GroES Human, His

What is GroES Human, His Tag and what are its key properties?

GroES Human, His Tag is a 12.0 kDa recombinant protein containing 111 amino acid residues of human GroES (HSP10) with an additional 10 amino acid N-terminal histidine tag. It belongs to the molecular chaperone family critical for efficient protein folding under both normal and stress conditions. The protein has several synonyms including CPN10, GROES, HSP10, HSPE1, Chaperonin-10, 10 kDa heat shock protein mitochondrial, 10 kDa chaperonin, and Early-pregnancy factor (EPF) .

The complete amino acid sequence is:
MKHHHHHHAS AGQAFRKFLP LFDRVLVERS AAETVTKGGI MLPEKSQGKV LQATVVAVGS GSKGKGGEIQ PVSVKVGDKV LLPEYGGTKV VLDDKDYFLF RDGDILGKYV D

How does human GroES function in protein folding?

Human GroES (HSP10) functions as a co-chaperonin that works in coordination with HSP60. The mechanism involves binding to HSP60 in the presence of ATP, causing a conformational change in HSP60 that encloses protein substrates within the complex. The ATP hydrolysis by HSP60 subsequently destabilizes the HSP10-HSP60 complex, allowing it to dissociate and release the properly folded substrate protein . Unlike the prokaryotic GroEL which operates as a double-ring structure, human Hsp60-Hsp10 functions as a single ring without transitioning through a double-ring intermediate .

What structural differences exist between prokaryotic GroEL-GroES and human Hsp60-Hsp10?

The main structural difference between prokaryotic GroEL-GroES and human Hsp60-Hsp10 is their quaternary structure arrangement. While GroEL functions through a two-stroke engine action with a double-ring structure where ATP binding and hydrolysis in one ring controls GroES release from the opposite ring, human Hsp60-Hsp10 operates as a single-ring system . Despite these structural differences, there is approximately 51% sequence identity between GroEL and human Hsp60, which contributes to functional similarities while maintaining distinct operational mechanisms .

What are the optimal storage and reconstitution conditions for GroES Human, His Tag?

For optimal storage, lyophilized GroES His Tag should be stored at -20°C. When reconstituting the protein, it is recommended to add deionized water to prepare a working stock solution of approximately 0.5mg/ml and allow the lyophilized pellet to dissolve completely .

After reconstitution, the protein should be aliquoted to avoid repeated freezing and thawing cycles. Reconstituted GroES His can be stored at 4°C for a limited period; stability tests show no significant changes after two weeks at 4°C . For applications requiring sterility, the product should be filtered through an appropriate sterile filter as it is not provided in sterile form .

How can researchers verify the purity and activity of GroES Human, His Tag?

The purity of GroES Human, His Tag should be verified using SDS-PAGE analysis, with commercially available preparations typically showing greater than 90% purity . Functional activity can be assessed through in vitro protein folding assays, such as measuring the ability of the Hsp60-Hsp10 complex to fold substrate proteins like malate dehydrogenase in the presence of ATP .

When comparing activity between different chaperonin systems, researchers should consider using standardized conditions (e.g., 37°C or 43°C) as demonstrated in previous studies where Hsp60-Hsp10 folded malate dehydrogenase with comparable efficiency to GroEL-GroES at these temperatures .

How can GroES Human, His be used in complementation studies?

GroES Human, His (as part of the Hsp60-Hsp10 system) can be employed in complementation studies to investigate chaperonin function across species. Expression of human Hsp60-Hsp10 has been shown to restore viability to E. coli cells lacking GroEL-GroES expression, demonstrating that the human mitochondrial chaperonin can carry out essential in vivo functions of the bacterial system .

For such experiments, researchers should use bacterial strains engineered to have their native groE operon under strict regulatory control, such as the arabinose-inducible promoter system used in strain MGM100. By expressing human Hsp60-Hsp10 in these strains and testing growth under conditions where native GroEL-GroES expression is repressed, researchers can assess the functional substitution capabilities of the human chaperonin system .

What are the temperature limitations of Hsp60-Hsp10 in experimental systems?

In heterologous expression systems, Hsp60-Hsp10 exhibits temperature-dependent functionality limitations. Experimental evidence indicates that E. coli strains expressing human Hsp60-Hsp10 instead of GroEL-GroES can grow at temperatures up to 42°C but fail to grow at 43°C .

Interestingly, this limitation doesn't appear to be due to intrinsic thermolability of the human chaperonin complex, as in vitro folding assays demonstrated that Hsp60-Hsp10 folds malate dehydrogenase with equivalent efficiency to GroEL-GroES at both 37°C and 43°C . The growth limitation at higher temperatures in vivo is more likely attributed to insufficient expression levels of Hsp60, potentially due to codon usage differences or protein instability in the bacterial host .

How do cochaperonin specificities differ between GroEL and Hsp60?

Research has revealed important differences in cochaperonin specificity between GroEL and Hsp60. GroEL demonstrates flexibility in its cochaperonin partners, capable of functioning with either its native cochaperonin GroES or the human mitochondrial cochaperonin Hsp10 both in vitro and in vivo .

In contrast, human Hsp60 displays stricter specificity, folding target proteins effectively only when paired with its cognate cochaperonin Hsp10 . This specificity difference has important implications for experimental design when studying these systems in heterologous environments or when creating chimeric chaperonin complexes for functional analysis.

