Recombinant Gloeobacter violaceus 60 kDa chaperonin 1 (groL1), partial

What is Gloeobacter violaceus and what makes it significant for research?

Gloeobacter violaceus is a unique genus of cyanobacteria that represents the sister group to all other photosynthetic cyanobacteria. Its evolutionary significance stems from its distinctive characteristic of lacking thylakoid membranes, which are present in all other cyanobacteria and chloroplasts. Instead, Gloeobacter conducts photosynthesis directly at the plasma membrane, with light-harvesting complexes (phycobilisomes) positioned on the cytoplasmic side of this membrane. This results in the formation of proton gradients across the plasma membrane rather than across thylakoid membranes as in other photosynthetic organisms . Gloeobacter violaceus produces several pigments, including chlorophyll a, β-carotene, oscillol diglycoside, and echinenone, with its purple coloration resulting from relatively low chlorophyll content . The species was originally isolated from limestone rock in Switzerland, and phylogenetic studies suggest that Gloeobacter may have diverged from other cyanobacteria between 3.7 and 3.2 billion years ago, making it invaluable for studying the early evolution of photosynthesis .

What is the 60 kDa chaperonin 1 (groL1) and what is its function?

The 60 kDa chaperonin 1 (groL1) from Gloeobacter violaceus is a protein belonging to the GroEL family of molecular chaperones. Chaperonins are essential proteins that assist in the proper folding of newly synthesized polypeptides. In bacteria like Escherichia coli, GroEL works in conjunction with its co-chaperonin GroES and ATP to form a cage-like structure that provides an isolated environment for protein folding . The GroEL/ES system typically functions post-translationally after polypeptides are released from ribosomes, though recent research suggests potential co-translational roles as well . In the specific context of Gloeobacter violaceus, which lacks thylakoid membranes and has unique photosynthetic machinery, chaperonins like groL1 likely play critical roles in ensuring the proper folding and assembly of photosynthetic and other essential proteins under the organism's unique cellular architecture .

How should recombinant Gloeobacter violaceus groL1 be stored and handled in laboratory settings?

For optimal preservation of recombinant Gloeobacter violaceus 60 kDa chaperonin 1 (groL1), storage conditions should be carefully maintained. The protein is available in both liquid and lyophilized forms, with different shelf-life characteristics. Liquid preparations generally remain stable for approximately 6 months when stored at -20°C or -80°C, while lyophilized forms maintain stability for up to 12 months at these temperatures .

For routine laboratory use, it is recommended to avoid repeated freeze-thaw cycles, as these can compromise protein integrity. Working aliquots should be stored at 4°C and used within one week . When reconstituting lyophilized protein, the vial should first be briefly centrifuged to bring contents to the bottom. Reconstitution should be performed using deionized sterile water to achieve a concentration of 0.1-1.0 mg/mL. For long-term storage of reconstituted protein, the addition of glycerol to a final concentration of 5-50% (with 50% being optimal) is recommended before aliquoting and storing at -20°C or -80°C .

What expression systems are optimal for producing functional recombinant Gloeobacter violaceus groL1?

Based on commercial production methods and research protocols, E. coli serves as the preferred expression system for recombinant Gloeobacter violaceus 60 kDa chaperonin 1 (groL1) . This bacterial expression system offers several advantages for chaperonin production, including rapid growth, high protein yields, and well-established protocols for induction and purification. For optimal expression, researchers should consider the following methodological approaches:

• Vector selection: pET-based expression vectors containing T7 promoters are typically effective for chaperonin expression, allowing tight regulation and high-level induction.

• E. coli strain selection: BL21(DE3) or Rosetta strains are recommended due to their deficiency in certain proteases and ability to provide rare codons that may be present in the cyanobacterial gene.

• Induction parameters: IPTG concentration (typically 0.1-1.0 mM), temperature (18-30°C), and duration (4-16 hours) should be optimized, with lower temperatures often yielding more soluble protein.

• Co-expression strategies: For functional studies, co-expression with the co-chaperonin GroES from Gloeobacter violaceus may enhance proper folding and assembly of the chaperonin complex.

Notably, the final product achieved through optimized E. coli expression typically reaches >85% purity as determined by SDS-PAGE analysis, making it suitable for most research applications .

