Recombinant Chicken Cytochrome b-c1 complex subunit Rieske, mitochondrial (UQCRFS1)

What is Chicken Cytochrome b-c1 complex subunit Rieske, mitochondrial (UQCRFS1)?

UQCRFS1 is the Rieske iron-sulfur protein component of the mitochondrial cytochrome bc1 complex (complex III) in the electron transport chain. It contains an iron-sulfur cluster that facilitates electron transfer during oxidative phosphorylation. The protein is also known by several other names including Complex III subunit 5, Cytochrome b-c1 complex subunit 5, Rieske iron-sulfur protein (RISP), and Ubiquinol-cytochrome c reductase iron-sulfur subunit .

What is the significance of the iron-sulfur cluster in Rieske proteins?

The iron-sulfur cluster in Rieske proteins is crucial for electron transfer during oxidative phosphorylation. It accepts electrons from ubiquinol and transfers them to cytochrome c1. The redox potential of this cluster significantly influences the efficiency of electron transfer. In high-potential Rieske proteins (≥ +300 mV), specific serine and tyrosine residues in crucial positions affect the coordination environment of the iron-sulfur cluster .

How can I establish a Uqcrfs1 knockout model for research?

To establish a Uqcrfs1 knockout model for research, you can follow these methodological approaches:

• For cell culture models:

  • Obtain primary Uqcrfs1fl/fl MPCs (muscle progenitor cells)

  • Treat cells with adenovirus CRE (Ad4697) at an MOI of 250 per flask

  • Incubate for 72 hours to allow complete infection

  • Confirm knockout via western blot analysis

  • Use adenovirus GFP (Ad4627) as a control

• For mouse models:

  • Cross C57BL/6J Uqcrfs1fl/fl (RISPfl/fl) mice with Cre-expressing strains such as B6.Cg-Pax7 tm1(cre/ERT2)Gaka/J

  • Validate genomic deletion via PCR and protein expression via western blot

What phenotypes are observed in Uqcrfs1 knockout models?

Uqcrfs1 knockout models display several characteristic phenotypes:

• Cellular effects:

  • Significant proliferation defects compared to control Uqcrfs1Flox/Flox cells

  • Marked reduction in expression of core electron transport chain proteins

  • Altered glucose metabolism and uptake

  • Compromised mitochondrial respiratory capacity

• Compensatory mechanisms:

  • Upregulation of RNA splicing and processing machinery

  • Increased expression of amino acid transporters

  • Enhanced glycolytic capacity to generate ATP in the absence of functional electron transport

How can small molecules rescue Uqcrfs1 deficiency phenotypes?

The small molecule oxybutynin has been shown to rescue the proliferative capacity of Uqcrfs1-deficient cells through the following mechanisms:

• Metabolic effects:

  • Significantly increases glucose uptake in Uqcrfs1-/- MPCs

  • Enhances glycolytic capacity to produce ATP

  • Improves cellular bioenergetics despite impaired electron transport chain function

• Molecular targets:

  • Binds to components of the RNA splicing and processing machinery

  • Alters gene expression patterns, particularly those related to metabolism

  • Creates a distinct transcriptomic profile that enables cellular adaptation to mitochondrial dysfunction

What are the optimal storage and handling conditions for recombinant UQCRFS1 protein?

For optimal results when working with recombinant UQCRFS1 protein:

• Storage recommendations:

  • Store lyophilized powder at -20°C/-80°C upon receipt

  • Aliquot reconstituted protein to avoid repeated freeze-thaw cycles

  • Working aliquots can be stored at 4°C for up to one week

• Reconstitution protocol:

  • Briefly centrifuge the vial prior to opening

  • Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

  • Add 5-50% glycerol (final concentration) for long-term storage

  • Default recommendation is 50% glycerol as the final concentration

• Quality control:

  • Verify protein purity via SDS-PAGE (should be greater than 90%)

  • Assess functional activity before experimental use

How can I determine the redox potential of recombinant Rieske proteins?

Determining the redox potential of Rieske proteins involves several methodological approaches:

• Spectroelectrochemistry:

  • Monitor absorbance changes at wavelengths characteristic of the iron-sulfur cluster

  • Use redox mediators to facilitate electron transfer

  • Apply controlled potentials and measure spectral shifts

• Sequence-based prediction:

  • Analyze the presence of serine and tyrosine residues in crucial positions

  • High-potential Rieske proteins typically have serine and tyrosine at specific conserved sites

  • These amino acids create a characteristic hydrogen bonding network that elevates the redox potential

• Structural analysis:

  • Examine the solvent accessibility of the iron-sulfur cluster

  • Evaluate the hydrogen bonding network around the cluster

  • Consider the effects of the protein environment on cluster properties

What approaches can be used for phylogenetic analysis of Rieske proteins?

For phylogenetic analysis of Rieske proteins, consider these methodological approaches:

• Multiple sequence alignment:

  • Focus on the well-conserved region around the Rieske cluster-binding motif

  • Remove N- or C-terminal extensions that occur in individual species before tree-building

  • Use alignment software that handles conservation patterns in iron-sulfur proteins

• Tree construction:

  • Employ maximum likelihood or Bayesian inference methods

  • Use appropriate substitution models for membrane proteins

  • Evaluate tree reliability through bootstrap analysis or posterior probabilities

• Comparative analysis:

  • Examine gene order and genomic context (e.g., canonical gene order Rieske ► cytochrome b)

  • Compare sequence conservation within and between different types of Rieske proteins

  • Identify specific residues that differentiate high and low potential Rieske proteins

How does the structure of Rieske proteins relate to their function across species?

