Recombinant Gloeobacter violaceus NAD (P)H-quinone oxidoreductase subunit M (ndhM)

What is Gloeobacter violaceus and why is it significant for evolutionary studies?

Gloeobacter violaceus is a primitive cyanobacterium that occupies a unique evolutionary position among photosynthetic organisms. Its most distinguishing feature is the complete absence of thylakoid membranes, which are otherwise present in all other oxygenic photosynthetic organisms . This makes G. violaceus an invaluable model for studying early evolution of photosynthetic mechanisms.

The genome of G. violaceus PCC 7421 is approximately 4.66 Mbp in size, while recent discoveries have expanded our understanding of the Gloeobacter genus with additional species including G. kilaueensis (4.72 Mbp) and the newly characterized G. morelensis (4.92 Mbp), which was isolated from a waterfall cave in Mexico . The G. morelensis genome is currently the largest among completely sequenced Gloeobacter species.

Average nucleotide identity (ANI) analyses have shown that G. morelensis shares only 92.6% identity with G. violaceus PCC 7421 despite having 99.93% 16S rRNA gene sequence identity, confirming its status as a distinct species . These genomic differences underscore the surprising diversity within this evolutionarily significant genus.

What is NAD(P)H-quinone oxidoreductase and what role does the ndhM subunit play?

NAD(P)H-quinone oxidoreductase (NDH-1) is a multi-subunit enzyme complex that catalyzes electron transfer from NAD(P)H to quinones in the respiratory and photosynthetic electron transport chains. This complex is analogous to Complex I (NADH:ubiquinone oxidoreductase) in mitochondria but has evolved additional subunits and functions in cyanobacteria.

The subunit M (ndhM) is a component of the NDH-1 complex that contributes to its stability and function. In cyanobacteria, NDH-1 complexes participate in several crucial processes:

• Cyclic electron flow around Photosystem I

• Respiratory electron transport

• CO₂ uptake mechanisms

• Stress responses

In G. violaceus, which lacks thylakoid membranes, NDH-1 complexes must be organized differently compared to other cyanobacteria, potentially giving ndhM unique properties or localization patterns. This makes the study of G. violaceus ndhM particularly valuable for understanding how electron transport chains functioned in early photosynthetic organisms before the evolution of thylakoid membranes.

How does G. violaceus' lack of thylakoid membranes affect ndhM function?

The absence of thylakoid membranes in G. violaceus represents a fundamental difference in cellular organization compared to all other known oxygenic photosynthetic organisms . This unique characteristic has significant implications for ndhM function:

• Altered subcellular localization: In typical cyanobacteria, NDH-1 complexes are primarily located in thylakoid membranes. In G. violaceus, these complexes must be integrated into the cytoplasmic membrane or organized in specialized membrane regions.

• Modified electron transport pathways: Without separate thylakoid compartments, the electron transport chains in G. violaceus likely operate with different spatial arrangements and possibly altered mechanisms.

• Unique protein-protein interactions: The interaction partners of ndhM in G. violaceus may differ from those in thylakoid-containing cyanobacteria, potentially resulting in specialized complex assemblies.

• Distinct regulatory mechanisms: The regulation of NDH-1 complex assembly and activity likely involves different signaling pathways compared to other cyanobacteria.

• Environmental response adaptations: The response of NDH-1 complexes to environmental stresses may have evolved unique characteristics in G. violaceus due to its distinct cellular organization.

This fundamental difference in cellular architecture makes G. violaceus ndhM an excellent subject for comparative studies to understand the evolution of photosynthetic and respiratory electron transport systems.

What genomic insights do we have about ndhM in Gloeobacter species?

Genomic analyses have provided valuable insights into the evolution and diversity of Gloeobacter species, which inform our understanding of ndhM:

Pangenomic comparisons revealed that G. morelensis encodes 759 unique genes not shared with other Gloeobacter species, highlighting the significant genetic diversity within this genus . While the specific details about ndhM weren't directly addressed in the search results, we can infer that proteins involved in electron transport chains, including ndhM, may show important variations across Gloeobacter species.

The Gloeobacter lineage represents one of the earliest branches of cyanobacterial evolution, with recent research identifying at least two major branches within Gloeobacterales . The complete genome of G. morelensis consists of one circular chromosome (4,921,229 bp), one linear plasmid (172,328 bp), and one circular plasmid (8,839 bp) . This genomic information provides the foundation for understanding the evolutionary context of ndhM.

Comparative genomic analyses between G. violaceus PCC 7421, G. kilaueensis, and G. morelensis can reveal how genes encoding components of electron transport chains, including ndhM, have evolved in this early-branching cyanobacterial lineage.

