Recombinant Thermomicrobium roseum Chaperone protein DnaK (dnaK), partial

What is the phylogenetic position of Thermomicrobium roseum and how does it affect our understanding of its DnaK protein?

T. roseum was originally assigned to its own phylum, Thermomicrobia, but subsequent phylogenetic analysis based on 16S rRNA gene sequences led to its reclassification within the phylum Chloroflexi in 2004 . This phylogenetic positioning has been confirmed through genome-scale analyses, which support its placement as a deep-branching member of the Chloroflexi phylum .

For researchers studying T. roseum DnaK, this phylogenetic context is critical as it provides evolutionary perspective on the protein's characteristics. As a deep-branching member of Chloroflexi, T. roseum DnaK represents an important reference point for understanding how heat shock proteins evolved in this lineage. Comparative analyses between T. roseum DnaK and homologs from other Chloroflexi members can reveal evolutionary patterns specific to this phylum.

Methodologically, researchers should approach evolutionary studies of T. roseum DnaK through:

• Comprehensive phylogenetic analysis using multiple sequence alignments

• Structural comparisons with DnaK homologs from various phylogenetic positions

• Gene synteny analysis to examine conservation of the genomic context

How does the genome structure of T. roseum relate to stress response genes like dnaK?

T. roseum possesses an unusual genome structure consisting of two circular DNA elements: a chromosome (2,006,217 bp) and a megaplasmid (919,596 bp) . The genomic data reveals several key insights:

This genomic organization may influence the expression and regulation of stress-response genes like dnaK. Researchers should determine whether dnaK is encoded on the chromosome or megaplasmid, as this location could affect its expression patterns, particularly during stress responses. The high G+C content (>63%) of both genomic elements may also influence codon usage in the dnaK gene, potentially affecting recombinant expression strategies.

How do the DnaK proteins from thermophiles like T. roseum compare to mesophilic homologs?

While specific comparisons for T. roseum DnaK are not directly provided in the search results, we can extrapolate from data on other heat shock proteins. The search results provide percentage identity/similarity data for Hsp70/DnaK proteins across different archaeal and bacterial species :

For T. roseum DnaK, researchers should expect:

• Higher sequence similarity with other thermophilic Chloroflexi members

• Conservation of key functional domains (nucleotide-binding domain and substrate-binding domain)

• Specific amino acid substitutions that contribute to thermostability

• Potential modifications in co-chaperone interaction regions

Methodological approach: Conduct comprehensive sequence alignments, generate phylogenetic trees, and identify signature sequences that distinguish thermophilic DnaK proteins from mesophilic homologs.

What expression systems are optimal for producing recombinant T. roseum DnaK?

For thermophilic proteins like T. roseum DnaK, selecting an appropriate expression system is crucial for obtaining properly folded, active protein. Researchers should consider:

• E. coli expression systems:

  • BL21(DE3) and derivatives for high-level expression

  • Arctic Express strains containing cold-adapted chaperonins to assist folding

  • Rosetta strains for addressing potential codon bias issues (important given T. roseum's high G+C content)

• Expression conditions optimization:

  • Reduced temperature (16-20°C) to slow protein folding and prevent inclusion body formation

  • Extended induction periods (overnight or longer)

  • IPTG concentration titration to control expression rate

• Vector selection:

  • pET series vectors with T7 promoter for high-level expression

  • Addition of solubility-enhancing tags (MBP, SUMO) if solubility issues are encountered

  • Inclusion of His6 or other affinity tags for purification

• Co-expression strategies:

  • Co-expression with E. coli chaperones (GroEL/ES) to enhance folding

  • Co-expression with T. roseum GrpE and DnaJ if available

The high thermostability of T. roseum DnaK can be advantageous during purification, as a heat treatment step (65-70°C) can be employed to remove many host cell proteins while leaving the target protein intact.

What purification protocol yields the highest activity for recombinant T. roseum DnaK?

Based on general principles for purifying thermophilic proteins and chaperones, the following protocol is recommended:

• Cell lysis:

  • Mechanical disruption (sonication or high-pressure homogenization)

  • Lysis buffer containing 50 mM HEPES pH 7.5-8.0, 300 mM NaCl, 5 mM MgCl₂, 1 mM DTT

  • Addition of protease inhibitors and nuclease

• Heat treatment:

  • Incubation at 65°C for 20 minutes to precipitate host proteins

  • Centrifugation to remove precipitated material

• Affinity chromatography:

  • Immobilized metal affinity chromatography (IMAC) for His-tagged constructs

  • ATP-agarose affinity chromatography exploiting DnaK's ATP-binding ability

• Ion exchange chromatography:

  • Anion exchange (Q Sepharose) at pH 8.0

  • Salt gradient elution (0-500 mM NaCl)

• Size exclusion chromatography:

  • Final polishing step

  • Assessment of oligomeric state

• Activity verification:

  • ATPase activity assay at elevated temperatures (70-75°C)

  • Substrate binding assays

Researchers should monitor protein purity by SDS-PAGE and verify folding status through circular dichroism spectroscopy. The purification buffer should contain ADP or ATP to stabilize the protein, along with Mg²⁺ as a cofactor for nucleotide binding.

