Protein-L Cys

What is L-Cysteine and what makes it unique among amino acids?

L-cysteine (L-Cys) is a non-essential amino acid characterized by a thiol group (-SH) at the end of its side chain. This thiol group is what distinguishes L-Cys from other amino acids and is responsible for its high reactivity and diverse biological functions. The unique chemical properties of the thiol group enable L-Cys to participate in redox reactions, form disulfide bridges, and coordinate with metal ions, making it one of the most functionally versatile amino acids in proteins . The reactivity of the thiol group is central to the structural roles of L-Cys in proteins, particularly in establishing covalent bonds that stabilize tertiary structures, and in catalytic mechanisms of various enzymes .

What role does L-Cysteine play in protein folding and stability?

L-Cysteine plays a fundamental role in protein folding and stability primarily through its ability to form disulfide bridges. These covalent bonds between the sulfur atoms of two cysteine residues are crucial for:

• Stabilizing tertiary protein structures

• Establishing and maintaining quaternary structural arrangements

• Providing mechanical strength to extracellular proteins

• Protecting proteins from harsh environmental conditions

Disulfide bonds formed by cysteine residues are particularly important for proteins that function extracellularly, where they help maintain structural integrity in pH-variable, protease-rich environments . The conservation of cysteine residues across evolutionarily diverse proteins underscores their essential role in protein folding, structure determination, and functional stability .

How prevalent are cysteine residues in the human proteome?

The human genome encodes approximately 214,000 cysteine-coding sequences, indicating the significant presence of this amino acid across the proteome . Analysis of protein structures in the Protein Data Bank reveals that around 22% of protein structures contain one or more disulfide-bonded cysteines. When removing entries with over 50% sequence identity to reduce redundancy, approximately 16% of unique protein structures still contain disulfide bonds . This high conservation rate across the proteome suggests that cysteine residues have been evolutionarily maintained due to their critical roles in protein structure and function .

What experimental approaches can determine if a cysteine residue is involved in a disulfide bond?

Determining whether a cysteine residue participates in a disulfide bond can be approached through several experimental techniques:

• Differential alkylation combined with mass spectrometry: This method involves sequential labeling of free and disulfide-bonded cysteines with different alkylating agents (such as iodoacetamide or maleimide derivatives). The mass differences between labels allow identification of the original redox state of each cysteine .

• Stable isotope labeling: Using reagents like ¹²C-iodoacetic acid and ¹³C-iodoacetic acid to differentially label free cysteines before and after reduction enables precise determination of disulfide-bonded cysteines through the 2 Da mass shift between labels .

• X-ray crystallography and NMR spectroscopy: These structural methods can directly visualize disulfide bonds when resolution is sufficient.

• Database-assisted analysis: Using resources such as Cys.sqlite database that contains comprehensive information about cysteine residues across protein structures, allowing researchers to analyze disulfide bond patterns with established parameters (e.g., bond distance threshold of 0.231 nm) .

How can researchers quantify free cysteine residues in proteins?

Quantification of free cysteine residues in proteins requires specific methodological approaches:

• Colorimetric assays: Use of 5,5'-dithiobis-(2-nitrobenzoic acid) (DNTB) which reacts with free thiols to produce a colored product measurable by spectrophotometry .

• Fluorescent labeling: Application of fluorescent probes such as Alexa Fluor C-5-coupled maleimide reagent (AF594) that specifically react with free thiols. After labeling, quantification can be performed by:

  • Measuring total fluorescence intensity

  • Using reverse-phase HPLC separation of labeled proteins/peptides

  • Comparing fluorescence intensity to a standard curve

• Mass spectrometry-based quantification: After differential alkylation of free cysteines, LC-MS/MS analysis can:

  • Identify the specific location of free cysteines in the protein sequence

  • Quantify the proportion of free vs. disulfide-bonded cysteines at each position

  • Provide absolute quantification when compared with isotopically labeled standards

What methodologies are available for studying cysteine modifications in therapeutic proteins?

For therapeutic proteins like monoclonal antibodies, several specialized methodologies have been developed:

• Fluorescent maleimide labeling under partial denaturing conditions: This approach uses 7M guanidine HCl as a partial denaturant while labeling free cysteines with fluorescent probes. After enzymatic digestion (e.g., with Lys-C), the labeled peptides can be analyzed by RP-HPLC with fluorescence detection .