The structure-function relationship in Rieske proteins reveals important evolutionary insights:

• Conserved structural elements:

  • The iron-sulfur cluster binding motif is highly conserved across species

  • The core structure remains recognizable despite sequence divergence

  • Transmembrane helices show higher conservation than soluble domains

• Functional adaptations:

  • Bacterial and archaeal Rieske proteins may have different cofactor arrangements

  • Variations in the number of transmembrane helices exist (typically six, but some species have seven)

  • Different gene organizations across species reflect diverse evolutionary paths

• Redox tuning mechanisms:

  • Sequence variations near the iron-sulfur cluster modulate redox potential

  • High-potential variants contain specific serine and tyrosine residues at key positions

  • These structural differences enable adaptation to different bioenergetic environments

What experimental approaches can assess the impact of UQCRFS1 dysfunction on mitochondrial bioenergetics?

To evaluate the impact of UQCRFS1 dysfunction on mitochondrial bioenergetics:

• Respiratory chain analysis:

  • Measure oxygen consumption rates in intact cells or isolated mitochondria

  • Evaluate specific complex III activity using spectrophotometric assays

  • Use inhibitors like antimycin A (100 μM) to confirm complex III-specific effects

• Metabolic profiling:

  • Assess glucose uptake and metabolism

  • Measure glycolytic rates and lactate production

  • Evaluate ATP production through various metabolic pathways

• Molecular adaptation assessment:

  • Analyze transcriptomic changes using methods like Principal Component Analysis (PCA)

  • Identify differentially regulated genes and pathways

  • Characterize compensatory mechanisms that emerge in response to UQCRFS1 deficiency

How can protein thermal shift assays be used to study UQCRFS1-drug interactions?

Protein thermal shift assays provide valuable insights into UQCRFS1-drug interactions:

• Experimental approach:

  • Monitor protein thermal stability in the presence and absence of potential binding compounds

  • Proteins significantly protected from thermal degradation are likely bound to the compound

  • This technique can identify novel binding partners for UQCRFS1

• Application to oxybutynin studies:

  • Thermal shift assays revealed that oxybutynin binds to components of the RNA splicing machinery

  • This binding correlates with altered gene expression patterns

  • The technique helped identify unexpected mechanisms of action for this compound

• Screening applications:

  • Systematically test libraries of compounds for UQCRFS1 binding

  • Identify molecules that stabilize the protein or its complexes

  • Develop potential therapeutic approaches for mitochondrial disorders

What biosafety requirements apply to research with recombinant UQCRFS1?

Research with recombinant UQCRFS1 requires adherence to specific biosafety guidelines:

• Institutional approval:

  • Work involving recombinant DNA typically requires Institutional Biosafety Committee (IBC) approval

  • Submit an IBC application detailing experimental procedures and safety protocols

• Risk assessment:

  • Evaluate potential hazards associated with expression systems (e.g., viral vectors)

  • Consider the biosafety level appropriate for your specific research activities

  • Implement appropriate containment measures based on risk assessment

• Documentation requirements:

  • Maintain detailed records of experiments

  • Ensure proper training of all personnel

  • Follow institutional protocols for waste disposal and accident reporting

What considerations apply when transferring UQCRFS1 materials between institutions?

When transferring UQCRFS1 materials between institutions:

• Material Transfer Agreements (MTAs):

  • Establish appropriate MTAs for sharing recombinant materials

  • Ensure compliance with intellectual property considerations

  • Clarify restrictions on material use and data publication

• Shipping requirements:

  • Follow proper packaging and labeling requirements for biological materials

  • Include appropriate documentation with shipments

  • Ensure recipient has appropriate approvals for receiving materials

• Regulatory compliance:

  • Verify that both sending and receiving institutions have appropriate IBC approvals

  • Consider any special requirements for international transfers

  • Document all transfer processes according to institutional guidelines

How can I address protein degradation issues when working with recombinant UQCRFS1?

To minimize protein degradation when working with recombinant UQCRFS1:

• Storage optimization:

  • Avoid repeated freeze-thaw cycles

  • Store at appropriate temperatures (-20°C/-80°C for long-term)

  • Use glycerol (5-50%) as a cryoprotectant

• Handling precautions:

  • Work with the protein on ice when possible

  • Add protease inhibitors to buffers

  • Minimize exposure to harsh conditions (extreme pH, detergents)

• Quality control measures:

  • Check protein integrity via SDS-PAGE before experiments

  • Verify functional activity through appropriate assays

  • Monitor for degradation products during storage

What strategies can overcome expression challenges for recombinant UQCRFS1?

For improved expression of recombinant UQCRFS1:

• Expression system optimization:

  • E. coli is commonly used for UQCRFS1 expression (as seen in the commercially available product)

  • Consider codon optimization for the expression host

  • Evaluate different fusion tags (the commercial product uses an N-terminal His tag)

• Culture conditions:

  • Optimize induction parameters (temperature, inducer concentration, duration)

  • Evaluate different media compositions

  • Consider co-expression of molecular chaperones to aid folding

• Purification strategies:

  • Implement multi-step purification protocols

  • Use affinity chromatography (e.g., IMAC for His-tagged proteins)

  • Include quality control steps to verify protein integrity and purity