Recombinant Haloquadratum walsbyi Putative metal transport protein HQ3622A (HQ3622A)

What is known about the putative metal transport protein HQ3622A in Haloquadratum walsbyi?

HQ3622A (UniProt ID: Q18EC3) is a putative metal transport protein from Haloquadratum walsbyi . Based on the available information, it is a relatively small protein with the mature form spanning amino acids 33-101 of the full sequence . While the specific metals transported by HQ3622A have not been definitively characterized in the provided search results, its classification as a metal transport protein suggests it may play a role in metal homeostasis in the extreme halophilic environment where Haloquadratum walsbyi thrives. The protein can be recombinantly expressed in E. coli with an N-terminal His tag, which facilitates purification and subsequent biochemical studies . Given the extremophilic nature of its source organism, HQ3622A likely has adapted features that enable it to function in high salt concentrations, making it potentially valuable for studies on protein adaptation to extreme environments.

What is the amino acid sequence of HQ3622A protein and what structural features does it have?

The amino acid sequence of the mature HQ3622A protein (amino acids 33-101) is:
ASIVGYAEPLENAAKMTGATDAAMNLNPGVLPDYTVGGFSGPIGTLISAGVGTVLTLIVAAFGAGRLLES

This 69-amino acid sequence likely contains hydrophobic regions that are characteristic of membrane transport proteins. The presence of glycine residues (G) may provide flexibility in protein structure, while the hydrophobic amino acids (such as A, I, V, L, F) suggest membrane-spanning domains. The charged and polar residues (such as E, D, N, T, S) could be involved in substrate binding or protein-protein interactions. Without experimental structural data, precise structural features cannot be definitively stated, but sequence analysis tools could predict secondary structure elements such as alpha-helices or beta-sheets. Based on other metal transport proteins, HQ3622A might have metal-binding motifs involving amino acids with electron-donating side chains (histidine, cysteine, aspartate, glutamate), though specific binding sites would need to be confirmed experimentally.

What is the function of metal transport proteins in extremophiles like Haloquadratum walsbyi?

Metal transport proteins in extremophiles like Haloquadratum walsbyi play crucial roles in maintaining cellular metal homeostasis under challenging environmental conditions. In hypersaline environments, the bioavailability of essential metals may be limited due to altered solubility, complex formation, or competition with abundant ions. Metal transporters help regulate the uptake, efflux, and intracellular distribution of both essential and potentially toxic metals.

Essential metals like zinc, iron, and copper serve as cofactors for many enzymes and are required for various cellular processes. For example, zinc is an essential trace element involved in numerous biological processes, as noted in the context of Zrt-/Irt-like proteins (ZIPs), a family of divalent metal transporters . The tight regulation of metal concentrations is particularly important in extreme environments where cellular systems operate under stress conditions.

Metal transporters in halophilic archaea like Haloquadratum walsbyi likely have adapted to function optimally at high salt concentrations, possibly with structural features that maintain stability and functionality despite the challenging ionic environment. These adaptations might include specific amino acid compositions favoring acidic residues on the protein surface, specialized metal coordination sites, or unique conformational change mechanisms during transport cycles.

How does the conformational change in HQ3622A compare to other ZIP transporters during metal binding?

While the search results don't provide specific information about conformational changes in HQ3622A, insights can be drawn from studies on related ZIP transporters. The prototypical ZIP from Bordetella bronchiseptica (BbZIP) has been extensively studied and shown to operate via an elevator transport mechanism . During metal binding, significant conformational changes occur that are essential for the transport process.

In BbZIP, metal binding induces a transition from an inward-facing conformation (IFC) to an outward-facing conformation (OFC) . Specific residues, such as L200 in BbZIP, change from being buried to exposed depending on the conformational state. Researchers have used this property to develop assays for monitoring conformational changes, such as the sandwich ELISA approach described for BbZIP .