• Dual isotope labeling with high-resolution LC-MS/MS: This approach involves:

  • Initial labeling of native free cysteines with one isotope form (e.g., ¹²C-IAA)

  • Denaturation and reduction of the protein

  • Labeling newly liberated cysteines with another isotope form (e.g., ¹³C-IAA)

  • Multi-enzyme digestion (using trypsin, Lys-C, chymotrypsin, Asp-N, or Glu-C)

  • LC-MS analysis to identify and quantify peptides containing different labels

• Stress-testing protocols: Application of mechanical agitation or other stressors to proteins followed by analysis of free cysteine formation and disulfide bond breakage .

What database resources facilitate cysteine residue research?

The Cys.sqlite database represents a significant resource for researchers studying cysteine residues in proteins. This database:

• Transforms over 30,000 Protein Databank files into a single SQLite database

• Contains a schema of six interconnected tables that preserve biological and structural information about cysteine residues

• Includes the PDB table and Entity_Cys table as the major parent tables

• Stores information specific to each PDB entry and all unique amino acid sequences containing cysteine residues

• Facilitates analysis of how a given entity with cysteine residues varies across different structures

The database design allows researchers to:

• Rapidly query information about disulfide bonds using predefined criteria (e.g., bond distance ≤ 0.231 nm)

• Compare cysteine residues across different protein structures

• Track sequence variations that might affect cysteine positioning and bonding

• Launch more elaborate sequence analyses from the Entity_Cys table

How can researchers differentiate between naturally occurring free cysteines and those resulting from experimental procedures?

Differentiating between naturally occurring free cysteines and those resulting from experimental procedures requires careful methodological approaches:

• Sequential labeling strategies: Researchers can use a dual-labeling approach:

  • Label native proteins under non-denaturing conditions to tag naturally free cysteines

  • Denature/reduce the protein and apply a different label to newly exposed cysteines

  • The differential labeling allows discrimination between original free cysteines and those resulting from reduction

• Isotope-coded alkylation: Using ¹²C-iodoacetic acid (¹²C-IAA) and ¹³C-iodoacetic acid (¹³C-IAA) allows researchers to:

  • Create a 2 Da mass shift between labels

  • Distinguish between free cysteines originally present in proteins and those liberated during sample processing

  • Quantify the relative abundance of each population

• Controlled experimental conditions: Minimizing oxidation or reduction during sample preparation by:

  • Using anaerobic conditions

  • Adding alkylating agents immediately after cell lysis

  • Controlling pH and temperature to minimize disulfide exchange reactions

How does L-Cysteine supplementation affect biochemical parameters in experimental models?

Studies examining L-Cysteine supplementation in experimental models have revealed several biochemical effects:

Table 1: Effects of L-Cysteine Supplementation on Biochemical Parameters

The data indicates that L-Cysteine supplementation can have beneficial effects on certain biochemical parameters while showing no evidence of hepatotoxicity or nephrotoxicity as assessed by liver and renal function tests .

What factors influence free cysteine residues in therapeutic monoclonal antibodies?

Several factors can influence the presence and levels of free cysteine residues in therapeutic monoclonal antibodies:

• Cellular processing factors:

  • Incorrect processing of disulfide bonds during protein synthesis

  • Variations in the redox environment of the endoplasmic reticulum

  • Limited access to protein disulfide isomerase enzymes

• Manufacturing process factors:

  • Reduction of disulfide bonds during harvest and purification

  • Exposure to reducing agents or redox-active impurities

  • Mechanical stress during processing (e.g., agitation, shear forces)

  • pH excursions during purification steps

• Storage and handling conditions:

  • Light exposure leading to photo-induced reduction

  • Temperature fluctuations affecting disulfide stability

  • Presence of trace metals catalyzing thiol-disulfide exchange

Research has demonstrated that mechanical agitation of monoclonal antibodies can result in breakage of disulfide bonds and subsequent covalent aggregation via the liberated cysteine residues . This has important implications for manufacturing processes and handling protocols for therapeutic proteins.

Why is monitoring free cysteine residues critical in biotherapeutic development?

Monitoring free cysteine residues is critical in biotherapeutic development for several reasons:

• Impact on product quality and function:

  • Free cysteines can affect the potency of therapeutic proteins

  • They may induce protein aggregation through aberrant intermolecular disulfide formation

  • They can decrease the stability of therapeutic proteins during storage

• Regulatory requirements:

  • The levels and positions of free cysteines in proteins are closely monitored by both manufacturers and regulators

  • This monitoring is essential to ensure safety and efficacy of biotherapeutic products

  • Changes in free cysteine levels may require additional characterization and potentially regulatory approval

• Risk management:

  • Understanding the relationship between free cysteines and product quality attributes allows for:

    • Development of appropriate control strategies

    • Establishment of meaningful specifications

    • Design of stability-indicating analytical methods

    • Implementation of appropriate storage conditions

What are the pharmacological benefits of L-Cysteine and its derivatives in human health?