How does the genomic context of groL1 in Gloeobacter violaceus compare to other Gloeobacter species?

The genomic context of groL1 provides valuable insights into the evolution and functional specialization of chaperonins across Gloeobacter species. Pangenomic analysis reveals significant differences between Gloeobacter violaceus and other members of the genus:

Gloeobacter morelensis, a recently discovered species isolated from a waterfall cave in Mexico, shares only 92.7% average nucleotide identity (ANI) with Gloeobacter violaceus, indicating substantial genomic divergence . Pangenomic comparisons demonstrate that G. morelensis encodes 759 genes not shared with other Gloeobacter species . While G. morelensis and G. violaceus share 798 unique gene clusters (comprising 1,717 genes total), their chaperone systems show distinctive features .

The chaperonin systems in these organisms likely evolved to accommodate the unique protein folding requirements imposed by the absence of thylakoid membranes. Comparative genomic analysis reveals that core gene clusters shared between Gloeobacter species and related sister taxa include approximately 580 genes, suggesting conservation of essential cellular machinery including basic chaperone functions .

The table below summarizes key genomic features across Gloeobacter species that influence chaperonin context:

This genomic diversity suggests potential functional specialization of chaperonins across Gloeobacter species that may correlate with their ecological adaptations and photosynthetic machinery configurations .

What structural features distinguish Gloeobacter violaceus groL1 from other bacterial chaperonins?

While detailed structural information specific to Gloeobacter violaceus groL1 is limited in the provided search results, comparative analysis with other bacterial chaperonins reveals several noteworthy features that likely apply to this protein:

• ATP binding pocket modifications: Given the unusual energetics of photosynthesis occurring at the plasma membrane rather than thylakoids in Gloeobacter, the ATP binding and hydrolysis domains may show adaptations to function optimally in this cellular context.

• Substrate binding region specialization: The apical domains of groL1 likely exhibit amino acid compositions tailored to interact with the unique proteome of Gloeobacter, particularly photosynthetic proteins that must assemble in the plasma membrane.

• Co-chaperonin interaction surface: The interface between groL1 and its presumed co-chaperonin may contain specific residues that ensure proper assembly of the functional chaperonin complex.

The structural adaptations of Gloeobacter violaceus groL1 would be expected to reflect its evolutionary position as part of one of the earliest diverging lineages of photosynthetic organisms. Comparative structural analysis between groL1 from Gloeobacter and chaperonins from organisms with thylakoid membranes could provide valuable insights into the co-evolution of protein folding machinery with photosynthetic systems .

How can researchers use recombinant Gloeobacter violaceus groL1 to study protein folding mechanisms?

Recombinant Gloeobacter violaceus 60 kDa chaperonin 1 (groL1) provides a valuable experimental tool for investigating fundamental aspects of protein folding, particularly in photosynthetic systems. Researchers can implement several methodological approaches:

• In vitro reconstitution assays: Purified recombinant groL1 can be combined with denatured substrate proteins to assess folding efficiency under controlled conditions. This approach typically involves:

  • Denaturing substrate proteins using chemical denaturants (urea or guanidinium hydrochloride)

  • Diluting the denatured protein into a buffer containing groL1, co-chaperonin, and ATP

  • Monitoring folding through enzymatic activity assays or spectroscopic methods (fluorescence, circular dichroism)

• GroEL trap experiments: Modified versions of groL1 can be engineered to "trap" substrate proteins, allowing researchers to identify natural binding partners. This involves:

  • Creating mutations in the ATP binding site to prevent release of substrate proteins

  • Incubating the trap variant with cellular extracts

  • Isolating the complex and identifying bound proteins through mass spectrometry

• Co-translational folding studies: Based on recent findings suggesting potential co-translational roles for GroEL systems , researchers can:

  • Establish in vitro translation systems supplemented with labeled groL1

  • Monitor the interaction with nascent polypeptide chains during translation

  • Compare the effects of groL1 from this evolutionarily ancient organism with conventional model systems

The unique evolutionary position of Gloeobacter violaceus makes its chaperonin system particularly valuable for comparative studies, potentially revealing fundamental principles about the co-evolution of protein folding mechanisms with photosynthetic machinery .