Key differences might exist due to the adaptation of HQ3622A to the hypersaline environment. The conformational changes in HQ3622A might be modified to maintain structural integrity in high salt conditions while still enabling metal transport.

What experimental approaches are optimal for studying the metal binding properties of HQ3622A?

Several experimental approaches can be employed to effectively study the metal binding properties of HQ3622A:

• Isothermal Titration Calorimetry (ITC): This technique can directly measure the thermodynamic parameters of metal binding to purified HQ3622A protein, including binding affinity (Kd), stoichiometry, enthalpy, and entropy changes.

• Fluorescence Spectroscopy: If HQ3622A contains tryptophan residues near potential metal binding sites, intrinsic fluorescence changes upon metal binding can be monitored. Alternatively, fluorescent metal probes or protein labeling can be used.

• Circular Dichroism (CD): This can detect conformational changes in secondary structure elements upon metal binding.

• Inductively Coupled Plasma Mass Spectrometry (ICP-MS): For quantitative analysis of metal content in purified protein samples.

• Site-Directed Mutagenesis: Based on sequence analysis, potential metal-coordinating residues can be mutated to assess their contribution to binding. Similar to the approach used for BbZIP where researchers introduced mutations to study the effects on metal binding .

• Sandwich ELISA: A method described for BbZIP could potentially be adapted for HQ3622A . This approach uses conformation-specific antibodies to probe metal-induced conformational changes.

• Stability Assays: Differential scanning fluorimetry or thermal shift assays to measure changes in protein stability upon metal binding.

Given the halophilic nature of Haloquadratum walsbyi, these experiments should be designed with consideration for the high salt requirements that might be necessary for proper protein folding and function.

How do the genomic features of Haloquadratum walsbyi affect the expression and function of HQ3622A?

The genomic context of HQ3622A within Haloquadratum walsbyi provides important insights into its expression regulation and potential functional relationships. Haloquadratum walsbyi has several notable genomic features that could influence HQ3622A:

• Genomic Conservation: The genome of Haloquadratum walsbyi shows remarkable conservation between geographically distant isolates, with 84% sequence similarity and 98.6% identity in the core sequence . This suggests that essential functions, potentially including metal transport systems like HQ3622A, may be well preserved across different strains.

• Synteny and Gene Organization: The open reading frames (ORFs) in the shared sequence between different Haloquadratum walsbyi strains are completely syntenic (conserved in genomic orientation and order) without inversions or rearrangements . If HQ3622A is located within this conserved region, its genomic context and potential operon structure would be preserved, suggesting consistent expression regulation across strains.

• Mobile Genetic Elements: Haloquadratum walsbyi contains numerous types of mobile genetic elements, most of which appear to be active . These elements could potentially influence the expression of HQ3622A if they are inserted near its genomic locus, either by disrupting regulatory elements or introducing new ones.

• Strain-Specific Insertions/Deletions: The genome analysis revealed strain-specific insertions/deletions, often associated with short direct repeats (4-20 bp) . Such genetic variations could affect HQ3622A expression if they occur in regulatory regions or alter the sequence of the gene itself.

• Deletion-Coupled Insertions: These have been observed to produce different sequences at identical genomic positions . If such events occurred within or near the HQ3622A gene, they could result in variant forms of the protein with potentially altered function.

What are the challenges in expressing and purifying functional HQ3622A protein?

Expressing and purifying functional HQ3622A protein presents several challenges that researchers should consider:

• Halophilic Adaptation: As a protein from a halophile requiring >14% salt for growth , HQ3622A likely has adapted to function in high salt environments. This adaptation often involves an abundance of acidic residues on the protein surface, which can cause folding issues in standard expression systems with lower salt concentrations.

• Membrane Protein Expression: If HQ3622A is an integral membrane protein, as suggested by its classification as a metal transport protein, it presents the typical challenges of membrane protein expression, including potential toxicity to host cells, inclusion body formation, and difficulties in extraction and purification.