L-Cysteine and its derivatives, particularly N-acetyl-L-cysteine (NAC), have demonstrated numerous pharmacological benefits:

• Antioxidant properties:

  • L-Cys promotes glutathione biosynthesis

  • NAC acts directly as a scavenger of free radicals, especially oxygen radicals

  • As a powerful antioxidant, it can treat disorders resulting from free oxygen radical generation

• Mucolytic effects:

  • NAC is a highly efficient mucolytic drug

  • It promotes the discharge of tenacious mucus, beneficial in respiratory conditions

• Metabolic effects:

  • L-Cys supplementation has been shown to lower insulin resistance

  • It can reduce glycemia and oxidative stress

  • It may influence markers of vascular inflammation

• Safety profile:

  • Clinical studies suggest L-Cys supplementation does not cause hepatotoxicity

  • No significant changes in transaminase levels have been observed

  • Renal function parameters remain within normal ranges during supplementation

The therapeutic applications of L-Cys have attracted considerable attention in medical research, particularly in personalized medicine approaches that integrate compounds of biological origin with clinical nutrition .

How do researchers characterize disulfide bond patterns in complex proteins?

Characterization of disulfide bond patterns in complex proteins requires sophisticated analytical approaches:

• Enzymatic digestion strategies:

  • Use of multiple proteolytic enzymes (trypsin, Lys-C, chymotrypsin, Asp-N, Glu-C) to generate overlapping peptide fragments

  • Partial digestion techniques to preserve disulfide-linked peptides

  • Non-reducing conditions during digestion to maintain disulfide integrity

• Mass spectrometry techniques:

  • LC-MS/MS analysis of non-reduced peptides to identify disulfide-linked peptide pairs

  • Electron capture dissociation (ECD) or electron transfer dissociation (ETD) to selectively cleave peptide bonds while preserving disulfide bonds

  • Comparison of MS/MS fragmentation patterns before and after reduction

• Database-assisted analysis:

  • Tools like Cys.sqlite to compare observed disulfide patterns with known structures

  • Analysis of internal coordinates for each cysteine disulfide to characterize bond geometry

  • Investigation of sequence context effects on disulfide formation probability

For example, the Cys_Cys table in the Cys.sqlite database contains internal coordinates for each cysteine disulfide, capturing spatial relationships with a bond distance threshold of 0.231 nm (which is 0.025 nm greater than twice the sulfur covalent radius of 0.103 nm) .

What is the evolutionary significance of cysteine conservation in proteins?

The evolutionary significance of cysteine conservation in proteins is substantial:

• Structural role conservation:

  • Cysteine residues are the most conserved amino acids throughout the entire proteome

  • The presence of conserved cysteines in protein motifs across all organisms suggests early evolutionary adoption

  • This conservation indicates crucial roles in enzyme catalysis, transcriptional regulation, protein folding, and three-dimensional structure

• Functional specialization:

  • The human genome encodes 214,000 cysteine-coding sequences

  • The reactivity and diverse functions of cysteines are mirrored by their roles in development, signal transduction, biological defenses, aging, and disease processes

  • Conservation patterns reflect adaptation to cellular compartments with different redox environments

• Vulnerability and adaptation:

  • The spectrum of susceptibilities and dysfunctions in cysteine-containing proteins highlights their central roles in biological processes

  • Conservation patterns reflect selective pressures that balance functional advantages against potential vulnerabilities

  • Extracellular proteins typically have more structural disulfides to withstand harsh environments

How do modifications of cysteine residues affect protein function and stability?

Modifications of cysteine residues can dramatically influence protein function and stability:

• Redox-based functional changes:

  • Oxidation of thiol groups can create disulfide bonds that stabilize protein structures

  • Reduction of disulfides can activate or inactivate protein functions

  • Reversible oxidation (e.g., to sulfenic acid) often serves as a regulatory mechanism

• Impact on therapeutic proteins:

  • Free cysteines in monoclonal antibodies can affect potency through altered tertiary structure

  • They can induce aggregation via formation of intermolecular disulfide bonds

  • This aggregation may reduce shelf-life and potentially increase immunogenicity

  • Mechanical agitation of monoclonal antibodies can break disulfide bonds, leading to covalent aggregation via liberated cysteines

• Experimental evidence:

  • Researchers have demonstrated that when free cysteines are present in the constant domains of IgG1 and IgG2 antibodies, they occur at levels of 1-2.7% and 1-2.8% respectively

  • These seemingly small percentages of free cysteines can significantly impact protein stability and function

  • The mechanical stress during manufacturing and handling can increase these percentages

What methodological approaches can assess the impact of cysteine modifications on protein aggregation?