What analytical techniques are most effective for characterizing the structure-function relationship of groL1?

To thoroughly characterize the structure-function relationship of Gloeobacter violaceus groL1, researchers should employ a complementary set of analytical techniques:

• Structural analysis approaches:

  • Cryo-electron microscopy (Cryo-EM): Optimal for visualizing the quaternary structure of assembled groL1 complexes, potentially in different nucleotide-bound states or with bound substrate proteins.

  • X-ray crystallography: Provides atomic-level resolution of the protein structure, though crystallization may be challenging.

  • Small-angle X-ray scattering (SAXS): Useful for examining conformational changes in solution under various conditions.

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS): Offers insights into dynamic regions and conformational changes upon substrate or co-chaperonin binding.

• Functional characterization methods:

  • ATPase activity assays: Measures ATP hydrolysis rates as a function of temperature, pH, or substrate presence.

  • Substrate binding assays: Employs fluorescence anisotropy or surface plasmon resonance to quantify binding kinetics.

  • Protein refolding assays: Monitors the recovery of enzymatic activity or native structure of model substrate proteins.

  • Thermal stability assessments: Differential scanning calorimetry (DSC) or differential scanning fluorimetry (DSF) to determine stability under various conditions.

• Computational approaches:

  • Molecular dynamics simulations: Models the dynamic behavior of groL1 in various states.

  • Sequence-structure-function analyses: Compares groL1 with chaperonins from related and distant species to identify conserved functional elements.

These methodologies can be particularly informative when applied comparatively to groL1 from different Gloeobacter species, potentially revealing adaptations associated with the distinct photosynthetic machinery and ecological niches of these organisms .

How can isothermal titration calorimetry be optimized for studying ATP binding and hydrolysis by groL1?

Isothermal titration calorimetry (ITC) provides valuable thermodynamic information about ATP binding and hydrolysis by Gloeobacter violaceus groL1. For optimal experimental design:

• Sample preparation considerations:

  • Protein concentration: Typically 10-20 μM of purified groL1 (oligomeric concentration) in the cell.

  • ATP concentration: 200-400 μM in the syringe for binding studies.

  • Buffer composition: Identical buffers for protein and ligand solutions, containing 20-50 mM HEPES or phosphate buffer (pH 7.4-7.8), 5-10 mM MgCl₂ (essential for ATP binding), and 50-100 mM KCl.

  • Protein quality: >85% purity by SDS-PAGE is sufficient, but higher purity yields more reliable results .

• Experimental parameters:

  • Temperature: 25°C is standard, but comparative measurements at different temperatures (10-40°C) provide enthalpy-entropy compensation information.

  • Injection volume: Initial injection of 0.5 μL followed by 1.5-2.5 μL injections.

  • Spacing between injections: 180-240 seconds to ensure return to baseline.

  • Stirring speed: 750-800 rpm for optimal mixing.

• Data analysis approaches:

  • Model selection: For ATP binding, a sequential binding sites model often best describes the cooperativity within the groL1 rings.

  • Control experiments: Protein-free injections to account for dilution effects.

  • Comparative analysis: Parallel experiments with other Gloeobacter chaperonins or E. coli GroEL to identify unique thermodynamic signatures.

• Advanced applications:

  • Coupling ITC with ATPase activity measurements at identical conditions.

  • Performing experiments in the presence of varying concentrations of potassium or substrate proteins to assess allosteric effects.

  • Examining the impact of reconstitution conditions on thermodynamic parameters .

This methodological approach provides comprehensive thermodynamic profiles (ΔH, ΔS, ΔG, and binding stoichiometry) that can reveal how the unique evolutionary history of Gloeobacter has shaped the energetics of its chaperonin system.

How does groL1 from Gloeobacter violaceus differ from analogous proteins in other cyanobacteria with thylakoid membranes?

The 60 kDa chaperonin 1 (groL1) from Gloeobacter violaceus presents distinctive features compared to analogous proteins in thylakoid-containing cyanobacteria, reflecting its unique evolutionary position and cellular architecture:

• Structural adaptations:

  • Substrate binding specificity: Given that Gloeobacter lacks thylakoid membranes, its groL1 likely evolved to handle a different spectrum of substrate proteins compared to typical cyanobacteria. The substrate-binding regions would be expected to show adaptations for interaction with proteins destined for the plasma membrane rather than thylakoids .