• Expression System Selection: While E. coli has been successfully used to express recombinant HQ3622A with an N-terminal His tag , optimal expression might require specialized strains or conditions. Alternative expression systems (yeast, insect cells) might be necessary if functional yields in E. coli are insufficient.

• Protein Stability: The search results indicate that repeated freeze-thaw cycles should be avoided for the purified protein , suggesting stability issues that could complicate experimental work.

• Proper Folding: Ensuring that recombinantly expressed HQ3622A adopts its native conformation is crucial for functional studies. This might require optimization of expression conditions, including temperature, induction parameters, and host cell chaperone co-expression.

• Metal Content Control: For a metal transport protein, controlling metal content during expression and purification is essential to obtain homogeneous protein preparations. Metal chelators or specific metal supplementation might be necessary.

What is the optimal protocol for reconstituting recombinant HQ3622A protein?

Based on the information provided in the search results, the optimal protocol for reconstituting recombinant HQ3622A protein is as follows:

• Initial Preparation:

  • Briefly centrifuge the vial containing lyophilized HQ3622A protein to bring the contents to the bottom .

• Reconstitution:

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

• Storage Preparation:

  • Add glycerol to a final concentration of 5-50% (with 50% being the default recommendation) .

  • Aliquot the solution for long-term storage to avoid repeated freeze-thaw cycles .

• Storage Conditions:

  • Store working aliquots at 4°C for up to one week .

  • For long-term storage, keep aliquots at -20°C or -80°C .

  • Avoid repeated freeze-thaw cycles as they can compromise protein integrity .

• Handling Considerations:

  • The protein is supplied in a Tris/PBS-based buffer containing 6% Trehalose at pH 8.0 .

  • For experiments requiring different buffer conditions, consider dialysis or buffer exchange methods.

• Functional Verification (recommended addition to basic protocol):

  • After reconstitution, verify protein integrity by SDS-PAGE (expected purity >90%) .

  • For functional studies, consider an initial assessment of metal binding capacity.

This protocol provides a starting point for working with recombinant HQ3622A protein. Researchers may need to optimize specific conditions based on their experimental requirements and the particular behavior of their protein preparation.

How can researchers effectively design experiments to determine the metal binding specificity of HQ3622A?

Designing experiments to determine the metal binding specificity of HQ3622A requires a systematic approach that examines various metal ions under controlled conditions. Based on insights from the study of other metal transporters like BbZIP , here is a comprehensive experimental strategy:

• Preliminary Metal Screening:

  • Test a panel of biologically relevant divalent metals (Zn²⁺, Cd²⁺, Cu²⁺, Fe²⁺, Ni²⁺, Co²⁺, Mn²⁺) using a fluorescence-based or colorimetric assay.

  • Include monovalent (Na⁺, K⁺) and trivalent (Fe³⁺) ions as controls to confirm specificity for divalent metals.

• Quantitative Binding Assays:

  • For metals showing positive results in the screening, perform Isothermal Titration Calorimetry (ITC) to determine binding affinities (Kd values).

  • Compare the binding constants to identify preferential metal substrates.

  • Example data format for presenting results:

• Competitive Binding Assays:

  • Perform competition experiments where one metal is used to displace another.

  • This can reveal preferential binding hierarchies among metals that bind to the same site.

• Conformational Change Assessment:

  • Adapt the sandwich ELISA approach used for BbZIP to determine if metal binding induces conformational changes in HQ3622A.

  • If applicable, introduce a cysteine residue at a strategic position to serve as a conformation indicator, similar to the L200C variant in BbZIP .

• Site-Directed Mutagenesis:

  • Identify potential metal-coordinating residues in HQ3622A based on sequence analysis.

  • Create mutants where these residues are replaced (e.g., with alanine or lysine, similar to the D208K and E240K mutations studied in BbZIP ).

  • Measure the impact on metal binding affinity and specificity.