What is known about light-sensing mechanisms in Gloeobacter and their relationship to electron transport?

Gloeobacter species exhibit unique light-sensing mechanisms that may interact with electron transport systems like those involving ndhM:

G. violaceus PCC 7421 contains a single cyanobacteriochrome (CBCR) of the DXCF type, which does not appear to be ancestral but may have been acquired through horizontal gene transfer . In contrast, CBCRs are completely absent in G. kilaueensis. This pattern suggests complex evolutionary dynamics of light-sensing mechanisms within the Gloeobacter lineage.

Recent research has identified characteristic lineages of DXCF and XRG CBCRs in Gloeobacterales associated with conserved photoreceptors, including a candidate phototaxis locus . These findings provide evidence for early evolution of CBCRs relative to cyanobacterial diversification.

Interestingly, some early CBCR lineages can integrate both light and pH cues, in contrast to other CBCR types . This dual-sensing capability could be particularly relevant in the context of electron transport chains, which often generate pH gradients across membranes.

Understanding these light-sensing mechanisms may provide insights into how electron transport components like ndhM are regulated in response to environmental conditions in these primitive cyanobacteria.

What expression systems are most effective for producing recombinant G. violaceus ndhM?

Selecting an appropriate expression system is crucial for obtaining functional recombinant ndhM from G. violaceus. Based on the properties of membrane-associated proteins and the unique characteristics of Gloeobacter proteins, several expression systems warrant consideration:

Escherichia coli-based systems:

• BL21(DE3) strains offer high yield but may result in inclusion bodies for membrane-associated proteins

• C41(DE3) and C43(DE3) strains are engineered specifically for membrane protein expression

• ArcticExpress strains allow low-temperature expression (10-13°C), reducing aggregation

• SHuffle strains facilitate proper disulfide bond formation in the cytoplasm

Yeast expression systems:

• Pichia pastoris provides eukaryotic post-translational modifications and secretion capabilities

• Saccharomyces cerevisiae offers membrane environments that may better accommodate membrane-associated proteins

Specialized expression approaches:

• Cell-free expression systems allow direct manipulation of the reaction environment

• Cyanobacterial hosts like Synechocystis sp. PCC 6803 provide a more native-like environment

For recombinant ndhM production, an effective strategy would be to start with C41(DE3) E. coli expression at 18-20°C with a solubility-enhancing tag like MBP or SUMO, and optimize induction conditions (IPTG concentration, induction time) to maximize yield of soluble, functional protein.

What purification strategies yield highest purity and activity of recombinant ndhM?

Purifying recombinant ndhM from G. violaceus requires careful consideration of its membrane association and enzymatic activity. The following comprehensive purification strategy addresses these challenges:

Initial extraction considerations:

• Buffer composition: 50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 1-5 mM DTT

• Detergent selection: Start with mild detergents like 0.5-1% n-dodecyl β-D-maltoside (DDM)

• Protease inhibitors: Complete protease inhibitor cocktail to prevent degradation

• Gentle cell disruption: Avoid excessive heating during lysis (sonication with cooling cycles)

Multi-step purification approach:

• Affinity chromatography: IMAC (Ni-NTA) for His-tagged constructs or amylose resin for MBP fusions

• Tag removal: Precision protease cleavage (if tag affects function)

• Ion exchange chromatography: Based on calculated pI of ndhM

• Size exclusion chromatography: Final polishing step, also enables buffer exchange

Activity preservation strategies:

• Maintain reducing conditions throughout purification

• Include stabilizing agents (glycerol, specific lipids) in all buffers

• Monitor protein activity at each purification step

• Keep temperature low (4°C) during all procedures

• Consider including natural substrates or substrate analogs

A crucial consideration for ndhM is that removal of the solubility tag may lead to aggregation if the native protein has poor solubility. In such cases, retaining the tag for functional studies or screening detergent conditions may be necessary to maintain protein stability and activity.

How can researchers verify the functional integrity of recombinant ndhM?