What analytical methods are essential for characterizing recombinant T. roseum DnaK?

Comprehensive characterization of recombinant T. roseum DnaK requires multiple analytical approaches:

• Structural integrity assessment:

  • Circular dichroism (CD) spectroscopy to confirm secondary structure content

  • Differential scanning calorimetry (DSC) to determine melting temperature and thermostability

  • Size exclusion chromatography with multi-angle light scattering (SEC-MALS) to verify oligomeric state

• Functional characterization:

  • ATPase activity assays at various temperatures (25-80°C)

  • Substrate binding assays using model peptides or denatured proteins

  • Nucleotide binding studies (isothermal titration calorimetry)

• Biophysical properties:

  • Thermofluor assays to determine buffer conditions that maximize thermal stability

  • Dynamic light scattering to assess homogeneity and aggregation propensity

  • Fluorescence spectroscopy to monitor conformational changes

• Comparative analyses:

  • Side-by-side functional comparisons with E. coli DnaK to highlight thermophilic adaptations

  • Temperature-dependent activity profiles

These methods will provide a comprehensive characterization profile necessary for understanding the unique properties of T. roseum DnaK and its potential applications in research and biotechnology.

What structural adaptations enable T. roseum DnaK to function at elevated temperatures?

T. roseum, growing optimally at 70-75°C , requires specialized adaptations in its molecular chaperones. While specific structural data for T. roseum DnaK is not provided in the search results, thermophilic proteins typically employ several thermostabilizing strategies:

• Increased internal hydrophobicity and improved core packing

• Enhanced secondary structure propensity with additional hydrogen bonding networks

• Increased number of salt bridges and electrostatic interactions

• Reduced number of thermolabile residues (Asn, Gln) and surface loops

• Strategic placement of proline residues to restrict conformational flexibility

For T. roseum DnaK specifically, researchers should investigate:

Methodological approaches should include X-ray crystallography or cryo-EM structural determination, molecular dynamics simulations at elevated temperatures, and mutational studies targeting predicted thermostabilizing features.

How does nucleotide binding affect T. roseum DnaK conformation and function at high temperatures?

The DnaK (Hsp70) chaperone cycle is driven by ATP binding and hydrolysis, which induces conformational changes that alter substrate binding affinity. For T. roseum DnaK, researchers should investigate:

• Nucleotide binding kinetics at elevated temperatures (65-80°C)

• Thermodynamic parameters of ATP/ADP binding using isothermal titration calorimetry

• Conformational changes upon nucleotide binding using techniques such as:

  • Hydrogen-deuterium exchange mass spectrometry

  • Fluorescence resonance energy transfer (FRET) with strategically placed fluorophores

  • Small-angle X-ray scattering (SAXS)

• Correlation between nucleotide-bound state and substrate affinity at various temperatures

A critical question is whether T. roseum DnaK maintains the canonical DnaK conformational cycle at its growth temperature or has evolved modifications to this mechanism to function efficiently in thermophilic conditions. Comparing the temperature dependence of these conformational changes with mesophilic homologs would provide valuable insights into thermophilic adaptations of molecular chaperones.

How does the unusual cell envelope of T. roseum influence DnaK function and localization?

The search results highlight T. roseum's unique cell envelope, featuring a membrane composed entirely of long-chain 1,2-diols instead of glycerolipids and a cell wall dominated by proteins . This unusual composition raises important questions about DnaK function:

• Membrane interaction: The absence of glycerolipids may affect how DnaK interacts with the membrane during stress conditions. Researchers should investigate:

  • Membrane association properties of T. roseum DnaK

  • Lipid binding assays using artificial membranes mimicking T. roseum composition

  • Localization studies using fluorescently labeled DnaK in native or reconstituted systems

• Cell wall protein folding: With a protein-rich cell wall, T. roseum DnaK may play specialized roles in:

  • Folding and assembly of cell wall proteins

  • Quality control of membrane-associated protein complexes

  • Transport of proteins across this unusual cell envelope

Methodological approaches should include membrane fractionation studies, identification of DnaK-interacting partners in membrane fractions, and analysis of DnaK's role in cell envelope integrity under stress conditions.

