Recombinant Trachypithecus cristatus Cytochrome c (CYCS)

What is Trachypithecus cristatus Cytochrome c and why is it significant for research?

Trachypithecus cristatus (silvered leaf monkey) Cytochrome c is a small heme-containing protein involved in electron transfer processes within the mitochondrial respiratory chain. This protein is of significant research interest due to its evolutionary conservation combined with primate-specific adaptations. Cytochrome c is an ancient protein that first appeared in anaerobic photosynthetic bacteria over 3 billion years ago and has since been highly conserved across species . The T. cristatus variant provides valuable insights into primate-specific evolutionary adaptations of this essential protein.

The significance of studying T. cristatus Cytochrome c lies in its position within primate evolution, which allows researchers to investigate evolutionary changes in protein structure and function. Like all cytochrome c proteins, it features a highly conserved ring of positively charged lysines surrounding the heme crevice that form electrostatic interactions with negatively charged Asp and Glu residues at the binding sites of its electron transfer partners . Studying the specific variations in T. cristatus Cytochrome c compared to human and other primate versions can elucidate evolutionary patterns and functional adaptations in this critical component of cellular respiration.

How does Trachypithecus cristatus Cytochrome c compare structurally to human Cytochrome c?

The structural comparison between T. cristatus and human Cytochrome c reveals both conservation and species-specific differences that reflect evolutionary adaptations. Both proteins maintain the characteristic folding pattern of cytochrome c with the heme group covalently attached to the protein via thioether bonds to cysteine residues. The heme iron in both species is liganded by a histidine nitrogen and a methionine sulfur, which is a hallmark of cytochrome c proteins across species .

T. cristatus Cytochrome c, like its human counterpart, possesses a distinctive ring of positively charged lysine residues surrounding the heme crevice, which are critical for electrostatic interactions with partner proteins in the electron transport chain. These lysine residues (typically including positions 8, 13, 27, 72, 79, 86, and 87) are highly conserved across species due to their functional importance . Any amino acid substitutions between the two species likely occur in regions less critical for function or may represent adaptive changes that maintain function while accommodating species-specific interaction partners. The chromosomal homologies between human and T. cristatus have been extensively studied, providing a genetic context for understanding protein-level differences .

What are the recommended expression systems for producing recombinant Trachypithecus cristatus Cytochrome c?

For recombinant expression of T. cristatus Cytochrome c, E. coli-based systems utilizing the System I (CcmABCDEFGH) bacterial cytochrome c biogenesis pathway are highly recommended as they provide a simple and efficient method for producing functional holocytochrome c . This approach involves co-expression of the T. cristatus CYCS gene with the necessary machinery for proper heme attachment and protein folding.

The recommended methodology involves cloning the CYCS gene from T. cristatus into an expression vector compatible with E. coli strains engineered to express the complete CcmABCDEFGH system. These specialized strains ensure proper covalent attachment of the heme group to the cysteine residues in the CXXCH motif of the protein. Researchers should optimize expression conditions including temperature (typically 25-30°C rather than 37°C), induction timing, and duration to maximize the yield of correctly folded holocytochrome c with properly attached heme. Following expression, purification typically involves cell lysis followed by a combination of ion-exchange chromatography and size exclusion chromatography, taking advantage of the protein's small size (approximately 12 kDa) and positive charge at physiological pH . Successful expression can be verified through heme staining of separated proteins, which provides a rapid assessment of properly formed holocytochrome c.

How should researchers design primers for PCR amplification of the Trachypithecus cristatus CYCS gene?

When designing primers for PCR amplification of the T. cristatus CYCS gene, researchers should consider several critical factors to ensure successful amplification and subsequent cloning. First, if working directly with T. cristatus samples, researchers should consult the chromosomal mapping information to locate the CYCS gene correctly. Studies have shown that T. cristatus has a unique chromosomal arrangement compared to humans, with 69 evolutionary conserved breakpoints identified in comparative analyses .

For optimal primer design, researchers should incorporate restriction enzyme sites compatible with their chosen expression vector, ensuring these sites are not present within the gene sequence itself. A 5-6 nucleotide overhang should be included before the restriction site to ensure efficient enzyme cutting. The primers should have complementarity of 18-25 nucleotides to the target sequence with a GC content between 40-60% for stable annealing. The melting temperatures (Tm) of both primers should be within 2-4°C of each other, typically in the range of 55-65°C for standard PCR protocols. Researchers should avoid primer designs that could form secondary structures or primer-dimers, which can reduce amplification efficiency.

