GroES Human
Description
Product Specs
Q&A
What is GroES and what is its human mitochondrial homolog?
GroES is an essential cochaperonin from E. coli that works with the chaperonin GroEL to assist in protein folding. The human homolog found in mitochondria is called mitochondrial heat shock protein 10 (mtHsp10), which exhibits approximately 33% sequence identity to bacterial GroES . mtHsp10 functions with mitochondrial heat shock protein 60 (mtHsp60), which is the human homolog of GroEL showing 51% sequence identity to its bacterial counterpart .
Both proteins are critical for cellular viability, with mtHsp60 being the only molecular chaperone required for cell growth under both normal and stressful conditions . The embryonic lethality observed in mice when the mtHsp60 gene is inactivated demonstrates the essential nature of this chaperonin system .
How does the bacterial GroEL-GroES system structurally differ from the human mtHsp60-mtHsp10 system?
The bacterial and human mitochondrial chaperonin systems exhibit significant structural differences despite their evolutionary relationship:
| Feature | Bacterial GroEL-GroES | Human mtHsp60-mtHsp10 |
|---|---|---|
| Complex shape | Asymmetric "bullet" | Symmetric "football" |
| Ring arrangement | Double-ring GroEL + single GroES | Double-ring with extensive interring contacts |
| Subunit symmetry | Preserved within each ring | Not preserved within rings |
| Nucleotide binding | Interring nucleotide asymmetry | Both rings can be in ADP-bound state |
| Interring contact area | Less extensive | More than twice as extensive (5,820 Ų) |
The human mtHsp60-mtHsp10 complex forms a symmetric double-ring, football-like structure [mtHsp60₁₄–(mtHsp10₇)₂] that displays extensive interring contacts . This stands in contrast to the asymmetric nature of the bacterial GroEL-GroES complex, suggesting distinct functional mechanisms between the two systems.
What is the molecular basis for GroES interaction with GroEL?
GroES functions as a lid for the GroEL barrel, containing a heptameric ring of ~10 kDa subunits with flexible loops that interact specifically with helices H and I in the apical domain of GroEL . This interaction is strictly ATP-dependent - when ATP binds to GroEL's subunit sites, it triggers a conformational change that enables GroES binding .
The isoleucine residue at position 25 (I25) in GroES plays a critical role in this interaction. Mutations of I25 can completely abolish GroES-GroEL interaction due to the seven-fold mutational amplification in the heptameric GroES structure . This amplification effect makes single residue mutations particularly impactful in multi-subunit protein complexes.
How do researchers visualize and analyze the conformational changes in chaperonin complexes?
Researchers employ several advanced techniques to study conformational changes in chaperonin complexes:
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X-ray crystallography to determine the static structure of complexes in different states
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Engineered mutations that arrest the complex at specific stages of the folding cycle
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Fluorescent protein fusions to monitor folding activity in real-time
For example, the mHsp60 E321K mutant provides valuable insights by breaking a salt bridge that normally disrupts during the transition from closed to open conformation . This mutant acquires the ability to function with GroES but loses functionality with its endogenous partner mtHsp10, creating a high-affinity complex useful for structural studies .
Such mutations can trap chaperonin complexes in specific conformational states, enabling researchers to dissect the mechanistic details of protein folding cycles that would otherwise be challenging to study due to their dynamic nature.
How can researchers design experiments to investigate potential individual functions of GroES?
To study potential autonomous functions of GroES independent of GroEL, researchers can employ several methodological approaches:
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Create an inducible GroES gene system using controlled expression systems such as the pTet-system
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Design co-expression systems with hard-to-fold, aggregation-prone proteins linked to fluorescent reporters
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Compare GroES overexpression effects to a control system containing the complete GroE machinery
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Utilize kinetic fluorescence spectroscopy to quantify protein folding in real-time
A specific experimental design reported in the literature includes expressing GroES under the control of a tetracycline-inducible promoter in a plasmid separate from the proteins being studied . Hard-to-fold proteins such as mNeonGreen-Aβ, EGFP-Aβ, EGFP-Tau, and EGFP-α-synuclein can be used as substrate proteins to assess GroES function . The fluorescence output serves as a quantifiable measure of properly folded proteins.
What strategies can be employed to study the specific contribution of individual amino acid residues in GroES?
Several innovative mutagenesis strategies have been developed to study residue-specific contributions to GroES function:
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Linked GroES heptamers: Using groES7, a gene linking seven copies of groES, researchers can incorporate specific mutations (like I25A or I25D) in selected modules . This approach enables precise control over the number and arrangement of mutated subunits within a single heptameric ring.
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Single-ring GroEL variants: GroEL SR (single-ring) variants can be paired with different GroES constructs to study their interactions without the complexity of inter-ring communications .
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Biochemical characterization: Systematically testing the ability of mutant GroES variants to support GroEL-mediated protein folding both in vitro and in vivo reveals the functional significance of specific residues .
These approaches have demonstrated that GroES7 variants with two mutated modules can still participate in GroEL SR–mediated protein folding in vitro, while variants with two or three mutated modules can collaborate with GroEL SR for chaperone function in vivo .
What evidence suggests GroES may have functions beyond its classical role as a passive co-chaperonin?
While GroES has traditionally been viewed as merely a "shielding" or lid-like component of the GroE system, recent research suggests more active roles:
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Studies by Moparthi et al. indicate that GroES can mediate protein folding dynamic remodeling individually in vitro
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GroES may actively contribute to protein unfolding and compression processes typically attributed solely to GroEL binding
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The finding that certain GroES7 variants with multiple mutated subunits can still function suggests a more nuanced role than previously thought
How do the reaction cycles differ between bacterial and human mitochondrial chaperonin systems?
