four-helix bundle, RHH domain, coiled-coil)

  • Conservation of DNA-binding residues that contact the matS sequence

• Pressure adaptations:

  • Potentially more flexible regions to accommodate high-pressure environments

  • Amino acid substitutions that enhance protein stability under pressure:

    • Fewer internal cavities

    • More hydrophilic residues on the surface

    • Altered salt bridge and hydrogen bonding networks

• Possible thermal adaptations:

  • Features enhancing stability at lower temperatures (15°C), as P. profundum grows optimally at this temperature

Comparative homology modeling using the known E. coli and Y. pestis MatP structures as templates would provide initial structural insights. Experimental validation would require determination of the P. profundum MatP structure.

What role does the coiled-coil domain play in P. profundum MatP function and how can it be structurally characterized?

The coiled-coil domain in MatP proteins is critical for tetramerization and DNA bridging functions . For P. profundum MatP:

• Functional importance:

  • Enables the bridging of distant matS sites through flexible tetramer formation

  • Critical for Ter macrodomain condensation

  • Allows adaptation to varying distances between matS sites

• Structural characterization methods:

  • Circular dichroism (CD) spectroscopy to confirm α-helical content

  • Size exclusion chromatography coupled with multi-angle light scattering (SEC-MALS) to determine oligomeric states

  • Cross-linking mass spectrometry to identify interaction interfaces

  • Crystallography of isolated coiled-coil domains

• Mutational analysis:

  • Introduce mutations at key residues in the heptad repeat pattern

  • Assess impact on tetramerization and DNA bridging function

  • Compare with coiled-coil mutants of E. coli MatP that disrupt looping

Understanding the coiled-coil domain is particularly important as it represents the mechanistic basis for MatP's ability to organize large chromosomal domains through flexible bridging of distant DNA sites.

How can recombinant P. profundum MatP be used to study chromosome organization under high pressure conditions?

Recombinant P. profundum MatP provides a unique tool for studying chromosome organization under high pressure conditions:

• In vitro systems:

  • High-pressure microscopy with fluorescently labeled MatP and DNA containing matS sites

  • Atomic force microscopy under varying pressure conditions to directly visualize pressure effects on DNA-bridging

  • Comparison of P. profundum MatP with E. coli MatP to identify pressure-specific adaptations

• In vivo applications:

  • Fluorescently tagged MatP to track Ter macrodomain organization in live cells under pressure

  • ChIP-seq experiments at different pressures to map genome-wide binding patterns

  • Cross-species complementation studies (P. profundum MatP in E. coli and vice versa)

• Hybrid systems:

  • Creation of chimeric MatP proteins combining domains from piezophilic and non-piezophilic organisms

  • Testing function under varying pressure conditions

These approaches would provide insights into how chromosome organization mechanisms adapt to extreme environmental conditions and the specific role MatP plays in these adaptations.

What can comparative studies of P. profundum MatP and E. coli MatP reveal about pressure adaptation mechanisms?

Comparative studies would provide valuable insights into bacterial pressure adaptation:

• Structural comparisons:

  • Identify amino acid substitutions that enhance stability and function under pressure

  • Analyze differences in flexibility and compactness between the proteins

  • Examine conservation versus divergence in key functional domains

• Functional comparisons:

  • DNA binding affinities under varying pressure conditions

  • Tetramerization and DNA bridging capabilities at different pressures

  • Temperature dependence of activity

• Cross-species functionality:

  • Can P. profundum MatP function in E. coli at atmospheric pressure?

  • Can E. coli MatP function in P. profundum under high pressure?

  • What domains are responsible for pressure adaptation?

Such comparative studies could reveal general principles of protein adaptation to high-pressure environments, extending beyond chromosome organization proteins to broader evolutionary adaptations.

How can P. profundum MatP be used as a tool for synthetic biology applications?

The unique properties of P. profundum MatP offer several potential synthetic biology applications:

• Pressure-responsive gene regulation systems:

  • Engineered MatP variants with altered pressure sensitivity

  • MatP-based transcription factors for pressure-dependent gene expression

  • Design of synthetic genetic circuits responsive to pressure changes

• Chromosome organization tools:

  • Designer MatP proteins with altered DNA specificity

  • Synthetic chromosome organization systems for non-native hosts

  • Tools for controlled DNA compaction and release

• Protein engineering platforms:

  • P. profundum MatP as a scaffold for creating pressure-stable protein fusions

  • Development of pressure-resistant enzymes for industrial applications

  • Structure-guided design of pressure-adapted proteins

• Bionanotechnology applications:

  • MatP-based DNA origami stabilization under varying pressure conditions

  • Creation of pressure-responsive DNA nanostructures

  • Development of pressure-sensitive biosensors

These applications would leverage the unique pressure-adapted properties of P. profundum proteins like MatP for both fundamental research and biotechnological innovations.