Verifying the functional integrity of recombinant ndhM from G. violaceus requires a multi-faceted approach that examines both structural integrity and enzymatic activity:

Primary activity assays:

• NADH/NADPH oxidation: Monitor decrease in absorbance at 340 nm

• Quinone reduction: Track changes in quinone absorbance (wavelength depends on specific quinone)

• Artificial electron acceptor assays: Using DCPIP (2,6-dichlorophenolindophenol) or ferricyanide

Structural integrity verification:

• Circular dichroism (CD) spectroscopy: Assess secondary structure composition

• Thermal shift assays: Determine protein stability (melting temperature)

• Limited proteolysis: Probe for correctly folded domains

• Dynamic light scattering: Check for monodispersity

Functional complex formation:

• Pull-down assays with other NDH-1 components

• Blue native PAGE to assess complex assembly

• Analytical size exclusion chromatography to determine oligomeric state

Substrate binding studies:

• Isothermal titration calorimetry (ITC) for binding thermodynamics

• Microscale thermophoresis (MST) for binding affinities

• Fluorescence-based binding assays if applicable

Since G. violaceus lacks thylakoid membranes, the functional verification of its ndhM should consider this unique cellular context. Comparisons with ndhM from thylakoid-containing cyanobacteria could reveal adaptations specific to the Gloeobacter lineage. Additionally, reconstitution experiments using liposomes or nanodiscs may provide insights into how the membrane environment affects ndhM function.

What experimental design considerations are important when studying ndhM function?

When designing experiments to study ndhM function from G. violaceus, researchers should consider several factors that can significantly impact results:

Buffer composition optimization:

• pH range: Test pH 6.5-8.5 to determine optimal conditions

• Ionic strength: Typically 50-200 mM NaCl or KCl

• Divalent cations: Include 1-5 mM Mg²⁺ or Mn²⁺

• Reducing agents: 1-5 mM DTT or β-mercaptoethanol

• Stabilizers: 10-20% glycerol to enhance protein stability

Experimental variables to control:

• Temperature: Test range between 20-40°C (consider G. violaceus' natural habitat)

• Light conditions: Some cyanobacterial enzymes show light-dependent activity

• Oxygen sensitivity: Consider anaerobic conditions if necessary

• Protein concentration: Test multiple concentrations to ensure linearity

• Reaction time: Ensure measurements are taken during linear phase

Substrate considerations:

• NADH vs. NADPH preference: Test both cofactors

• Quinone selection: Natural (plastoquinone) vs. synthetic (decylubiquinone)

• Alternative electron acceptors for in vitro assays

Experimental designs for specific research questions:

As G. violaceus represents an early-branching cyanobacterial lineage lacking thylakoid membranes , experimental designs should consider this unique evolutionary context. Comparative studies with homologous proteins from thylakoid-containing cyanobacteria can provide valuable insights into functional adaptations. Additionally, the integration of light and pH sensing observed in early cyanobacterial photoreceptors suggests that testing ndhM function under various light and pH conditions may reveal regulatory mechanisms specific to Gloeobacter.

What approaches can address solubility challenges with recombinant ndhM?

Membrane-associated proteins like ndhM from G. violaceus often present solubility challenges during recombinant expression. Here are strategic approaches to address these issues:

Expression optimization strategies:

• Reduce induction temperature (16-20°C)

• Lower inducer concentration (0.01-0.1 mM IPTG)

• Use auto-induction media for gradual expression

• Co-express with molecular chaperones (GroEL/GroES, DnaK/DnaJ/GrpE)

• Utilize weak promoters to slow expression rate

Fusion partners to enhance solubility:

• Maltose-binding protein (MBP): Highly effective solubilizing tag (~40 kDa)

• SUMO: Enhances solubility and can be precisely removed (~11 kDa)

• Thioredoxin (Trx): Small tag that promotes disulfide bond formation (~12 kDa)

• Glutathione S-transferase (GST): Dual function as solubility tag and purification handle (~26 kDa)

Buffer optimization for extraction:

• Detergent screening: Test panel of non-ionic (DDM, Triton X-100), zwitterionic (CHAPS, LDAO), and mild ionic detergents

• Salt concentration: Test range from 150-500 mM NaCl

• Stabilizing additives: Glycerol (10-25%), arginine (50-100 mM), sucrose (5-10%)

• pH optimization: Test extraction efficiency at different pH values

Alternative approaches for recalcitrant proteins:

• Cell-free expression systems

• Periplasmic targeting to facilitate proper folding

• Inclusion body solubilization followed by refolding

• Co-expression with interaction partners

For ndhM specifically, a recommended approach would combine several strategies: expression as an MBP fusion protein at 18°C with co-expression of GroEL/GroES chaperones, followed by extraction in a buffer containing 50 mM Tris-HCl pH 7.5, 300 mM NaCl, 10% glycerol, 0.05% DDM, and 1 mM DTT. This multi-faceted approach addresses the membrane association of ndhM while promoting proper folding and preventing aggregation.

How can researchers compare ndhM function across different cyanobacterial species?

