Recombinant Photobacterium profundum Na (+)-translocating NADH-quinone reductase subunit D (nqrD)
Description
Photobacterium profundum: Organism Background
Photobacterium profundum is a deep-sea Gammaproteobacterium belonging to the family Vibrionaceae, a marine bacterial group with significant ecological and biotechnological importance. This gram-negative rod-shaped bacterium has remarkable adaptive capabilities that allow it to thrive in extreme marine environments . The organism possesses two circular chromosomes, a characteristic shared with other members of its genus . With cell dimensions ranging from 2-4μm in length and 0.8-1.0μm in width, P. profundum exhibits a single unsheathed flagellum for motility in its deep-sea habitat .
The bacterium demonstrates extraordinary adaptability to varying environmental conditions, with growth capabilities spanning temperatures from 0°C to 25°C and pressures from 0.1 MPa (atmospheric pressure) to an impressive 70 MPa, depending on the strain . This remarkable pressure tolerance classifies certain strains as piezophiles—organisms that grow optimally under high hydrostatic pressure conditions. The organism requires salt for survival and can metabolize a wide variety of simple and complex carbohydrates, making it metabolically versatile .
Strain Diversity and Growth Characteristics
Several strains of P. profundum have been identified, each with distinct optimal growth conditions that reflect adaptation to specific marine ecological niches. The most extensively studied strain, SS9, demonstrates optimal growth at 15°C and 28 MPa, classifying it as both a psychrophile (cold-loving) and a piezophile (pressure-loving) . Other notable strains include 3TCK (isolated from San Diego Bay), which grows optimally at 9°C and atmospheric pressure (0.1 MPa), and strain DSJ4 (isolated from the Ryukyu Trench at a depth of 5110m), which prefers 10°C and 10 MPa conditions .
Research has demonstrated a fascinating relationship between osmotic and hydrostatic pressure responses in P. profundum SS9. The bacterium shows a dramatic decrease in growth at NaCl concentrations below 200 mM, regardless of hydrostatic pressure conditions . This suggests a fundamental salt requirement that is independent of pressure adaptation mechanisms. When comparing growth at atmospheric pressure (0.1 MPa) versus optimal pressure (28 MPa), the bacterium displays similar growth rates at intermediate salt concentrations (200-400 mM NaCl), but exhibits inhibited growth at higher salt concentrations when under elevated pressure .
Table 1: Growth Characteristics of P. profundum Strains
| Strain | Optimal Temperature | Optimal Pressure | Isolation Source |
|---|---|---|---|
| SS9 | 15°C | 28 MPa | Sulu Sea (2.5 km depth) |
| 3TCK | 9°C | 0.1 MPa | San Diego Bay |
| DSJ4 | 10°C | 10 MPa | Ryukyu Trench (5.11 km depth) |
| 1230 | Variable | Variable | Marine environment |
Na(+)-translocating NADH-quinone reductase (NQR) Complex
The Na(+)-translocating NADH:quinone oxidoreductase (NQR) represents a sophisticated membrane protein complex that serves a critical function in the respiratory chain of numerous marine and pathogenic bacteria, including P. profundum and its close relative Vibrio cholerae . This enzyme complex catalyzes the electron transfer from NADH to ubiquinone, coupling this redox reaction with the translocation of sodium ions across the bacterial membrane . This process is fundamental to energy conservation and maintenance of ion gradients in these organisms.
The NQR complex consists of six distinct subunits designated NqrA through NqrF, which are encoded by consecutive structural genes in the bacterial genome . These subunits work in concert to facilitate electron transport and ion translocation. The peripheral subunit NqrF initiates the electron transport process by catalyzing NADH oxidation, utilizing a flavin adenine dinucleotide (FAD) and a 2Fe-2S cluster as essential cofactors . Subunits NqrB and NqrC each contain a covalently attached flavin mononucleotide (FMN), which requires the flavin insertase ApbE for proper attachment .
Role in Bacterial Metabolism and Adaptation
Recent research has highlighted the influence of the NQR complex on iron metabolism in bacteria, suggesting its significance beyond mere energy production . This connection to iron metabolism positions the NQR complex as a potential drug target for antibiotics, particularly against pathogenic bacterial species . For extremophiles like P. profundum, which must adapt to challenging environmental conditions, the NQR complex may play additional roles in maintaining cellular homeostasis under varying pressure and salt conditions.
Recombinant Production and Characteristics
The recombinant form of P. profundum NqrD has been successfully expressed in Escherichia coli expression systems with an N-terminal histidine tag to facilitate purification . This approach yields a highly pure protein (>90% as determined by SDS-PAGE) that can be used for various biochemical and structural studies . The recombinant protein is typically supplied as a lyophilized powder, which requires reconstitution in deionized sterile water to a concentration of 0.1-1.0 mg/mL for experimental use .