If the complete genome sequence of T. cristatus is not available or accessible, researchers may need to design degenerate primers based on conserved regions of CYCS genes from closely related primate species. Alternatively, if working with synthesized gene constructs, codon optimization for E. coli expression should be considered while maintaining critical amino acid residues, particularly those involved in heme binding and protein-protein interactions as identified in structural studies of cytochrome c .

What purification strategy yields the highest activity for recombinant Trachypithecus cristatus Cytochrome c?

An optimal purification strategy for recombinant T. cristatus Cytochrome c should maximize both yield and functional activity. Based on established protocols for cytochrome c purification, a multi-step approach is recommended. Following recombinant expression in E. coli using the System I cytochrome c biogenesis pathway, cells should be harvested and lysed using gentle methods such as freeze-thaw cycles or mild detergent treatment to preserve protein structure and heme attachment .

The initial purification step should utilize ion-exchange chromatography, typically with a cation exchange resin (e.g., CM-Sepharose) since cytochrome c is positively charged at physiological pH. A gradient elution with increasing salt concentration (typically 0-500 mM NaCl) effectively separates cytochrome c from many bacterial proteins. This step should be followed by size exclusion chromatography to remove any remaining impurities based on molecular size. For applications requiring extremely high purity, an additional chromatographic step such as hydrophobic interaction chromatography may be beneficial.

Throughout the purification process, researchers should monitor both protein concentration (Bradford or BCA assay) and heme content (absorbance at 410 nm for oxidized cytochrome c). The ratio of A410/A280 provides a useful measure of heme incorporation, with values approaching 5 indicating highly pure holocytochrome c. Functional activity should be assessed through standard electron transfer assays, such as reduction by ascorbate and oxidation by cytochrome c oxidase or artificial electron acceptors. For maximum stability, the purified protein should be stored in phosphate buffer (pH 7.0-7.4) with minimal exposure to freeze-thaw cycles, and when possible, maintained in the oxidized state which is generally more stable .

How can researchers verify the correct folding and heme incorporation in recombinantly expressed Trachypithecus cristatus Cytochrome c?

Verification of correct folding and heme incorporation is crucial for ensuring the functional integrity of recombinant T. cristatus Cytochrome c. Researchers should implement several complementary analytical techniques to comprehensively assess protein quality. The primary verification method is UV-visible spectroscopy, which provides characteristic absorption spectra for properly folded holocytochrome c. The oxidized (ferric) form typically shows a strong Soret band at approximately 410 nm and weaker α and β bands at approximately 530 nm and 550 nm respectively . Upon reduction (ferrous form), the Soret band shifts to approximately 415 nm, while the α and β bands become more pronounced at approximately 550 nm and 520 nm.

Heme incorporation can be specifically assessed using a heme stain technique following SDS-PAGE separation. This technique relies on the peroxidase activity of heme-containing proteins when exposed to chemiluminescent substrates, allowing visualization of bands containing properly incorporated heme . Circular dichroism (CD) spectroscopy provides additional confirmation of proper folding by analyzing the secondary structure content of the protein, which should show the characteristic alpha-helical pattern of cytochrome c.

For definitive assessment of functional integrity, researchers should conduct electron transfer activity assays. These may include reduction kinetics with ascorbate or cytochrome b5, and oxidation kinetics with cytochrome c oxidase or artificial electron acceptors such as ferricyanide. The measured rate constants can be compared with those reported for other mammalian cytochrome c proteins; properly folded T. cristatus Cytochrome c should exhibit rate constants on the order of 10^4-10^6 s^-1 for these reactions, similar to those observed with other mammalian cytochrome c proteins .

How does phosphorylation affect the function of Trachypithecus cristatus Cytochrome c compared to human Cytochrome c?

Phosphorylation represents a critical post-translational modification that can significantly alter cytochrome c function in primates, including T. cristatus. Human cytochrome c can be phosphorylated at five different residues - Tyr97, Tyr48, Thr28, Ser47, and Thr58 - in different tissues, suggesting tissue-specific regulation of its functions . For T. cristatus Cytochrome c, the conservation of these phosphorylation sites would be expected due to the general conservation of cytochrome c structure across primates, but potential species-specific differences might exist that could affect regulation.

Studies with phosphomimetic mutants of human cytochrome c (where glutamate replaces the phosphorylated residue to mimic the negative charge) have demonstrated significant functional consequences. The T28E, S47E, and Y48E mutations increase the dissociation rate constant from binding partners, suggesting that phosphorylation at these sites regulates interaction dynamics in the electron transport chain . In the context of T. cristatus Cytochrome c, researchers should investigate whether the same phosphorylation sites exist and if the functional consequences mirror those observed in human cytochrome c.

