Affinity Measurement Methods (+ DSF) Flashcards
Why can’t we see individual biological molecules with a microscope using visible light?
- The wavelength of visible light is around 10⁻⁷ meters, which is much larger than the building blocks of biological molecules. Because of this, visible light doesn’t interact with the molecules in a way that allows us to form an image of them.
- Even though microscopes use visible light, it doesn’t solve this issue since we still can’t see individual molecules.
- A solution is to use a fluorescent tag. The tag emits photons, which can be detected, allowing us to observe the biological molecules indirectly.
Why are affinity measurements important?
- Affinity measurements are crucial because they help us understand how molecules interact with each other and their environment.
- They are fundamental to how nature works, as molecular interactions drive processes like enzyme-substrate binding, antigen-antibody interactions, and receptor-ligand binding.
- Understanding affinity allows scientists to study biological processes and design better drugs, therapies, and materials.
What does “observing a proxy for the actual binding event” mean?
- When studying molecular interactions, we often can’t see the actual molecules or binding events directly, so we measure something that represents (or acts as a “proxy” for) these interactions.
(1) Example (simplest case): Gel shift assay
- In a gel shift assay, the “proxy” is the label (like a dye) bound to a component of the complex you’re studying.
- You can measure the intensity and position of the label to infer what is interacting.
- On a gel: The label (e.g., dye) will show where the proteins are located, allowing us to see if binding occurred based on where the label is in the gel.
- Native gel: The label on the protein shows where it is in the gel lane, telling you whether there is an interaction or not, based on its position.
- This method uses light interaction (like dye staining) as a proxy to visualize and track proteins during the experiment.
What is Native-PAGE and how does it separate its proteins?
- Native-PAGE is a type of gel electrophoresis that separates proteins based on their charge to mass ratio.
- The protein maintains its native structure and charge density in a nonreducing and nondenaturing environment (i.e., it doesn’t lose its functional shape during the process).
- Proteins are polyampholytes, meaning their charge depends on the pH and the local environment.
- If the pH > pI (protein’s isoelectric point), the protein is negatively charged.
- If the pH < pI, the protein is positively charged.
How does the protein binding to DNA experiment work to estimate the equilibrium dissociation constant (Kd)?
- The DNA is labeled with a radioactive isotope to track binding interactions.
- DNA concentration is kept constant and is much lower than Kd.
- The protein concentration is increased from below Kd to above Kd, gradually.
- By tracking how much DNA is bound to the protein at each concentration, you can estimate the Kd (the concentration at which half of the DNA is bound).
- At 50% binding, the Kd is equal to the concentration of free protein ([B]).
- The significance of this experiment lies in its ability to quantify the strength of the interaction between a protein and DNA, which is crucial for understanding biological processes like gene regulation, DNA replication, and repair.
What are the pros of Native Polyacrylamide Gel Electrophoresis (Native-PAGE) Shift Assay?
- Simple yet robust technique.
- Can accommodate a wide range of binding conditions (anything compatible with the gel).
- Measures many variations of binding interactions.
- Flexible with the size of molecules.
- Offers flexibility in the quantitative proxy (radioisotope, fluorescent, chemiluminescence, immunohistochemistry).
- Can be performed with low protein and nucleic acid concentrations (0.1 nM or less, depending on label).
- Works with highly purified proteins or crude cell extracts.
What are the cons of Native Polyacrylamide Gel Electrophoresis (Native-PAGE) Shift Assay?
(1) Samples are not at chemical equilibrium during electrophoresis.
- Rapid dissociation can prevent detection of complexes.
- Moderate dissociation can result in underestimation of binding.
- Many complexes are more stable in the gel than in free solution.
(2) Electrophoretic mobility of a complex depends on factors other than size: The shift does not always represent molecular weight (MW) or stoichiometry.
(3) Often done manually, leading to: Low time resolution/Low throughput.
In Surface Plasmon Resonance, what proxy for the actual binding event are you observing?
- Proxy: Measuring disturbances of a chip under your protein which occurs on complex formation.
What is the basic setup of SPR?
(1) Biosensor: A sensor surface that is coated with a metal layer (typically gold). This is where the interaction between the protein ligand and analyte occurs.
(2) Protein Ligand: A molecule (usually a protein) that is immobilized on the biosensor surface. It is the molecule that will interact with the analyte (usually a different protein or small molecule).
(3) Analyte: The molecule that will bind to the immobilized protein ligand. It flows over the biosensor surface and interacts with the ligand, causing a change in the sensor surface.
(4) Laser: A light source directed at the biosensor surface. The angle at which the laser light is reflected from the surface is used to measure the changes in refractive index, which occurs due to the binding of the analyte to the protein ligand.
(5) Detector: A device that measures the angle of the reflected light (from the laser). This change in angle correlates with changes in the refractive index at the biosensor surface, providing real-time data on the binding interaction between the protein ligand and analyte.
How does SPR work?
1) When the analyte binds to the protein ligand on the biosensor surface, the refractive index near the sensor changes. This alters the angle at which light is reflected from the biosensor surface.
2) The laser shines light onto the biosensor, and the detector measures changes in the angle of reflection, which corresponds to changes in the refractive index caused by the binding event.
3) The data collected helps to determine the binding kinetics (e.g., affinity, rate of binding and dissociation) between the protein ligand and the analyte.
What is plasma in the context of Surface Plasmon Resonance (SPR)?
Plasma refers to the electron cloud around a thin metal film, which behaves similarly to plasma, where electrons and ions are free to move.