How is dnaK gene expression regulated in T. roseum compared to other bacteria?

While the search results don't provide specific information about dnaK regulation in T. roseum, they do discuss regulatory patterns in archaeal systems that may provide insights:

• The search results indicate that archaeal hsp70 loci have different structural organization compared to bacterial operons, suggesting different modes of transcription and regulation . Researchers investigating T. roseum dnaK should:

  • Analyze the genomic context surrounding the dnaK gene

  • Identify potential regulatory elements in the promoter region

  • Determine if dnaK is expressed monocistronically or as part of an operon

• In archaea, the regions upstream of hsp70 genes contain unique features:

  • M. mazei S-6 and M. thermophila TM-1 have longer intergenic regions than bacterial equivalents

  • Some contain repeats and palindromes that might serve as binding sites for regulatory factors

To study T. roseum dnaK regulation, researchers should employ:

• Promoter mapping through primer extension and 5' RACE

• Reporter gene assays to identify regulatory elements

• Chromatin immunoprecipitation to identify transcription factors

• RNA-seq analysis under various stress conditions to characterize expression patterns

What is the relationship between T. roseum's thermophilic lifestyle and constitutive versus inducible expression of dnaK?

This question addresses a fundamental aspect of thermophilic biology: how do organisms that live at consistently high temperatures regulate heat shock proteins that are typically stress-induced in mesophiles?

For T. roseum, which grows optimally at 70-75°C , researchers should investigate:

• Baseline expression levels of dnaK at optimal growth temperature

• Expression changes when exposed to:

  • Temperatures above optimal growth (e.g., 80-85°C)

  • Temperatures below optimal growth

  • Other stressors (oxidative stress, pH changes)

• Comparison with expression patterns in mesophilic bacteria

Methodological approaches include:

• qRT-PCR analysis of dnaK transcription under various conditions

• Western blot analysis of DnaK protein levels

• Promoter-reporter fusion studies to visualize expression dynamics

• Ribosome profiling to assess translational regulation

Understanding this aspect of T. roseum biology would provide insights into how thermophiles have adapted their stress response systems to function in environments that would be stressful for mesophiles.

What co-chaperones interact with T. roseum DnaK and how are they co-regulated?

DnaK typically functions in concert with co-chaperones, primarily DnaJ (Hsp40) and GrpE. In analyzing the T. roseum chaperone network:

• Researchers should identify T. roseum homologs of:

  • DnaJ (Hsp40) proteins

  • GrpE nucleotide exchange factor

  • Potential novel co-chaperones specific to thermophiles

• Investigate co-expression patterns:

  • Are co-chaperone genes located in the same genomic region as dnaK?

  • Do they share regulatory elements?

  • Are expression levels coordinated under different stress conditions?

• Characterize physical interactions:

  • Affinity purification followed by mass spectrometry to identify interaction partners

  • Surface plasmon resonance to determine binding kinetics at different temperatures

  • In vitro reconstitution of the complete chaperone system

The search results mention that in some archaea, the grpE-hsp70 intergenic regions are longer than in bacteria, suggesting different modes of transcription and regulation . Similar analyses in T. roseum would help understand how thermophilic bacteria organize and regulate their chaperone networks.

What are the optimal buffer conditions for assaying T. roseum DnaK activity in vitro?

For thermophilic proteins like T. roseum DnaK, buffer optimization is critical for meaningful functional assays. Researchers should consider:

• Temperature stability: Buffers should maintain pH at elevated temperatures (70-75°C)

  • HEPES buffer (pKa has lower temperature dependency)

  • Phosphate buffer (if pH >7.0 is required)

  • Avoid Tris buffer due to significant temperature-dependent pH shifts

• Essential components:

  • Magnesium ions (3-5 mM MgCl₂) for nucleotide binding

  • Potassium ions (50-100 mM KCl) for optimal activity

  • ATP or ADP (0.1-1 mM) depending on the specific assay

  • Reducing agent (1-2 mM DTT or TCEP) thermostable at assay temperature

• Stabilizing additives:

  • Glycerol (5-10%) to prevent aggregation

  • Nucleotides (ADP often stabilizes DnaK proteins)

• pH considerations:

  • Optimal pH should be determined experimentally, but typically between pH 7.5-8.5

  • Remember that T. roseum grows optimally at pH 8.2-8.5

Methodologically, researchers should systematically test different buffer components through thermal shift assays (Thermofluor) to identify conditions that maximize thermostability while maintaining activity.