To experimentally address this question, researchers should consider generating phosphomimetic mutants of T. cristatus Cytochrome c (e.g., T28E, S47E, Y48E, Y97E) and comparing their electron transfer kinetics with the wild-type protein. Laser kinetic studies similar to those performed with human cytochrome c would be particularly informative for measuring association and dissociation rate constants with electron transfer partners. Additionally, structural studies using X-ray crystallography or NMR spectroscopy could reveal how phosphorylation-induced conformational changes might differ between human and T. cristatus Cytochrome c, potentially providing insights into primate-specific regulatory mechanisms of this essential protein.

What are the key considerations when designing electron transfer experiments with Trachypithecus cristatus Cytochrome c?

Designing electron transfer experiments with T. cristatus Cytochrome c requires careful consideration of several factors to ensure meaningful and reproducible results. First, researchers must select appropriate electron transfer partners that interact with cytochrome c. These typically include upstream components like cytochrome bc1 complex and downstream components like cytochrome c oxidase. Due to the conserved nature of the electron transport chain, mammalian (bovine or rat) complexes are often suitable partners for T. cristatus Cytochrome c, though species-specific differences in binding interfaces may affect kinetics .

The methodology should include preparation of ruthenium-labeled derivatives of T. cristatus Cytochrome c (such as Ru-39-Cc or Ru-72-Cc) for laser-activated electron transfer studies. This approach allows precise measurement of intracomplex electron transfer rates, which can provide insights into the evolutionary adaptations of T. cristatus Cytochrome c . The experimental design should control for factors that affect electron transfer rates, including temperature (typically 25°C), ionic strength (which modulates electrostatic interactions), pH (physiological range 7.0-7.4), and redox potential.

A comprehensive experimental plan should include:

Analysis of results should focus on identifying any significant differences in electron transfer properties between T. cristatus Cytochrome c and human or other primate cytochromes, which could reveal evolutionary adaptations specific to this species .

How can researchers investigate the role of specific lysine residues in Trachypithecus cristatus Cytochrome c function?

Investigating the role of specific lysine residues in T. cristatus Cytochrome c function requires a strategic approach combining mutagenesis, binding studies, and electron transfer measurements. The conserved ring of lysine residues (typically including positions 8, 13, 27, 72, 79, 86, and 87) surrounding the heme crevice plays a crucial role in electrostatic interactions with negatively charged residues on partner proteins . These interactions are essential for proper orientation and efficient electron transfer.

A systematic research approach should begin with site-directed mutagenesis to create single lysine-to-alanine substitutions for each lysine residue in the conserved ring. Additionally, researchers should consider creating lysine-to-arginine mutants, which maintain the positive charge but alter the structure, to distinguish between charge-dependent and structure-dependent effects. Following expression and purification using the System I cytochrome c biogenesis pathway in E. coli , each mutant should undergo comprehensive functional characterization.

Binding assays with physiological partners (cytochrome bc1 complex and cytochrome c oxidase) using techniques such as isothermal titration calorimetry, surface plasmon resonance, or fluorescence anisotropy can quantify the effects of mutations on binding affinity and kinetics. Laser flash photolysis experiments with ruthenium-labeled derivatives can directly measure intracomplex electron transfer rates for each mutant . Chemical modification studies using reagents specific for lysine residues (e.g., N-hydroxysuccinimide esters) provide complementary information about surface accessibility.

Based on studies with other cytochrome c proteins, researchers should expect mutations of lysines 8, 13, 27, 72, 79, 86, and 87 to significantly decrease the rate of complex formation with cytochrome bc1 and potentially alter electron transfer kinetics . Any species-specific differences in the effects of these mutations between T. cristatus and human cytochrome c could reveal evolutionary adaptations in the electron transport chain of this primate species.

What are common challenges in expressing recombinant Trachypithecus cristatus Cytochrome c and how can they be addressed?

Recombinant expression of T. cristatus Cytochrome c presents several challenges that researchers should anticipate and address methodically. One of the most common issues is incomplete heme incorporation, resulting in a mixture of apo- and holocytochrome c. This problem typically manifests as a lower than expected A410/A280 ratio (below 4.5) and reduced functional activity. To address this issue, researchers should optimize the expression of the CcmABCDEFGH system components by adjusting inducer concentrations or using different promoter systems . Additionally, supplementing the growth medium with δ-aminolevulinic acid (a heme precursor) at 1-2 mM can enhance heme biosynthesis.