What are the major technical challenges in studying the function of P. profundum MatP under high pressure conditions?

Several significant technical challenges must be addressed:

• Equipment limitations:

  • Specialized high-pressure vessels compatible with various experimental techniques

  • High-pressure microscopy systems for live-cell imaging

  • Adaptation of biochemical and biophysical methods for high-pressure conditions

• Experimental design challenges:

  • Maintaining consistent temperature while manipulating pressure

  • Separating pressure effects from other variables

  • Real-time measurements under pressure

• Biological system challenges:

  • Creating genetic tools functional in P. profundum

  • Developing pressure-compatible reporter systems

  • Establishing growth and expression protocols that account for pressure effects

• Data interpretation challenges:

  • Distinguishing direct pressure effects on MatP from indirect cellular responses

  • Accounting for pressure effects on experimental equipment and reagents

  • Normalizing results across different pressure conditions

Methodological innovations such as microfluidic high-pressure systems and pressure-compatible imaging techniques are needed to overcome these challenges.

How does the interaction of P. profundum MatP with other nucleoid-associated proteins differ from E. coli MatP interactions?

This complex question requires investigation of multiple protein-protein interactions:

• Potential interaction partners:

  • ZapB protein (known to interact with E. coli MatP to anchor the Ter domain at midcell)

  • Other nucleoid-associated proteins (NAPs) like H-NS, HU, and Fis

  • Components of the divisome

• Differential interaction analysis methods:

  • Co-immunoprecipitation followed by mass spectrometry

  • Bacterial two-hybrid screening

  • Microscopy-based co-localization studies under varying pressure

  • Crosslinking mass spectrometry

• Functional implications:

  • How pressure affects MatP's protein interaction network

  • Whether P. profundum has evolved pressure-specific interactions

  • How these interactions contribute to chromosome organization under pressure

The protein interactome of P. profundum MatP may reveal unique adaptations that facilitate chromosome organization and segregation under high-pressure conditions, potentially involving novel interaction partners not present in E. coli.

What is the evolutionary relationship between MatP proteins in piezophilic and non-piezophilic bacteria?

Investigating the evolutionary relationship would involve:

• Phylogenetic analysis:

  • Comprehensive sequence analysis of MatP proteins across bacterial phyla

  • Correlation of MatP sequence features with habitat pressure

  • Identification of convergent adaptations in distantly related piezophilic species

• Structural comparison:

  • Mapping pressure-adaptive mutations onto protein structure

  • Identification of structurally conserved regions versus variable regions

  • Analysis of coevolution between MatP and matS sequences

• Experimental approaches:

  • Ancestral sequence reconstruction and protein resurrection

  • Testing ancestral and intermediate MatP variants under varying pressures

  • Directed evolution experiments under pressure selection

• Bioinformatic analysis:

  • Positive selection analysis to identify adaptively evolving residues

  • Correlation of MatP sequence features with depth distribution of host organisms

  • Comparative genomics of matS site distribution in different species

This evolutionary perspective would provide insights into how essential chromosome organization mechanisms adapt to extreme environmental pressures while maintaining core functionality.

How does pressure influence the kinetics and thermodynamics of P. profundum MatP-DNA interactions?

This complex biophysical question can be approached through multiple methods:

• Equilibrium binding studies:

  • Fluorescence polarization under varying pressures

  • Isothermal titration calorimetry in pressure-compatible cells

  • Electrophoretic mobility shift assays with samples prepared under pressure

• Kinetic analysis:

  • Stopped-flow fluorescence with pressure cells

  • Surface plasmon resonance under varying pressures

  • Single-molecule approaches adapted for high pressure

• Thermodynamic profiling:

  • Determination of ΔH, ΔS, and ΔG as functions of pressure

  • Van't Hoff analysis across pressure ranges

  • Volume change (ΔV) calculations from pressure-dependent binding data

Anticipated findings might include pressure-dependent changes in association/dissociation rates, altered binding specificity at high pressure, or pressure-dependent conformational selection. The activation volume for binding could provide insights into the molecular mechanism of pressure adaptation.

Table of Comparative Properties