The reaction cycles of bacterial and human mitochondrial chaperonin systems show fundamental differences:
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The bacterial GroEL/GroES system undergoes two distinct cycles:
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The human mtHsp60-mtHsp10 system functions differently:
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The interring contacts between the two systems differ significantly:
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The mHsp60 rings in the football structure have more than twice the contact surface area compared to GroEL rings
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Key contact residues are altered, including the replacement of A109 in GroEL with a K109-E105 salt bridge in mHsp60
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New symmetric interactions form between A10 residues (hydrophobic) and D11 residues (hydrogen bonding) in mHsp60
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These structural and mechanistic differences likely contribute to the unique functional adaptations of the human mitochondrial chaperonin system compared to its bacterial evolutionary ancestor.
How are mutations in human mitochondrial chaperonins linked to neurodegenerative disorders?
Mutations in human mtHsp60 have been identified in three neurodegenerative genetic disorders . The essential nature of this chaperonin system is demonstrated by:
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Embryonic lethality in mice resulting from inactivation of the mtHsp60 gene
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The identification of specific disease-causing mutations with neurological implications
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Mitochondrial dysfunction as a common feature in multiple neurodegenerative diseases
Understanding the structural and functional consequences of these mutations provides insights into disease mechanisms. For example, mutations that affect the ability of mtHsp60 to properly fold client proteins or interact with mtHsp10 could lead to protein misfolding, aggregation, and eventual neurodegeneration.
What are the potential applications of single-ring chaperonin research for understanding human mitochondrial chaperonins?
Studies on functional single-ring bacterial chaperonin systems provide valuable insights into the human mitochondrial chaperonin system:
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The GroEL SR (single-ring) variants and their interactions with GroES serve as simplified models for understanding mtHsp60-mtHsp10 interactions
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Evolutionary insights can be gained by studying how the double-ring bacterial system has evolved to the functionally distinct mitochondrial system
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The development of functional single-ring bacterial chaperonin systems may provide experimental tools for:
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Testing substrate protein folding mechanisms
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Screening for compounds that modulate chaperonin function
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Developing potential therapeutic approaches for chaperonin-related disorders
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This cross-species comparative approach bridges the gap between bacterial model systems and human mitochondrial chaperonins, potentially accelerating progress in understanding and treating neurodegenerative disorders associated with chaperonin dysfunction.
What are the best experimental systems for studying human mtHsp60-mtHsp10 interactions?
Researchers new to the field should consider these experimental approaches:
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Heterologous expression systems in E. coli for producing recombinant mtHsp60 and mtHsp10
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Mutational analysis targeting key residues identified through comparative analysis with bacterial systems
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In vitro reconstitution of chaperonin-mediated folding using fluorescent substrate proteins
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Cell-based assays in mammalian systems to validate findings in a more physiologically relevant context
When designing experiments, it's important to consider the differences between bacterial and mitochondrial systems, particularly in terms of oligomeric state, nucleotide requirements, and substrate specificity.
How can researchers address the challenges of studying dynamic chaperonin complexes?
Studying dynamic protein complexes like chaperonins presents several methodological challenges:
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The transient nature of certain conformational states makes them difficult to capture
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The complexity of multi-subunit assemblies with multiple nucleotide binding sites
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The challenge of distinguishing between different functional states
To address these challenges, researchers can:
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Use mutants that arrest the chaperonin cycle at specific points (like the mHsp60 E321K mutant)
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Employ time-resolved techniques to capture transient states
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Develop novel fusion proteins that enable real-time monitoring of conformational changes
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Combine structural approaches with functional assays to correlate structure with function
These methodological considerations are essential for new researchers entering the field and seeking to make meaningful contributions to our understanding of these complex molecular machines.
Product Science Overview
GroES, also known as Heat Shock Protein 10 (HSP10), is a molecular chaperone that plays a crucial role in protein folding under both normal and stress conditions. It is a co-chaperonin that works in conjunction with GroEL (HSP60) to ensure proper protein folding, preventing misfolding and aggregation that can lead to cellular dysfunction .
GroES is composed of a single heptameric ring of 10 kDa subunits that binds to the ends of the GroEL cylinder. The interaction between GroES and GroEL is ATP-dependent. When ATP binds to GroEL, it induces a conformational change that allows GroES to bind, forming a complex that encloses the substrate protein within the GroEL cavity. This encapsulation provides an isolated environment for the substrate protein to fold correctly .
The GroES-GroEL complex operates through a cycle of binding and release driven by ATP hydrolysis. Initially, GroES binds to GroEL in the presence of ATP, causing a conformational change in GroEL that encloses the substrate protein. ATP hydrolysis then destabilizes the GroES-GroEL complex, leading to the release of GroES and the properly folded substrate protein .
Recombinant human GroES is typically produced in Escherichia coli (E. coli) using recombinant DNA technology. The GroES gene is amplified by polymerase chain reaction (PCR) and cloned into an expression vector. This vector is then introduced into E. coli cells, which express the GroES protein. The protein is subsequently purified using conventional chromatographic techniques to achieve high purity levels .
Recombinant GroES is used extensively in research to study protein folding mechanisms and chaperone functions. It is also employed in various biotechnological applications where proper protein folding is critical. Additionally, understanding the function and mechanism of GroES can provide insights into diseases caused by protein misfolding and aggregation .
