Recombinant Miniopterus schreibersii Cytochrome c (CYCS)

What is Miniopterus schreibersii Cytochrome c and why is it used in research?

Miniopterus schreibersii Cytochrome c is an electron carrier protein derived from the Schreiber's bent-winged bat (Miniopterus schreibersii). Like other cytochrome c proteins, it plays dual roles in cellular metabolism and apoptosis. In the electron transport chain, the oxidized form of the cytochrome c heme group accepts electrons from the cytochrome c1 subunit of cytochrome reductase and transfers them to the cytochrome oxidase complex, serving as a critical component in mitochondrial respiration . Additionally, it functions as an apoptotic signaling molecule when released from mitochondria. When anti-apoptotic Bcl-2 family members are suppressed or pro-apoptotic members are activated, mitochondrial membrane permeability changes, causing cytochrome c release into the cytosol, where it binds to Apaf-1 to trigger caspase-9 activation and subsequent apoptotic cascade .

Researchers use this protein as an experimental model for several reasons:

• It serves as an excellent model for studying protein structure, folding mechanisms, and electron transfer processes

• Its role in apoptosis makes it valuable for cell death research

• As a highly conserved protein across species, it provides insights into evolutionary relationships

• The availability of recombinant forms enables controlled experimental conditions

What expression systems are commonly used for producing recombinant Miniopterus schreibersii Cytochrome c?

Several expression systems can be employed for the production of recombinant Miniopterus schreibersii Cytochrome c, each with specific advantages depending on research requirements:

For bacterial expression specifically, the System I cytochrome c biogenesis pathway (CcmABCDEFGH) is crucial for proper heme attachment when producing holocytochrome c species in E. coli . This pathway ensures the covalent attachment of heme to the CXXCH motif in the protein sequence, which is essential for proper folding and function of the recombinant cytochrome c .

How can researchers validate the functionality of recombinant Miniopterus schreibersii Cytochrome c after expression?

Validating the functionality of recombinant Miniopterus schreibersii Cytochrome c requires a multi-faceted approach to ensure both structural integrity and biological activity:

Spectroscopic Analysis:

• UV-visible spectroscopy to confirm proper heme incorporation (characteristic absorption peaks at ~410 nm (Soret band) and ~530 nm (α-band))

• Circular dichroism to assess secondary structure integrity

• Fluorescence spectroscopy to evaluate tertiary structure

Heme Attachment Verification:

• Heme staining following SDS-PAGE separation can confirm the covalent attachment of heme to the protein, which is essential for functionality

• Peroxidase activity assay using enhanced chemiluminescence (ECL) substrates can verify the catalytic activity of the heme group

Electron Transfer Capability:

• Cyclic voltammetry to measure redox potential and electron transfer rates

• Oxygen consumption assays when included in reconstituted electron transport chain components

Apoptotic Activity Assessment:

• Cell-free apoptosis assays using isolated cytosolic extracts to verify Apaf-1 binding and caspase-9 activation

• Mitochondrial cytochrome c release assays in permeabilized cells

A comprehensive validation protocol should include both structural characterization (SDS-PAGE, HPLC, mass spectrometry) and functional assays to ensure that the recombinant protein behaves similarly to the native form in experimental conditions .

What are the key considerations when designing experiments using Miniopterus schreibersii Cytochrome c for apoptosis studies?

When designing experiments using Miniopterus schreibersii Cytochrome c for apoptosis studies, researchers should consider several critical factors:

Protein Preparation:

• Ensure high purity (≥85% as determined by SDS-PAGE) to minimize confounding factors

• Verify proper folding and heme incorporation prior to experiments

• Determine appropriate protein concentration ranges (typically 0.5-10 μM) for different cell types and experimental conditions

Experimental Design Considerations:

• Include appropriate positive controls (e.g., human or mouse cytochrome c) and negative controls (denatured protein or buffer only)

• Consider the delivery method for exogenous cytochrome c (cell permeabilization, microinjection, or liposome encapsulation)

• Account for potential species-specific interactions with apoptotic machinery components

Measurement Parameters:

• Establish clear readouts for apoptotic activation (caspase activity, PARP cleavage, DNA fragmentation)

• Define appropriate timepoints for measurements (typically 1-24 hours post-treatment)

• Include complementary assays to confirm apoptotic pathway engagement (e.g., Bax/Bak activation, mitochondrial membrane potential)

Data Analysis:

• Establish dose-response relationships for cytochrome c concentration vs. apoptotic markers

• Account for cell type-specific differences in apoptotic sensitivity

• Consider potential differences between bat cytochrome c and other mammalian orthologs in apoptotic potency

Researchers should be aware that while cytochrome c's apoptotic function is generally conserved across species, subtle sequence differences might affect its interaction with Apaf-1 and subsequent caspase activation, potentially impacting experimental outcomes when using bat-derived cytochrome c in human or rodent cellular systems.

How does the heme attachment differ between bacterial and eukaryotic expression systems for Miniopterus schreibersii Cytochrome c?