Comparative analysis of ndhM across diverse cyanobacterial species provides valuable insights into evolutionary adaptations and functional conservation. Here are comprehensive approaches for such comparative studies:

Sequence-based analyses:

• Multiple sequence alignment to identify conserved and variable regions

• Phylogenetic analysis to establish evolutionary relationships

• Conservation scoring to identify functionally important residues

• Coevolution analysis to detect co-evolving residues in protein networks

Experimental comparative approaches:

• Heterologous expression of ndhM from multiple species using identical protocols

• Standardized activity assays under identical conditions

• In vitro reconstitution with subunits from various species

• Cross-species complementation in model organisms

Structural comparative methods:

• Homology modeling based on available structures

• Circular dichroism to compare secondary structure content

• Thermal stability comparisons across homologs

• Structural dynamics analysis using hydrogen-deuterium exchange mass spectrometry

Specialized comparative techniques:

• Chimeric proteins with domains from different species

• Site-directed mutagenesis to convert species-specific residues

• Cross-species protein-protein interaction studies

A comprehensive comparative analysis would involve:

• Expression and purification of ndhM from multiple species using identical protocols

• Detailed biochemical characterization (substrate specificity, kinetic parameters)

• Protein-protein interaction studies to identify species-specific partners

• Complementation experiments in model organisms

Given that Gloeobacter species lack thylakoid membranes while all other cyanobacteria possess them , comparing ndhM function between these groups could reveal how this protein has adapted to different cellular architectures throughout cyanobacterial evolution. The significant genomic diversity observed within the Gloeobacter genus itself suggests that even closely related species may show important functional variations in their electron transport components.

What are the methodological approaches for studying ndhM interactions with other proteins?

Understanding how ndhM from G. violaceus interacts with other proteins is essential for elucidating its function within the NDH-1 complex. Here are methodological approaches for investigating these interactions:

In vitro protein-protein interaction methods:

• Pull-down assays using affinity-tagged ndhM

• Surface plasmon resonance (SPR) for real-time interaction kinetics

• Isothermal titration calorimetry (ITC) for thermodynamic parameters

• Microscale thermophoresis (MST) for interactions in solution

• Blue native PAGE to visualize intact complexes

• Analytical ultracentrifugation for complex stoichiometry

• FRET-based assays for proximity detection

Structural approaches to identify interaction interfaces:

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS)

• Crosslinking coupled with mass spectrometry (XL-MS)

• Cryo-electron microscopy of reconstituted complexes

• X-ray crystallography of co-crystallized components

Computational and bioinformatic methods:

• Molecular docking simulations

• Coevolution analysis to predict interacting residues

• Comparative genomics across cyanobacterial species

• Protein-protein interaction network analysis

In vivo approaches:

• Bacterial two-hybrid systems

• Split-GFP complementation assays

• Co-immunoprecipitation from cyanobacterial lysates

• In vivo crosslinking followed by pulldown

For ndhM from G. violaceus, the unique cellular context lacking thylakoid membranes means that its interaction partners and complex assembly may differ from other cyanobacteria. A comprehensive investigation would begin with identifying potential interaction partners through pull-down experiments or co-purification, followed by detailed characterization of specific interactions using biophysical methods like SPR or ITC. Blue native PAGE could then verify complex formation, while structural techniques like HDX-MS or XL-MS would provide detailed information about interaction interfaces.

The significant genomic diversity observed within Gloeobacter species suggests that comparative studies of protein-protein interactions across different members of this genus could reveal important evolutionary adaptations in NDH-1 complex assembly.

What spectroscopic methods are most suitable for studying ndhM activity?

Spectroscopic techniques provide powerful tools for investigating the activity and electron transfer properties of ndhM from G. violaceus. Here's a comprehensive analysis of applicable spectroscopic methods:

UV-visible absorption spectroscopy:

• NADH/NADPH oxidation monitored at 340 nm

• Quinone reduction tracked at wavelengths specific to the quinone (typically 270-290 nm)

• Real-time kinetic measurements to determine reaction rates

• Stopped-flow spectroscopy for pre-steady-state kinetics

• Difference spectroscopy to detect subtle spectral changes

Fluorescence spectroscopy:

• Intrinsic protein fluorescence to monitor conformational changes

• NADH/NADPH fluorescence (excitation ~340 nm, emission ~460 nm)

• Förster resonance energy transfer (FRET) for interaction studies

• Fluorescence quenching to investigate binding events

• Time-resolved fluorescence for dynamic processes

Electron paramagnetic resonance (EPR) spectroscopy:

• Detection of iron-sulfur clusters in the NDH-1 complex

• Characterization of semiquinone radical intermediates

• Spin-labeling for structural dynamics

• Pulsed EPR techniques for detailed electronic structure

Advanced spectroscopic techniques:

• Resonance Raman spectroscopy for cofactor vibrations

• Fourier transform infrared (FTIR) spectroscopy for protein structure

• Circular dichroism for secondary structure analysis

• Magnetic circular dichroism for electronic structure of cofactors

For ndhM from G. violaceus, a comprehensive spectroscopic approach would begin with standard UV-visible spectroscopy to characterize basic enzymatic activity, followed by stopped-flow measurements to determine pre-steady-state kinetics. EPR spectroscopy would be valuable for characterizing any iron-sulfur clusters involved in electron transfer, while fluorescence techniques could monitor binding events and conformational changes.

