Folding and Unfolding Kinetics Analysis

Protein folding and unfolding kinetics analysis is a critical component of protein biopharmaceutical testing in the pharmaceutical industry, especially when developing protein-based therapeutics. Understanding the rate at which a protein folds from a denatured state into its functional three-dimensional structure, or unfolds under different conditions, provides valuable insights into drug stability, formulation design, and quality control.

Illustration of protein from unfolded to folded. (STEMart Original) Fig.1 Illustration of protein folding. (STEMart Original)

Protein Folding and Unfolding Kinetics

Protein folding is the process by which a polypeptide chain adopts its native three-dimensional structure, which is essential for its biological function. Unfolding refers to the disruption of this structure, leading to a loss of function. The kinetics of these processes refers to the rates at which folding and unfolding occur, which are influenced by factors such as temperature, pH, and the presence of ligands or denaturants. For example, folding kinetics studies the speed at which a protein recovers from a denatured state to a functional state, while unfolding kinetics focuses on the rate at which a protein loses its structure under denaturing conditions.

Our Services

We offer comprehensive protein folding and unfolding kinetics analysis services, using advanced technologies and a team of experienced scientists to provide detailed kinetic data.

Stopped-Flow Spectroscopy

Principle: Stopped-flow spectroscopy is a rapid kinetics technique that involves quickly mixing protein solutions with denaturing or refolding buffers and monitoring structural changes in real-time, typically using fluorescence, absorbance, or circular dichroism (CD) measurements.

Application: In folding studies, stopped-flow is used to monitor structural recovery by rapidly diluting denatured proteins into buffers that promote folding, thereby determining folding rates and folding intermediates. In unfolding studies, the protein is mixed with a denaturant, and structural loss is observed. This technique is particularly suited for protein folding and unfolding on a millisecond to second time scale.

Temperature-Jump Experiments

Principle: Temperature-jump experiments involve rapidly changing the temperature of a protein solution by mixing it with a pre-heated buffer or using laser-induced temperature changes, while spectral techniques are used to monitor the response.

Application: By suddenly increasing or decreasing the temperature, the protein is pushed out of equilibrium, and the folding or unfolding response can be measured. This helps study temperature-dependent kinetics, evaluate thermal stability, and determine activation energy, making it particularly useful for understanding protein behavior at different temperatures.

pH-Jump Experiments

Principle: pH-jump experiments involve rapidly changing the pH of a protein solution (e.g., by mixing buffers of different pH) to initiate folding or unfolding, followed by measurement of structural changes.

Application: pH is crucial for protein stability and function. By studying how proteins respond to sudden pH changes, the impact of protonation and deprotonation events on folding and unfolding kinetics can be understood. This is particularly relevant for pH-sensitive proteins, such as those involved in intracellular transport.

Single-Molecule Techniques

Principle: Single-molecule techniques, such as atomic force microscopy (AFM), optical tweezers, and fluorescence resonance energy transfer (FRET), allow observation of individual protein molecules during folding and unfolding, providing high-resolution mechanical and conformational data.

Application: These techniques reveal folding pathways, energy landscapes, and the dynamics of individual folding events. For example, AFM can be used to mechanically unfold a protein and measure the forces involved, while FRET can monitor conformational changes in real-time. These methods are critical for understanding the heterogeneity and complexity of protein folding.

Our Workflow

We employ a customized approach to ensure each project meets the specific needs of our clients:

Illustration of STEMart’s customized approach. (STEMart Original)

Custom Protocols: Experiments are designed based on the protein's characteristics (e.g., size, stability, folding time scales), which may involve selecting appropriate denaturants, buffers, and spectral probes.
Data Collection: Advanced instruments are used to collect high-quality kinetic data, ensuring sufficient time resolution to capture folding and unfolding events.
Data Analysis: Our team employs sophisticated analytical methods, such as nonlinear least-squares fitting and global analysis, to extract kinetic parameters, including rate constants, half-lives, and activation energy.
Reporting: We provide detailed reports that include raw data, analysis results, and scientific interpretations, formatted for regulatory submission or internal research use.

Our comprehensive reports include:

Detailed methods used
Raw data and processed kinetic curves
Kinetic parameters such as rate constants, half-lives, and activation energy
Graphs and figures illustrating folding and unfolding behavior
Discussion of results in the context of protein stability and function
Recommendations for further study or optimization, if applicable

By leveraging our expertise and advanced technologies, STEMart offers unparalleled protein folding and unfolding kinetics analysis services. Our commitment to quality and innovation makes us the partner of choice for pharmaceutical companies and research institutions dedicated to understanding and optimizing the properties of their protein-based products.

For more information on our Folding and Unfolding Kinetics Analysis service or to discuss your specific requirements, please contact us today.