The mechanisms of heme attachment to cytochrome c differ significantly between bacterial and eukaryotic expression systems, which can impact the quality and functionality of the recombinant protein:

Bacterial Expression (E. coli):

• Naturally lacks the machinery for cytochrome c maturation

• Requires co-expression of the System I (CcmABCDEFGH) bacterial cytochrome c biogenesis pathway

• The Ccm system facilitates:

  • Heme transport to the periplasm

  • Reduction of the CXXCH motif cysteines

  • Stereospecific attachment of heme to the reduced cysteines

  • Release of the mature holocytochrome c

Yeast Expression Systems:

Mammalian and Baculovirus Systems:

• Employ System III similar to yeast but with species-specific CCHL variants

• Provide more authentic post-translational modifications

• May result in more native-like heme environment and protein conformation

The bacterial System I pathway involves multiple proteins and is more complex but can be efficiently engineered in E. coli for high-yield production. Analysis of heme attachment can be performed via heme staining following SDS-PAGE, which allows visualization of covalently bound heme groups . Research has shown that proper heme attachment is critical not only for electron transfer activity but also for the protein's stability and ability to interact with apoptotic machinery components.

What methodological approaches can be used to study the electron transfer capabilities of Miniopterus schreibersii Cytochrome c compared to other mammalian cytochromes?

Advanced methodological approaches for comparative electron transfer studies of Miniopterus schreibersii Cytochrome c include:

Kinetic Analysis Techniques:

• Stopped-flow spectroscopy to measure real-time electron transfer rates between cytochrome c and its redox partners

• Flash photolysis with ruthenium complexes as photoinducible electron donors to trigger and measure electron transfer events

• Pulse radiolysis to generate and monitor rapid electron transfer reactions

Electrochemical Methods:

• Protein film voltammetry for direct measurement of electron transfer kinetics at electrode surfaces

• Square wave voltammetry to determine formal reduction potentials with high precision

• Impedance spectroscopy to characterize electron transfer resistance parameters

Computational and Structural Approaches:

• Molecular dynamics simulations to model electron transfer pathways between cytochrome c and its binding partners

• Quantum mechanical/molecular mechanical (QM/MM) calculations to predict electron tunneling probabilities

• NMR paramagnetic relaxation enhancement experiments to map electron transfer pathways within the protein structure

Comparative Experimental Design:

These methodologies allow researchers to quantitatively compare the electron transfer properties of Miniopterus schreibersii Cytochrome c with other mammalian orthologs, potentially revealing evolutionary adaptations in this metabolically demanding bat species .

How can researchers troubleshoot inconsistent results in apoptosis assays using recombinant Miniopterus schreibersii Cytochrome c?

When encountering inconsistent results in apoptosis assays using recombinant Miniopterus schreibersii Cytochrome c, researchers should systematically investigate several potential sources of variability:

Protein Quality Issues:

• Assess protein degradation using SDS-PAGE and Western blotting before each experiment

• Verify heme attachment status using absorbance spectroscopy (A410/A280 ratio should be >4 for properly folded holocytochrome c)

• Evaluate oxidation state of the protein, as oxidized and reduced forms may have different apoptotic potencies

• Check for batch-to-batch variations by comparing spectral properties and activity of different preparations

Experimental Variables:

• Standardize cell permeabilization techniques (digitonin concentration and exposure time are critical parameters)

• Control cytosolic pH and ionic strength, as these significantly affect cytochrome c-Apaf-1 interactions

• Ensure consistent dATP/ATP concentrations, which are essential cofactors for apoptosome formation

• Monitor potential inhibitors present in the experimental system (e.g., IAPs, XIAP)

Species-Specific Considerations:

• Determine compatibility between bat cytochrome c and the experimental cellular system (human, mouse, etc.)

• Consider testing interaction efficiency between Miniopterus schreibersii Cytochrome c and Apaf-1 from your experimental system using pull-down assays

• Perform side-by-side comparisons with human cytochrome c under identical conditions

Analytical Approach to Troubleshooting:

• Perform systematic titration experiments to establish dose-response relationships

• Implement time-course studies to identify potential kinetic differences

• Use defined, cell-free systems to eliminate cellular variables before moving to intact cells

• Consider structural analysis (CD spectroscopy, limited proteolysis) to confirm protein conformation

The binding of cytochrome c to Apaf-1 triggers the activation of caspase-9, which then accelerates apoptosis by activating other caspases. Any inconsistencies in this pathway can lead to variable experimental outcomes .

What advanced analytical techniques can be employed to study the structural dynamics of Miniopterus schreibersii Cytochrome c under varying experimental conditions?

Advanced analytical techniques for studying the structural dynamics of Miniopterus schreibersii Cytochrome c include:

High-Resolution Spectroscopic Methods:

• Multidimensional NMR spectroscopy to map residue-specific conformational changes and dynamics (15N-HSQC, NOESY, TOCSY)

• Resonance Raman spectroscopy to characterize the heme environment and its response to changing conditions

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to identify regions of structural flexibility and solvent accessibility

Biophysical Characterization Techniques:

Advanced Computational Approaches:

• Molecular dynamics simulations with enhanced sampling techniques to explore conformational landscapes

• Markov state modeling to identify metastable states and transition pathways

• Machine learning analysis of structural ensembles to identify patterns in conformational changes

Experimental Conditions Matrix:

These techniques can reveal how the structural dynamics of Miniopterus schreibersii Cytochrome c respond to environmental changes, providing insights into its functional adaptations. For instance, bat cytochrome c may exhibit unique structural stability characteristics that reflect evolutionary adaptations to the high metabolic demands of flight and potential hypoxic conditions experienced by these mammals .