How can researchers design experiments to study the interaction between Hsp60 and Hsp10?

To study Hsp60-Hsp10 interactions, researchers can employ several approaches:

• Protein-protein interaction assays: Using techniques such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), or co-immunoprecipitation to measure binding affinities and kinetics.

• ATP hydrolysis assays: Measuring the ATPase activity of Hsp60 in the presence and absence of Hsp10 to understand how the cochaperonin regulates the chaperonin's enzymatic activity.

• Structural biology approaches: Employing cryo-electron microscopy or X-ray crystallography to visualize the Hsp60-Hsp10 complex at various stages of the folding cycle.

• FRET-based assays: Using fluorescently labeled Hsp60 and Hsp10 to monitor real-time assembly and disassembly of the complex under different conditions .

The experimental design should account for the single-ring structure of human Hsp60-Hsp10, which differs from the double-ring structure of bacterial GroEL-GroES .

What considerations are important when designing complementation studies with GroEL-GroES and Hsp60-Hsp10?

When designing complementation studies comparing GroEL-GroES and Hsp60-Hsp10 systems, researchers should consider:

• Expression system optimization: Human Hsp60 expression in bacterial systems may be limited by codon usage differences. Optimizing codons for bacterial expression can improve protein yields .

• Temperature conditions: Experiments should be conducted at temperatures below 43°C when using Hsp60-Hsp10 in E. coli systems, as growth limitations occur at higher temperatures .

• Mixed chaperonin effects: The potential formation of inactive mixed multimers between GroEL and Hsp60 subunits can affect results. Experimental designs should control for this by ensuring complete suppression of native chaperonin expression when testing heterologous systems .

• Quantification methods: Accurate quantification of expressed chaperonins using techniques like Western blotting with standardized controls is essential for comparative studies .

How should researchers interpret growth differences between bacterial strains expressing GroEL-GroES versus Hsp60-Hsp10?

When interpreting growth differences between bacterial strains expressing different chaperonin systems, researchers should consider multiple factors:

Researchers should differentiate between effects caused by intrinsic functional differences between the chaperonin systems versus those resulting from expression level variations or host compatibility issues .

What controls are essential when evaluating the functional substitution of GroEL-GroES by Hsp60-Hsp10?

When evaluating functional substitution between chaperonin systems, essential controls include:

• Positive control: Strains expressing native GroEL-GroES from the same promoter/vector system used for Hsp60-Hsp10 expression to establish baseline growth parameters.

• Negative control: Strains with the native groE operon under strict regulatory control but without complementing plasmids to confirm the essentiality of chaperonin function.

• Individual component controls: Testing GroEL alone, GroES alone, Hsp60 alone, and Hsp10 alone to confirm the requirement for both components of each system.

• Mixed system controls: Testing GroEL with Hsp10 and Hsp60 with GroES to assess cross-compatibility between chaperonins and cochaperonins.

• Phage infection susceptibility: As shown in the research data, phage susceptibility testing (using phages like λ, T4, T5) provides an additional functional readout for chaperonin activity .

The collected data from these controls allows for comprehensive interpretation, as demonstrated in previous studies where MGM100 strains expressing different chaperonin combinations were evaluated for growth on different media at varying temperatures .

What are promising research directions for studying GroES Human, His in relation to disease mechanisms?

Promising research directions include:

• Mitochondrial disease models: Investigating how mutations in human Hsp10 contribute to mitochondrial disorders by affecting protein folding efficiency within mitochondria.

• Neurodegenerative disease connections: Exploring the role of Hsp60-Hsp10 in preventing protein aggregation associated with neurodegenerative conditions like Alzheimer's and Parkinson's diseases.

• Cancer biology: Examining altered expression patterns of Hsp10 in various cancer types and determining whether these changes contribute to cancer cell survival under stress conditions.

• Autoimmune response modulation: Studying how Hsp10 may function in immunomodulation, given previous associations between heat shock proteins and autoimmune responses.

These investigations would benefit from the use of purified GroES Human, His in biochemical assays, structural studies, and cellular models to understand disease-relevant molecular mechanisms .

How might comparing single-ring versus double-ring chaperonin systems advance our understanding of protein folding mechanisms?

The natural divergence between single-ring (human Hsp60-Hsp10) and double-ring (bacterial GroEL-GroES) chaperonin systems provides a unique opportunity to understand fundamental aspects of protein folding mechanisms. Future research could:

• Identify substrate specificity determinants: Comparing the spectrum of proteins folded by each system to identify features that determine chaperonin specificity.

• Elucidate evolutionary adaptations: Investigating how the single-ring structure evolved and what advantages it might confer in eukaryotic cells versus the double-ring structure in prokaryotes.

• Develop hybrid systems with novel properties: Creating chimeric chaperonins that combine domains from both systems to understand the functional contributions of each domain and potentially engineer chaperonins with enhanced or specialized activities.

• Resolve the energetic efficiency question: Determining whether single-ring systems operate with different ATP efficiency compared to double-ring systems .

These comparative studies could lead to significant insights into the fundamental principles of chaperonin-mediated protein folding and potentially inspire novel therapeutic approaches targeting protein misfolding diseases.