Another frequent challenge is protein misfolding or aggregation, particularly when expression levels are high. This can be mitigated by lowering the expression temperature to 16-25°C, reducing inducer concentration, and extending the expression duration. Co-expression with molecular chaperones like GroEL/GroES may also improve folding efficiency. If inclusion bodies form despite these measures, researchers can attempt refolding procedures specific for cytochrome c, though these are generally less successful than optimizing conditions for soluble expression.

Purification challenges include co-purification of bacterial cytochromes or heme-binding proteins. This can be addressed by incorporating additional purification steps such as hydroxyapatite chromatography, which effectively separates different cytochrome species. Consistent monitoring of spectral properties (A410/A280 ratio, α/β band ratios) throughout purification helps identify fractions containing pure, correctly folded holocytochrome c.

How should researchers interpret and resolve contradictory kinetic data when comparing Trachypithecus cristatus Cytochrome c with human Cytochrome c?

When researchers encounter contradictory kinetic data in comparative studies of T. cristatus and human Cytochrome c, a systematic approach to interpretation and resolution is essential. Contradictions may arise from experimental variables, intrinsic properties of the proteins, or a combination of factors. The first step should be a thorough examination of experimental conditions, as parameters such as pH, ionic strength, temperature, and buffer composition significantly influence cytochrome c kinetics. Even small differences in these conditions can lead to apparent contradictions in measured rate constants.

Another common source of contradictory data is protein heterogeneity. Researchers should verify that both protein preparations have comparable purity (>95%), complete heme incorporation (A410/A280 ratio above 4.5), and are in the same redox state. Post-translational modifications, particularly phosphorylation, can dramatically alter cytochrome c kinetics . Therefore, researchers should characterize the modification status of both proteins using mass spectrometry or phosphorylation-specific detection methods.

The interpretation of kinetic differences should consider evolutionary context. If consistent differences are observed under controlled conditions, these may represent genuine species-specific adaptations. Researchers should evaluate whether the observed differences correlate with known sequence variations between T. cristatus and human Cytochrome c, particularly those affecting the lysine ring around the heme crevice or interaction interfaces with electron transfer partners .

To resolve contradictory data, researchers should:

• Establish standardized experimental conditions and apply them consistently across comparative studies

• Perform measurements using multiple complementary techniques (e.g., steady-state kinetics, stopped-flow, and laser flash photolysis)

• Generate concentration-dependent kinetic profiles rather than single-point measurements

• Include appropriate controls, such as well-characterized cytochrome c variants from other species

• Apply statistical analysis to determine whether differences are significant

By applying this systematic approach, researchers can determine whether kinetic differences represent experimental artifacts or genuine evolutionary adaptations in T. cristatus Cytochrome c compared to the human protein .

What statistical approaches are most appropriate for analyzing evolutionary rate differences in Trachypithecus cristatus Cytochrome c compared to other primates?

For specific hypothesis testing regarding accelerated evolution in T. cristatus Cytochrome c, likelihood ratio tests comparing nested models (e.g., one with uniform rates across all branches versus one allowing the T. cristatus branch to have a different rate) provide statistical support for lineage-specific rate variations. Bayes factors can also be calculated to compare non-nested models. Researchers should be cautious about multiple testing issues and apply appropriate corrections (e.g., Bonferroni or false discovery rate methods) when testing multiple hypotheses.

Codon-based models that distinguish between synonymous (dS) and nonsynonymous (dN) substitution rates are particularly informative for detecting selection. A dN/dS ratio (ω) significantly greater than 1 in the T. cristatus lineage would indicate positive selection, while ω significantly less than 1 indicates purifying selection. Site-specific models can identify specific amino acid positions under selection, which should be mapped to the protein structure to evaluate functional implications.

To provide context for these analyses, researchers should draw connections to the chromosomal evolutionary patterns observed in T. cristatus. The species exhibits unique chromosomal arrangements with 69 evolutionary conserved breakpoints compared to humans , and understanding whether these genomic rearrangements correlate with protein evolution patterns could provide insights into the mechanisms driving cytochrome c evolution in this lineage.

How does the electron transfer efficiency of Trachypithecus cristatus Cytochrome c compare with other primate species?

The electron transfer efficiency of T. cristatus Cytochrome c compared to other primate species provides valuable insights into the functional evolution of this protein in different lineages. Based on the structural conservation observed in cytochrome c across species, electron transfer efficiencies would generally be expected to be similar, but subtle evolutionary adaptations may lead to measurable differences. To comprehensively assess these differences, researchers should measure electron transfer parameters with standardized partners (typically bovine cytochrome c oxidase and cytochrome bc1 complex) under identical conditions.