What techniques can be used to study the structural changes in HQ3622A during metal transport?

Multiple complementary techniques can be employed to study the structural changes in HQ3622A during metal transport, drawing inspiration from approaches used for other transporters like BbZIP :

• Sandwich ELISA for Conformational Change Detection:

  • Develop a sandwich ELISA using conformation-specific antibodies, similar to the approach used for BbZIP .

  • This technique can detect metal-induced conformational changes in the native membrane environment.

  • The assay can quantitatively measure binding affinities for different metals by analyzing the dose-dependent conformational response.

• Cysteine Accessibility Assays:

  • Introduce cysteine residues at strategic positions in HQ3622A to serve as reporters of local environments.

  • Use membrane-impermeable sulfhydryl reagents to probe the accessibility of these cysteines in different conformational states.

  • This approach successfully identified L200 in BbZIP as a residue that changes from buried to exposed during conformational transitions .

• FRET-Based Approaches:

  • Introduce fluorophore pairs at key positions to monitor distance changes upon metal binding.

  • This can provide real-time information about conformational dynamics during the transport cycle.

• EPR Spectroscopy:

  • Spin-label specific residues and use EPR to monitor local environment changes.

  • Particularly useful for detecting changes in mobility or solvent exposure of specific protein regions.

• Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS):

  • Compare the rate of hydrogen-deuterium exchange in the presence and absence of metals.

  • Regions undergoing conformational changes typically show altered exchange rates.

• Limited Proteolysis:

  • Expose the protein to proteases in different conformational states.

  • Changes in proteolytic patterns can reveal regions that become more or less accessible.

• Structural Biology Approaches:

  • Cryo-EM studies of HQ3622A in different conformational states (metal-bound, metal-free).

  • X-ray crystallography of the protein captured in different conformations.

How can researchers compare HQ3622A with other metal transporters in evolutionary studies?

Comparing HQ3622A with other metal transporters in evolutionary studies requires a multifaceted approach that integrates sequence analysis, structural comparisons, functional characterization, and genomic context. Here's a comprehensive methodology:

• Sequence-Based Phylogenetic Analysis:

  • Construct multiple sequence alignments of HQ3622A with diverse metal transporters, including ZIP family proteins like BbZIP .

  • Generate phylogenetic trees using maximum likelihood, Bayesian, or distance-based methods.

  • Identify key conserved residues and motifs across evolutionary lineages.

  • Example phylogenetic approach:

    • Collect homologs using BLAST or HMM searches against diverse genomic databases

    • Align sequences using MUSCLE, MAFFT, or T-Coffee algorithms

    • Trim alignments to remove poorly aligned regions

    • Construct trees using appropriate evolutionary models

• Comparative Structural Analysis:

  • If structural data is available, compare the fold and metal binding sites of HQ3622A with other transporters.

  • For proteins lacking experimental structures, use homology modeling to predict structures based on related transporters.

  • Analyze conservation patterns by mapping sequence conservation onto structural models.

• Functional Comparative Studies:

  • Compare metal binding specificities and affinities across different transporters.

  • Examine conservation of transport mechanisms (e.g., elevator transport as observed in BbZIP ).

  • Create chimeric proteins by swapping domains between HQ3622A and other transporters to identify functionally equivalent regions.

• Genomic Context Analysis:

  • Compare the genomic neighborhood of HQ3622A in Haloquadratum walsbyi with those of related transporters in other organisms.

  • Identify conserved gene clusters that might indicate functional associations.

  • Analyze the presence of regulatory elements that have been conserved or diverged.

• Adaptation to Extreme Environments:

  • Examine specific adaptations of HQ3622A to the hypersaline environment of Haloquadratum walsbyi.

  • Compare these adaptations with those of metal transporters from other extremophiles.

  • Analyze amino acid composition biases characteristic of halophilic proteins (e.g., enrichment in acidic residues).