Assessing the impact of cysteine modifications on protein aggregation requires specialized methodological approaches:

• Stress testing protocols:

  • Application of mechanical agitation to induce disulfide bond breakage

  • Analysis of resulting aggregation through size-exclusion chromatography

  • Correlation of free cysteine formation with aggregation kinetics

• Fluorescent labeling and quantification:

  • Use of Alexa Fluor C-5-coupled maleimide reagent (AF594) as a probe for free cysteines

  • Analysis under partial denaturing conditions (7M guanidine HCl)

  • Quantification of free cysteines in aggregated vs. non-aggregated protein fractions

• Peptide-level analysis:

  • Enzymatic digestion (e.g., with Lys-C) of labeled proteins

  • RP-HPLC separation of labeled peptides

  • Identification of free-cysteine-containing peptides by comparing experimental and theoretical masses

  • Quantification using fluorescence standard curves

• Molecular dynamics simulations:

  • Computational modeling of how free cysteines influence protein dynamics

  • Prediction of aggregation-prone regions in the presence of free thiols

  • Simulation of disulfide reshuffling events during aggregation processes

How has research interest in L-Cysteine evolved over time?

Research interest in L-Cysteine has shown a significant upward trajectory:

• Publication trends:

  • There has been a meaningful and relevant increase in the number of publications about L-Cys during the last two decades

  • The greatest number of scientific-technical publications has been within the field of pharmacology (52,873 publications)

• Factors driving increased interest:

  • Rising interest in personalized medicine globally

  • Discontent with traditional treatments in certain population segments

  • High demand for personalized treatments in developed countries

  • Growing rejection of chemically synthesized drugs

  • Recognition of nutrition as a key factor for maintaining and restoring health

• Research diversity:

  • Despite the large scientific and technical production on L-Cys, no single author, institution, or journal significantly dominates the field

  • This suggests L-Cys is a topic of broad interest analyzed from multiple perspectives for diverse applications

The data indicates that L-Cys research represents a highly active and expanding field with contributions from diverse scientific disciplines and applications.

What are the current methodological challenges in studying cysteine modifications?

Current methodological challenges in studying cysteine modifications include:

• Sample preparation issues:

  • Preventing oxidation or reduction artifacts during protein isolation

  • Maintaining native disulfide bond patterns throughout analytical workflows

  • Ensuring complete labeling of free thiols without disrupting native structures

• Analytical limitations:

  • Distinguishing between different oxidation states of cysteine (e.g., thiol, disulfide, sulfenic, sulfinic, sulfonic acid)

  • Quantifying low-abundance cysteine modifications with high precision

  • Analyzing complex mixtures of proteins with diverse cysteine modifications

• Data interpretation challenges:

  • Correlating observed cysteine modifications with functional consequences

  • Determining whether modifications are causative or consequential in disease states

  • Establishing quantitative relationships between free cysteine content and protein stability

• Standardization needs:

  • Developing validated reference materials for cysteine modification analysis

  • Establishing standardized protocols for sample preparation and analysis

  • Creating consensus guidelines for reporting cysteine modification data

Researchers continue to develop new approaches to address these challenges, including advanced mass spectrometry techniques, improved labeling strategies, and enhanced computational tools for data analysis.

What innovations are emerging for therapeutic applications of L-Cysteine research?

Emerging innovations in therapeutic applications of L-Cysteine research include:

• Personalized medicine approaches:

  • Integration of L-Cys in personalized treatment protocols

  • Tailoring L-Cys supplementation based on individual metabolic profiles

  • Consideration of genetic factors affecting cysteine metabolism

• Biopharmaceutical manufacturing improvements:

  • Development of cell lines with enhanced capacity for correct disulfide bond formation

  • Process modifications to minimize disulfide bond reduction during manufacturing

  • Novel formulation approaches to stabilize cysteine-containing therapeutics

• Analytical method advancements:

  • High-sensitivity detection of free cysteines in biotherapeutics

  • Improved methods to predict stability based on free cysteine content

  • Better understanding of the relationship between free cysteines and protein aggregation

• Clinical applications:

  • Expansion of NAC usage beyond traditional applications

  • Development of L-Cys derivatives with enhanced bioavailability or specificity

  • Combined therapies leveraging the antioxidant properties of L-Cys with other therapeutic modalities

The integration of these innovations reflects the growing recognition of L-Cysteine's importance in both fundamental protein science and therapeutic applications.