Table 2: Physical and Biochemical Properties of Recombinant P. profundum NqrD
| Property | Characteristic |
|---|---|
| Amino acid length | 210 amino acids |
| Molecular weight | Approximately 23 kDa (estimated) |
| Tag | N-terminal His-tag |
| Expression system | E. coli |
| Form | Lyophilized powder |
| Purity | >90% by SDS-PAGE |
| Storage buffer | Tris/PBS-based buffer, 6% Trehalose, pH 8.0 |
| Reconstitution recommendation | Deionized sterile water (0.1-1.0 mg/mL) |
| Storage conditions | -20°C/-80°C, avoid repeated freeze-thaw cycles |
Electron Transport Function
Within the electron transport chain of the NQR complex, NqrD works in conjunction with NqrE to form an iron-binding domain within the membrane portion of the complex . This iron center serves as an electron carrier, accepting electrons from upstream components and transferring them to subsequent acceptors in the respiratory pathway. The precise arrangement of this iron center and its coordination within the protein structure is crucial for efficient electron flow through the complex.
Sodium Translocation Mechanism
The primary function of the NQR complex is to couple electron transport with the translocation of sodium ions across the bacterial membrane, generating an electrochemical gradient that can drive various cellular processes. The NqrD subunit likely contributes to forming the sodium transport pathway through the membrane, with its transmembrane domains creating portions of the channel through which sodium ions are translocated. This process is energetically coupled to the redox reactions occurring within the complex.
Research Applications
The availability of recombinant NqrD enables various research applications, including:
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Structural studies to determine three-dimensional protein configurations
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Functional assays to assess electron transport capabilities
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Binding studies to identify potential inhibitor compounds
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Crystallography attempts to resolve atomic-level structures
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Antibody production for immunological detection methods
Potential as Antimicrobial Target
The NQR complex has been identified as a potential drug target for antibiotics due to its influence on iron metabolism and its essential role in bacterial respiration . As a component of this complex, NqrD represents a potential target for antimicrobial development, particularly against marine pathogens and related organisms like Vibrio cholerae. The structural and functional characterization of NqrD could therefore contribute to novel therapeutic approaches against bacterial infections.
Adaptation to Extreme Environments
The NQR complex, including the NqrD subunit, may play a role in the adaptation of P. profundum to extreme pressure and temperature conditions in its deep-sea habitat. The ability of P. profundum strains to grow under varying pressure conditions (from atmospheric to 70 MPa) suggests specialized adaptations in membrane-associated proteins like NqrD .
Research has shown that P. profundum modifies its cell membrane composition in response to pressure and temperature changes, increasing the abundance of mono- and polyunsaturated fatty acids at low temperature and high pressure to maintain membrane fluidity . As a membrane-embedded protein, NqrD must function effectively within this dynamic membrane environment, suggesting potential structural adaptations that enable operation under extreme conditions.
The observed relationship between osmotic and hydrostatic pressure responses in P. profundum further implies that membrane-associated systems like the NQR complex may contribute to the organism's remarkable adaptability . Understanding these adaptation mechanisms could provide insights into bacterial evolution and survival strategies in extreme environments.
Product Specs
Note: We will prioritize shipping the format we have in stock. However, if you have a specific format requirement, please indicate it in your order notes. We will prepare the product according to your request.
Note: All our proteins are shipped with standard blue ice packs. If you require dry ice shipping, please notify us in advance as additional fees will apply.
Generally, the shelf life of the liquid form is 6 months at -20°C/-80°C. For the lyophilized form, the shelf life is 12 months at -20°C/-80°C.
Target Background
KEGG: ppr:PBPRA0826
STRING: 298386.PBPRA0826
Q&A
What is the biological significance of Na(+)-translocating NADH-quinone reductase in Photobacterium profundum?
The Na(+)-translocating NADH-quinone reductase (NQR) complex plays a critical role in the bioenergetics of Photobacterium profundum, particularly as an adaptation to its deep-sea environment. P. profundum is a marine Gammaproteobacterium belonging to the Vibrionaceae family that has been isolated from deep ocean environments, including the Sulu Sea . As a psychrophile and piezophile (strain SS9 has optimal growth at 15°C and 28 MPa), this organism has developed specialized membrane proteins to maintain cellular function under high pressure conditions .
The NQR complex, of which nqrD is a subunit, functions as a primary sodium pump in the respiratory chain, converting the energy from NADH oxidation into an electrochemical sodium gradient across the cell membrane. This gradient is subsequently utilized for various cellular processes, including ATP synthesis, nutrient transport, and flagellar rotation. The ability to use a sodium motive force rather than a proton motive force represents an important adaptation for survival in extreme deep-sea environments with high pressure and low temperature.