  • Oligomeric stability: The absence of thylakoids may influence the stability requirements of groL1 complexes, potentially resulting in amino acid substitutions at subunit interfaces.

• Functional specialization:

  • ATP utilization: The energetics of protein folding in Gloeobacter may differ from other cyanobacteria due to the unique localization of photosynthetic machinery, potentially leading to differences in ATP binding and hydrolysis properties.

  • Co-chaperonin interactions: The interface between groL1 and its co-chaperonin partner may show adaptations specific to the Gloeobacter lineage.

• Genomic context:

  • While specific data on groL1 gene organization is limited in the search results, the genomic analysis of Gloeobacter species reveals substantial divergence from other cyanobacteria. For instance, Gloeobacter morelensis, closely related to G. violaceus, encodes 759 unique genes not found in other Gloeobacter species .

  • The genome of G. violaceus lacks many genes for photosystem I and II components present in thylakoid-containing cyanobacteria, suggesting co-evolution of its chaperone system with this reduced photosynthetic machinery .

These differences make groL1 from Gloeobacter violaceus an important model for understanding the evolution of molecular chaperones in relation to major cellular innovations like thylakoid membranes, which are central to modern photosynthesis .

What insights does the study of groL1 provide about the evolution of protein folding mechanisms in early photosynthetic organisms?

The study of Gloeobacter violaceus groL1 offers a unique window into the evolution of protein folding mechanisms in early photosynthetic organisms, yielding several key insights:

• Evolutionary timing and divergence:

  • Gloeobacter is estimated to have diverged from other cyanobacteria between 3.7 and 3.2 billion years ago, with different Gloeobacter species diverging approximately 280 million years ago . This places its chaperonin system as a representative of very ancient protein folding machinery.

  • The groL1 protein likely represents one of the earliest adaptations of chaperonin systems to support oxygenic photosynthesis, predating the evolution of thylakoid membranes.

• Co-evolution with photosynthetic machinery:

  • The absence of thylakoids in Gloeobacter suggests that its chaperonin system evolved to support protein folding for a simpler photosynthetic apparatus localized to the plasma membrane .

  • Comparative genomic analyses reveal that Gloeobacter species have distinctive patterns in genes encoding photosystem components, including the D1 protein of Photosystem II. For example, G. morelensis encodes six distinct D1 proteins, some identical to each other, suggesting specific evolutionary processes like concerted evolution .

  • These patterns indicate that the chaperonin system likely co-evolved with these specialized photosynthetic components to ensure their proper folding and assembly.

• Fundamental mechanisms of protein folding:

  • The functional conservation of chaperonins across billions of years suggests that certain fundamental mechanisms of protein folding were established very early in evolutionary history.

  • The transition from plasma membrane-based to thylakoid-based photosynthesis would have required adaptations in chaperonin systems to accommodate new substrate proteins and cellular compartments.

• Potential primitive features:

  • The groL1 protein from Gloeobacter may preserve features of ancestral chaperonins that were subsequently modified in lineages that evolved thylakoids.

  • Analysis of these features could reveal which aspects of chaperonin function are fundamental to all photosynthetic organisms versus those that arose specifically to support thylakoid-based photosynthesis .

This evolutionary perspective makes groL1 an invaluable model for understanding how protein folding mechanisms co-evolved with major transitions in cellular architecture during the early history of photosynthetic life.

How can phylogenetic analysis of groL1 sequences contribute to understanding cyanobacterial evolution?

Phylogenetic analysis of groL1 sequences provides a powerful approach for investigating cyanobacterial evolution, offering several methodological advantages and insights:

• Molecular clock applications:

  • The groL1 gene, being essential for cellular function, evolves under strong selective pressure, making it useful for calibrating molecular clock models.

  • By comparing substitution rates in groL1 sequences across Gloeobacter species (which diverged approximately 280 million years ago) and between Gloeobacter and other cyanobacteria (divergence estimated at 3.2-3.7 billion years ago), researchers can calibrate evolutionary rates .