The unique cellular organization of G. violaceus, lacking thylakoid membranes , might result in distinctive spectroscopic properties for its ndhM compared to homologous proteins from other cyanobacteria, potentially revealing adaptations in electron transfer mechanisms specific to this early-branching cyanobacterial lineage.

How can site-directed mutagenesis be used to study ndhM structure-function relationships?

Site-directed mutagenesis provides a powerful approach for investigating structure-function relationships in ndhM from G. violaceus. Here's a comprehensive guide to applying this technique:

Strategic selection of mutation targets:

• Conserved residues identified through multiple sequence alignments

• Predicted active sites or substrate-binding regions

• Charged or polar residues at potential interface regions

• Residues unique to Gloeobacter compared to other cyanobacteria

• Putative cofactor binding sites or electron transfer pathways

Types of mutations to consider:

• Conservative substitutions (e.g., Asp to Glu) to test specific functional groups

• Alanine scanning to remove side chain functionality

• Cysteine substitutions for subsequent labeling studies

• Introduction of spectroscopic probes (e.g., Trp) for monitoring changes

• Charge reversal mutations to test electrostatic interactions

Experimental design for mutagenesis studies:

• Create multiple mutants in parallel for comprehensive analysis

• Include positive and negative controls (wild-type and inactive variants)

• Verify protein folding and stability for each mutant

• Consider double or triple mutants to test coupled functions

Functional analysis of mutants:

• Enzymatic activity compared to wild-type

• Substrate binding studies

• Protein-protein interaction assays

• Structural stability assessments

• In vivo complementation studies

For ndhM from G. violaceus, site-directed mutagenesis could provide particular insights into how this protein functions in a cellular environment lacking thylakoid membranes . Mutations targeting residues involved in membrane association or interaction with other NDH-1 components could reveal adaptations specific to the Gloeobacter lineage.

A systematic mutagenesis approach would involve:

• Bioinformatic analysis to identify targets based on conservation and predicted function

• Generation of mutants using standard molecular biology techniques

• Expression and purification of mutant proteins

• Comprehensive characterization using activity assays, binding studies, and structural analyses

• Comparison with equivalent mutations in homologous proteins from thylakoid-containing cyanobacteria

This approach would significantly advance our understanding of the structure-function relationships in ndhM and how they have evolved in this early-branching cyanobacterial lineage.

What methodological approaches are appropriate for experimental and quasi-experimental designs in ndhM research?

Designing rigorous experimental approaches for ndhM research requires careful consideration of methodological frameworks. Based on implementation science principles, researchers can employ various experimental and quasi-experimental designs:

Randomized experimental designs:

• Factorial designs to test multiple variables simultaneously

• Randomized block designs to control for known sources of variation

• Split-plot designs for experiments with hard-to-change factors

• Optimization trials to identify ideal conditions for ndhM expression or activity

Quasi-experimental approaches:

• Interrupted time series designs for tracking changes in experimental parameters

• Non-equivalent control group designs when full randomization is not possible

• Stepped wedge designs for sequential implementation of experimental conditions

Implementation considerations for ndhM research:

• Standard protocols for expression and purification to ensure reproducibility

• Systematic variation of experimental conditions (pH, temperature, substrate concentration)

• Comprehensive controls at each experimental stage

• Rigorous validation of methodology before full-scale experiments

Key considerations for experimental design quality:

As noted in implementation science literature, "implementation-focused RCTs usually differ from traditional efficacy- or effectiveness-oriented RCTs on key parameters" . For ndhM research, this translates to designs that focus not just on the protein's basic properties but on optimizing conditions for its expression, purification, and functional analysis.

When full experimental control is not possible, quasi-experimental designs provide rigorous alternatives. For example, interrupted time series designs could be valuable for monitoring ndhM stability over time under various storage conditions .

Building on the concept of methodological research phases , ndhM studies should progress from initial characterization (phase I) through empirical evidence in targeted settings (phase II) to extended investigations across various conditions (phase III) and finally to comprehensive understanding of when specific methods are preferred (phase IV).