How does the structure of nqrD contribute to the function of the NQR complex?
The nqrD subunit is one of six subunits (NqrA-F) that form the complete NQR complex. The structural analysis reveals that nqrD is a hydrophobic membrane protein containing transmembrane helices that participate in forming the sodium translocation pathway. Research indicates that nqrD, together with nqrB and nqrE, constitutes the membrane domain of the complex that is directly involved in Na+ transport.
Key structural features of nqrD include:
| Structural Element | Description | Functional Significance |
|---|---|---|
| Transmembrane helices | 3-4 membrane-spanning α-helical segments | Forms part of the Na+ channel |
| Conserved acidic residues | Asp and Glu residues in transmembrane regions | Potentially involved in Na+ binding and transport |
| Interaction domains | Protein-protein interaction surfaces | Enables assembly with other NQR subunits |
| Cofactor binding sites | Regions that may interact with redox cofactors | Participates in electron transfer pathway |
The precise arrangement of these structural elements enables nqrD to participate in the coordinated conformational changes that couple electron transfer to sodium translocation.
What are the optimal conditions for expressing recombinant P. profundum nqrD in heterologous systems?
Expression of recombinant P. profundum nqrD presents significant challenges due to its hydrophobic nature and membrane localization. Based on experimental data and the organism's native growth conditions, the following protocol has been optimized:
Expression System Selection:
E. coli C41(DE3) or C43(DE3) strains are recommended as they are engineered specifically for membrane protein expression. These strains contain mutations that prevent cell death associated with toxic membrane protein overexpression.
Expression Conditions:
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Growth temperature: 15-20°C (reflecting P. profundum's psychrophilic nature)
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Induction: 0.1-0.5 mM IPTG at OD600 of 0.6-0.8
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Post-induction growth: 16-24 hours at reduced temperature (15°C)
Like other P. profundum proteins, nqrD expression may be affected by pressure and temperature stress response pathways. Research has shown that several stress response genes in P. profundum strain SS9 (including htpG, dnaK, dnaJ, and groEL) are upregulated in response to atmospheric pressure . Co-expression of these chaperones may improve folding and stability of recombinant nqrD.
What experimental design is most appropriate for studying nqrD function in vitro?
A Completely Randomized Design (CRD) is often suitable for initial in vitro studies of nqrD function, particularly when working with purified protein preparations where experimental units can be made relatively homogeneous . The CRD approach allows for flexible comparison of multiple experimental conditions.
CRD Implementation for nqrD Functional Studies:
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Determine the total number of experimental units required based on treatments and replications .
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Assign treatments (e.g., different substrate concentrations, inhibitors, or pH conditions) completely at random to ensure each unit has an equal chance of receiving any treatment .
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Ensure homogeneity among experimental units (e.g., protein preparation, buffer conditions) .
The statistical model for CRD with one observation per unit is:
Yij = m + ti + eij
Where:
For more complex in vivo studies or when dealing with inherent variability, other designs such as randomized block design may be more appropriate.
How can Q-methodology be applied to systematically analyze contradictory results in nqrD research?
When faced with conflicting findings in nqrD research literature, Q-methodology offers a structured approach to systematically analyze subjective viewpoints within the scientific community . This hybrid qualitative-quantitative method can help identify patterns in how researchers interpret experimental results and approach methodological challenges.
Implementation Steps:
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Collection of statements (Q-set): Gather diverse perspectives on nqrD function, methodology, and interpretation from published literature.
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Participant selection (P-set): Recruit researchers with expertise in membrane proteins, bioenergetics, or Photobacterium physiology.
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Q-sorting: Ask participants to rank statements based on their agreement/disagreement.
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Factor analysis: Apply statistical techniques to identify shared viewpoints and areas of consensus/disagreement.
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Interpretation: Analyze emergent factors to reveal underlying patterns in research approaches or interpretations .
Q-methodology is particularly valuable when research communities face interpretive challenges, as it can "reveal connections between accounts that other techniques may overlook" . For nqrD research, this approach could illuminate why certain experimental protocols yield contradictory results or identify implicit theoretical assumptions that influence data interpretation.
How can anti-pattern analysis be used to detect inconsistencies in computational models of nqrD function?
When developing computational models of nqrD function or integrating nqrD data into larger knowledge graphs, researchers may encounter logical inconsistencies or contradictions. Anti-pattern analysis provides a framework for detecting and resolving these inconsistencies.