What controls are essential when assessing the chaperone activity of T. roseum DnaK?

Rigorous experimental design for T. roseum DnaK functional studies requires several control conditions:

• Nucleotide state controls:

  • Nucleotide-free (achieved by EDTA treatment)

  • ATP-bound (non-hydrolyzable analogs like AMP-PNP)

  • ADP-bound

  • Comparison between different nucleotide states

• Temperature controls:

  • Activity assessment across a range of temperatures (30-80°C)

  • Comparison with mesophilic DnaK (e.g., E. coli) at various temperatures

  • Controls for temperature effects on substrate proteins and assay components

• Functional controls:

  • DnaK mutants defective in ATP hydrolysis

  • DnaK mutants defective in substrate binding

  • Assays with and without co-chaperones (DnaJ, GrpE)

• System controls:

  • Spontaneous refolding/aggregation of substrate proteins

  • Non-specific effects of high protein concentrations

  • Buffer components contribution to observed effects

These controls help distinguish the specific contributions of T. roseum DnaK from non-specific effects and provide crucial comparative data to understand its thermophilic adaptations.

What techniques can be used to study the interaction between T. roseum DnaK and client proteins at high temperatures?

Studying chaperone-substrate interactions at elevated temperatures presents technical challenges but is essential for understanding T. roseum DnaK function. Recommended approaches include:

• Binding affinity measurements:

  • Isothermal titration calorimetry in thermostable cells

  • Microscale thermophoresis with temperature control

  • Surface plasmon resonance using thermostable sensor chips

  • Fluorescence anisotropy with labeled model peptides

• Structural characterization of complexes:

  • Hydrogen-deuterium exchange mass spectrometry

  • Crosslinking coupled with mass spectrometry

  • Cryo-electron microscopy of DnaK-substrate complexes

• Functional interaction assays:

  • Prevention of thermal aggregation (light scattering)

  • Protein refolding assays with thermolabile model substrates

  • Protection of enzyme activity during thermal stress

• Client protein identification:

  • Pull-down assays at elevated temperatures

  • In vivo crosslinking followed by immunoprecipitation

  • Comparative proteomics under stress conditions

These methods should be adapted for high-temperature work, with appropriate controls to account for temperature effects on the experimental system itself.

How can T. roseum DnaK be engineered to enhance its biotechnological applications?

The thermostable nature of T. roseum DnaK makes it an excellent candidate for protein engineering aimed at biotechnological applications:

• Stability enhancement:

  • Further increasing thermostability through rational design

  • Improving solvent tolerance for industrial applications

  • Engineering pH tolerance for broader application range

• Substrate specificity modification:

  • Altering the substrate binding domain to recognize specific targets

  • Creating variants with broadened substrate specificity

  • Developing variants that preferentially bind to aggregation-prone industrial enzymes

• Functional modifications:

  • Creating ATP-independent variants

  • Engineering controllable substrate release mechanisms

  • Developing immobilized versions for biotechnology applications

• Fusion protein development:

  • Creating bifunctional chaperones with combined DnaK and other chaperone activities

  • Developing DnaK-enzyme fusions for enhanced enzyme stability

Methodological approaches should include structure-guided mutagenesis, directed evolution techniques optimized for thermophilic proteins, and high-throughput screening assays to identify variants with desired properties.

What insights can comparative genomics of thermophilic Chloroflexi provide about the evolution of dnaK genes?

Now that T. roseum has been reclassified as a deep-branching member of the Chloroflexi phylum , comparative genomic analyses can yield valuable insights:

• Researchers should analyze:

  • dnaK gene sequences across Chloroflexi members

  • Genomic context and synteny around dnaK genes

  • Co-evolution patterns with co-chaperones and client proteins

  • Selection pressure indicators in different lineages

• Specific questions to address:

  • Is there evidence for horizontal gene transfer of dnaK genes?

  • How do dnaK genes in photosynthetic vs. non-photosynthetic Chloroflexi compare?

  • Are there lineage-specific adaptations in thermophilic vs. mesophilic members?

• Phylogenetic approaches:

  • Reconciliation of gene trees with species trees

  • Dating of gene duplication/divergence events

  • Analysis of positive selection signatures

Such analyses would contribute to understanding broader evolutionary patterns of molecular chaperones and provide context for T. roseum DnaK's specific adaptations.

What role might T. roseum DnaK play in the organism's adaptation to its extreme environment beyond protein folding?

While protein folding is the canonical function of DnaK/Hsp70 chaperones, they often play additional roles, particularly in extremophiles:

Understanding these non-canonical functions would provide a more complete picture of how molecular chaperones contribute to extremophile adaptation.