  • This allows more accurate dating of important evolutionary events in cyanobacterial history, such as the acquisition of specific photosynthetic innovations.

• Co-evolutionary analysis:

  • Parallel phylogenetic analysis of groL1 and photosystem components can reveal instances of co-evolution.

  • For example, comparative analysis of groL1 with D1 protein variants (such as the Group 0 D1 found in G. kilaueensis and G. morelensis but not G. violaceus) may identify correlated evolutionary changes .

  • Such correlations could indicate functional adaptations in the chaperonin system to accommodate specific substrate proteins.

• Horizontal gene transfer detection:

  • Incongruence between groL1 phylogeny and organismal phylogeny based on other markers can identify potential horizontal gene transfer events.

  • The presence of multiple groL1 paralogs in some cyanobacterial genomes allows investigation of duplication and specialization events.

• Methodological approach for phylogenetic analysis:

  • Sequence alignment: MUSCLE or MAFFT algorithms optimized for protein sequences.

  • Model selection: Perform model testing to identify appropriate substitution models (typically LG or WAG for proteins).

  • Tree building: Maximum likelihood (RAxML, IQ-TREE) and Bayesian inference (MrBayes) methods with bootstrap values >70% or posterior probabilities >0.95 considered significant.

  • Comparative analysis: Integration with whole-genome phylogenetic approaches based on average nucleotide identity (ANI) metrics, as used in comparing Gloeobacter species (e.g., 92.7% ANI between G. violaceus and G. morelensis) .

This multifaceted phylogenetic approach using groL1 sequences can provide robust insights into the timing and nature of major evolutionary transitions in cyanobacteria, complementing whole-genome analyses and studies of photosynthetic components.

What methodologies are most effective for studying interactions between groL1 and substrate proteins in Gloeobacter violaceus?

For investigating interactions between Gloeobacter violaceus groL1 and its substrate proteins, researchers should employ a multi-method approach:

• Co-immunoprecipitation strategies:

  • Antibody development: Generate antibodies specific to G. violaceus groL1 using purified recombinant protein (>85% purity) .

  • Pull-down protocol: Lyse G. violaceus cells under mild conditions (non-ionic detergents, physiological pH) to preserve protein-protein interactions.

  • Mass spectrometry identification: Analyze co-precipitated proteins using LC-MS/MS followed by database searching against the G. violaceus proteome.

  • Validation: Perform reciprocal co-IPs with antibodies against identified substrate proteins.

• Crosslinking mass spectrometry (XL-MS):

  • In vivo crosslinking: Treat intact G. violaceus cells with membrane-permeable crosslinkers such as DSS or formaldehyde.

  • In vitro reconstitution: Mix purified recombinant groL1 with candidate substrate proteins followed by crosslinking.

  • MS analysis: Digest crosslinked complexes and identify crosslinked peptides using specialized search algorithms.

  • Structural mapping: Map identified crosslinks onto structural models to determine interaction interfaces.

• Surface plasmon resonance (SPR) and bio-layer interferometry (BLI):

  • Sensor preparation: Immobilize purified groL1 (>85% purity) on sensor surfaces .

  • Binding kinetics: Measure association and dissociation rates of potential substrate proteins under various conditions (ATP presence/absence, temperature variation).

  • Competition assays: Determine whether different substrate proteins compete for binding sites.

• Fluorescence-based approaches:

  • FRET pairs: Label groL1 and substrate proteins with appropriate fluorophores.

  • Single-molecule FRET: Monitor individual binding and release events to capture transient interactions.

  • Fluorescence anisotropy: Measure changes in rotational diffusion upon complex formation.

These methodologies can be particularly powerful when applied to photosynthetic proteins unique to Gloeobacter's plasma membrane-based photosynthetic machinery, potentially revealing how this ancient chaperonin system accommodates substrates without the compartmentalization provided by thylakoid membranes in other cyanobacteria .

How does ATP binding and hydrolysis regulate the chaperone function of groL1?

The ATP-driven conformational cycle is central to the chaperone function of Gloeobacter violaceus groL1, though specific details must be inferred from studies of related chaperonins:

• ATP binding and conformational changes:

  • ATP binding to groL1 likely induces substantial conformational changes in the apical and intermediate domains of each subunit.