Key parameters to measure include the second-order rate constant for association (kon), the first-order rate constant for dissociation (koff), the equilibrium dissociation constant (Kd = koff/kon), and the first-order rate constant for intracomplex electron transfer (ket). Based on studies with human cytochrome c and other mammalian variants, ket values with cytochrome c oxidase typically fall in the range of 10^4-10^6 s^-1, while association rate constants are typically in the range of 10^6-10^8 M^-1s^-1 .

A comparative experimental design should include cytochrome c from humans, T. cristatus, and other primates representing different evolutionary lineages (e.g., great apes, Old World monkeys, New World monkeys). The following table illustrates the type of data that would be collected and compared:

Variations in these parameters among primate species should be interpreted in light of sequence differences, particularly those affecting the conserved lysine ring around the heme crevice. These lysines (typically at positions 8, 13, 27, 72, 79, 86, and 87) are critical for electrostatic interactions with electron transfer partners . Species-specific substitutions affecting charge distribution or heme environment could explain observed differences in electron transfer efficiency.

What insights can be gained from studying the evolution of Trachypithecus cristatus Cytochrome c in the context of primate divergence?

Studying T. cristatus Cytochrome c evolution in the context of primate divergence offers unique insights into protein evolution under different selective pressures. Cytochrome c is generally highly conserved due to its critical role in cellular respiration, making any accepted mutations potentially significant for understanding adaptive evolution. T. cristatus represents the Colobinae subfamily of Old World monkeys, which diverged from Cercopithecinae (including macaques and baboons) approximately 15-20 million years ago, providing sufficient evolutionary time for functional adaptations to emerge.

One particularly valuable approach is to analyze the rate of cytochrome c evolution along different primate lineages. Previous studies have identified accelerated evolution of cytochrome c in higher primates , suggesting potential adaptive changes. Researchers should determine whether T. cristatus follows this pattern or shows lineage-specific acceleration or conservation. Mapping amino acid substitutions onto the three-dimensional structure can reveal whether changes cluster in functionally significant regions, such as the lysine ring involved in protein-protein interactions or regions affecting redox potential.

The chromosomal context of the CYCS gene in T. cristatus provides additional evolutionary insights. T. cristatus has a unique chromosomal arrangement compared to humans, with 69 evolutionary conserved breakpoints identified . Understanding whether the CYCS gene is located near such breakpoints could reveal how genomic rearrangements might influence gene expression and protein evolution. Additionally, this species possesses a multiple sex chromosome system with a male karyotype 44,XY1Y2 , which presents an opportunity to study potential sex-linked effects on cytochrome c expression or function.

Comparative studies should extend beyond sequence analysis to include expression patterns, post-translational modifications, and functional parameters. This integrative approach can reveal whether evolutionary changes in T. cristatus Cytochrome c represent neutral drift, adaptive evolution in response to specific environmental pressures, or co-evolution with interacting proteins in the electron transport chain.

How can structural modeling help predict functional differences between Trachypithecus cristatus Cytochrome c and human Cytochrome c?

Structural modeling represents a powerful approach for predicting functional differences between T. cristatus and human Cytochrome c, particularly when experimental structures are unavailable. Homology modeling using existing crystal structures of mammalian cytochrome c (typically horse or human) as templates can generate reliable structural models of T. cristatus Cytochrome c with high confidence due to the protein's high degree of conservation. Researchers should employ modern modeling software such as MODELLER, SWISS-MODEL, or AlphaFold2, with careful template selection and model validation using tools like PROCHECK and MolProbity.

Molecular dynamics (MD) simulations can extend static structural models to provide insights into dynamic properties and conformational flexibility. Typical MD simulations should run for at least 100 ns under physiological conditions (310 K, pH 7.4, 150 mM ionic strength) to capture relevant dynamics. Analysis of trajectory data can reveal differences in protein flexibility, solvent accessibility of key residues, and transient pocket formation that might influence function.

For more specific functional predictions, researchers should employ protein-protein docking simulations between the T. cristatus Cytochrome c model and structures of interaction partners (cytochrome bc1 complex and cytochrome c oxidase). These simulations can predict binding modes, contact residues, and estimate binding affinities. Comparison with equivalent simulations using human cytochrome c can highlight species-specific differences in interaction networks. Electrostatic calculations using methods such as Poisson-Boltzmann surface analysis are particularly valuable for cytochrome c due to the importance of charge-charge interactions in its function .