Anti-patterns represent generalized structures of contradictions that may appear in knowledge representations . For nqrD research, common anti-patterns might include:
| Anti-Pattern Type | Description | Example in nqrD Research |
|---|---|---|
| Disjoint Class Violation | Assigning an entity to classes defined as disjoint | Classifying nqrD as both a peripheral and integral membrane protein |
| Functional Property Violation | Assigning multiple values to a property defined as functional | Multiple contradictory values for nqrD redox potential |
| Domain/Range Constraint Violation | Using properties with entities outside their defined domain/range | Applying electron transfer metrics to non-redox regions of nqrD |
| Cardinality Constraint Violation | Exceeding allowed number of relations | Specifying too many transmembrane helices for nqrD |
The detection of these anti-patterns follows a structured approach:
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Extract justifications for contradictions in the knowledge base
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Generalize these justifications into anti-patterns
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Classify detected anti-patterns to identify systematic modeling errors
This method is scalable even for large datasets and can help identify recurring errors in computational representations of nqrD function or structure.
What statistical approaches are most appropriate for analyzing pressure-dependent changes in nqrD expression?
Given that P. profundum is a piezophile with distinct adaptations to high-pressure environments, researchers often investigate how pressure affects nqrD expression and function. Statistical analysis of such data requires careful consideration of experimental design and data characteristics.
For comparing nqrD expression levels across different pressure conditions, analysis of variance (ANOVA) following a completely randomized design is appropriate if the assumptions of normality and homogeneity of variance are met . The ANOVA model allows researchers to determine if observed differences in nqrD expression between pressure treatments are statistically significant.
| Source of Variation | Degrees of Freedom | Sum of Squares | Mean Square | F-value |
|---|---|---|---|---|
| Treatments (Pressure levels) | t-1 | TrSS | TrMS | TrMS/EMS |
| Error | n-t | ESS | EMS | |
| Total | n-1 | TSS |
Where:
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t = number of treatments (pressure levels)
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n = total number of observations
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TrSS = treatment sum of squares
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ESS = error sum of squares
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TSS = total sum of squares
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TrMS = treatment mean square
If the calculated F-value exceeds the critical F-value at the chosen significance level, researchers can reject the null hypothesis and conclude that pressure significantly affects nqrD expression. Post-hoc tests (e.g., Tukey's HSD) can then identify which specific pressure conditions differ significantly from each other.
How should researchers address contradictions in experimental data on nqrD function?
Contradictory results regarding nqrD function are not uncommon in the scientific literature, particularly when comparing results across different experimental systems or conditions. A systematic approach to handling such contradictions involves:
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Justification Retrieval: Identify the minimal set of experimental conditions or assumptions that lead to each contradictory result . This process, analogous to justification retrieval in knowledge graphs, helps isolate the specific factors contributing to inconsistency.
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Anti-pattern Identification: Generalize these justifications into patterns that might explain the contradictions . For example, differences in lipid composition of expression systems might systematically affect nqrD activity measurements.
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Methodological Reconciliation: Design experiments specifically to test hypotheses about why contradictions occur. This might involve systematically varying conditions identified in the anti-pattern analysis.
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Meta-analysis Approaches: When sufficient data exists across multiple studies, formal meta-analysis techniques can help quantify heterogeneity and identify moderating variables that explain apparently contradictory results.
This structured approach transforms contradictions from frustrations into research opportunities, potentially revealing important context-dependencies in nqrD function that might have biological significance.
What methodological innovations might advance understanding of nqrD's role in pressure adaptation?
Future research on nqrD's role in pressure adaptation could benefit from several methodological innovations:
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High-Pressure Protein Expression Systems: Development of expression systems that can operate under high pressure conditions would allow researchers to study nqrD folding and assembly under native-like conditions. This approach would build on our understanding of P. profundum's natural pressure adaptations, where strain SS9 shows optimal growth at 28 MPa .
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Pressure-Modulated Structural Analysis: Techniques that allow structural characterization under varying pressure conditions, such as high-pressure NMR or pressure-modulated X-ray crystallography, could reveal conformational changes in nqrD that might be critical for its function in deep-sea environments.
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Comparative Functional Genomics: Systematic comparison of nqrD sequences and expression patterns across P. profundum strains adapted to different pressures (such as strains SS9, 3TCK, and DSJ4, which have different optimal growth pressures ) could identify specific amino acid changes or regulatory mechanisms associated with pressure adaptation.
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Integration of Stress Response Networks: Given that P. profundum upregulates several stress response genes (htpG, dnaK, dnaJ, and groEL) in response to pressure changes , future research could explore how these chaperone systems interact with nqrD folding and function.
These methodological approaches would help address fundamental questions about how membrane proteins adapt to extreme environmental conditions while maintaining essential bioenergetic functions.