  • These conformational changes alter the nature of the central cavity, transitioning from a hydrophobic (substrate-binding) state to a hydrophilic (folding-permissive) state.

  • The binding of the co-chaperonin (presumed GroES equivalent) to the ATP-bound form creates an enclosed folding chamber.

• Experimental approaches to characterize ATP regulation:

  • Site-directed mutagenesis: Create variants with mutations in ATP binding pocket residues.

  • ATPase activity assays: Measure ATP hydrolysis rates under varying conditions (temperature, pH, substrate presence).

  • Structural analyses: Use cryo-EM or crystallography to capture different nucleotide-bound states.

  • Kinetic analyses: Employ stopped-flow techniques to measure rates of conformational changes following ATP binding.

• ATP hydrolysis and substrate release:

  • ATP hydrolysis likely triggers a second set of conformational changes that ultimately lead to substrate release.

  • The timing of ATP hydrolysis across the seven subunits of each ring may exhibit negative cooperativity, creating an ordered sequence of events.

  • The complete ATP cycle includes binding of ATP to one ring, binding of co-chaperonin, substrate protein folding, ATP hydrolysis, and release of products.

• Unique aspects in Gloeobacter:

  • Given Gloeobacter's ancient evolutionary position and unique cellular architecture (lacking thylakoids), its groL1 may exhibit distinctive ATP utilization patterns optimized for its specific substrate profile .

  • The ATP cycle of groL1 may be adapted to function optimally under the energy availability conditions created by plasma membrane-localized photosynthesis rather than thylakoid-based photosynthesis .

The ATP-driven cycle is essential for the functioning of chaperonins like groL1, and detailed characterization of this cycle can provide insights into how this ancient protein folding machine operates in one of the earliest diverging lineages of photosynthetic organisms .

How does the function of groL1 relate to the unique photosynthetic machinery in Gloeobacter violaceus?

The functional relationship between groL1 and the unique photosynthetic machinery in Gloeobacter violaceus represents a fascinating example of co-evolution between protein folding systems and cellular architecture:

• Membrane localization adaptations:

  • Unlike all other cyanobacteria, Gloeobacter lacks thylakoid membranes and instead localizes its photosynthetic complexes directly to the plasma membrane .

  • The groL1 chaperonin system likely evolved specific adaptations to facilitate the folding and assembly of photosynthetic proteins destined for this alternative membrane system.

  • This may include specialized substrate binding regions that recognize features of plasma membrane-targeted proteins rather than thylakoid-targeted proteins.

• Photosystem component handling:

  • Genome analysis of Gloeobacter species reveals unique patterns in photosystem genes. For example, G. morelensis encodes six distinct D1 proteins, with some groups showing evidence of concerted evolution .

  • The groL1 chaperonin likely plays a crucial role in folding these diverse photosystem components, particularly those that differ significantly from counterparts in other cyanobacteria.

  • The genome of G. violaceus lacks many genes for photosystem I and II that are present in thylakoid-containing cyanobacteria, suggesting co-evolution of its chaperone system with this reduced photosynthetic machinery .

• Experimental approaches for investigating this relationship:

  • Proteomic identification of groL1 substrates in vivo, with particular attention to photosynthetic components.

  • In vitro folding assays comparing groL1-mediated folding of photosynthetic proteins from Gloeobacter versus those from thylakoid-containing cyanobacteria.

  • Localization studies to determine whether groL1 exhibits specific subcellular distribution patterns relative to plasma membrane photosynthetic complexes.

  • Comparative expression analysis of groL1 under different light conditions to assess co-regulation with photosynthetic machinery.

• Evolutionary significance:

  • As part of one of the earliest diverging lineages of cyanobacteria (estimated divergence 3.2-3.7 billion years ago), Gloeobacter's groL1-photosystem relationship may preserve features of ancient interactions that were subsequently modified in lineages that evolved thylakoids .

  • This system provides a window into how protein folding machinery co-evolved with major transitions in cellular architecture during the early history of photosynthetic life.

Understanding this relationship is not merely of academic interest but could provide insights into fundamental principles of protein folding that might be applicable to synthetic biology approaches for engineering novel photosynthetic systems .