What is a plasmon?
A plasmon is a basic unit of oscillation of the electron cloud (plasma), where electrons oscillate in response to a field.
What is a surface plasmon?
A surface plasmon is a plasmon that exists on the surface of a material, specifically on a thin metal film. The thinness of the metal film means surface effects are important.
A surface plasmon is a collective oscillation of electrons on the surface of a metal, like gold. When the light in TIR conditions hits the gold surface, it makes these electrons oscillate, generating an evanescent field.
What is resonance in Surface Plasmon Resonance (SPR)? How does it occur? What happens during resonance?
Resonance occurs when the frequency (or energy, or wavelength) of an external factor matches the frequency of an oscillating system. In SPR, resonance happens when the frequency of surface plasmon oscillations matches the frequency of the evanescent wave.
At a specific angle of incident light, the energy of the light wave matches the energy of the oscillating surface plasmon. This is called resonance, and it leads to a transfer of energy from the light to the metal’s electrons.
At the resonance angle, the intensity of the reflected light decreases because energy is transferred from the light wave to the surface plasmon. This attenuation is the key to detecting molecular interactions in SPR.
How does total internal reflection relate to SPR?
Total internal reflection creates an evanescent wave, which is a decaying light wave traveling along the surface. This evanescent wave interacts with the surface plasmon when their frequencies align, resulting in resonance.
What is total internal reflection (TIR)? Why is TIR important in SPR?
- Total Internal Reflection happens when light strikes an interface (like glass and air or liquid) at an angle greater than the critical angle, causing the light to reflect entirely, with no transmission through the surface.
- When light undergoes TIR, it creates an evanescent wave. This is a special kind of wave that decays exponentially as it moves away from the surface, but it can interact with materials near the surface, like the gold film in SPR.
In SPR, what is the critical angle?
The critical angle refers to the angle of incidence at which light traveling from a medium with a higher refractive index (e.g., glass or water) to a medium with a lower refractive index (e.g., air) undergoes total internal reflection. At this angle, no light passes through the interface, and all of it is reflected back into the higher-index medium. In SPR, the critical angle is important because it determines the angle at which surface plasmon waves can be excited by light. This angle is typically used to monitor binding events in SPR experiments.
How does the evanescent wave behave (SPR)?
The evanescent wave decreases exponentially as you move away from the surface. Its intensity is strongest right at the interface and quickly decays with distance.
What is the SPR angle?
The SPR angle is the angle at which the light undergoes total internal reflection and resonates with the surface plasmon. This angle is sensitive to changes in the material or molecules attached to the metal surface because those changes affect the way the evanescent wave interacts with the surface plasmon.
What does SPR detect?
- SPR is used to detect molecular interactions at the surface of the sensor. When molecules, like proteins or DNA, bind to the metal surface (gold in this case), the mass and electronic properties of the surface change. This alters the SPR angle.
- The difference in the SPR angle due to binding can be measured to infer information about the binding event, such as the amount of analyte binding to the surface.
What are the characteristics of what directly immobilizes the ligand in SPR?
- covalent chemistry
- often heterogenous orientation
- higher binding capacity
- examples of functional groups or chemical groups that are used to modify or functionalize the sensor surface to facilitate specific interactions with target molecules (such as proteins, DNA, or other analytes): amine, ligand thio, surface thiol, maleimide, aldehyde
What are the characteristics of capturing the analyte and ligand binding on the surface?
- orientation-specific
- selective ligand capture from crude samples
- lower binding capacity
- examples: streptavidin- biotin/ Anti-GST-GST / Anti-his-6His
What is the role of Carboxymethylated dextran (CM) sensor chips in SPR?
Carboxymethylated dextran (CM) sensor chips enable amine-coupling using EDC/NHS chemistry (activates electrophiles). This interaction is irreversible and cannot reuse the surface.
How does thiol-coupling work in SPR, and can the surface be reused?
Thiol-coupling using PDEA after EDC/NHS activation can be used. The surface can be regenerated using a reducing agent, allowing reuse.
How does biotinylation work in SPR, and can the surface be reused?
Biotinylation and binding to streptavidin-coated chips create a robust interaction, but the surface cannot be reused for another ligand after this binding.
How does His-tagged protein binding to a Ni-NTA chip work in SPR?
His-tagged proteins bind to a Ni-NTA chip with a weak interaction. The ligand eventually dissociates over time.
How does GST-tagged protein binding work in SPR, and can the surface be reused?
GST-tagged proteins bind to amine-coupled anti-GST antibodies with a robust interaction. The surface can be regenerated using glycine (pH 2).
What is the role of bilayer or liposome adsorption on HPA or L1 chips in SPR?
Bilayer or liposome adsorption on HPA or L1 chips is used for protein:lipid or membrane protein:protein interactions.
How does the SPR sensogram work?
- A sensogram plots the resonance units (RU) measured as a function of time.
- When the analyte is injected, it starts associating with the bound ligand. This is the association phase. It is a function of both kon and koff.
- After a period of time, the association rate equals the dissociation rate, and we have reached equilibrium, or steady-state. Function of KD
- At the end of the injection, buffer is injected and the analyte dissociates. The dissociation phase is a function of koff only.
- The surface can then be regenerated for another injection.
What are the pros of SPR?
-“Label free”
-SPR has been applied in a wide range of settings, even mimicking biological environments
-Generates equilibrium data and kinetic data, plus stoichiometry
-Real-time, continuous measurement
-Relatively rapid
-Relatively small sample amounts & volumes
-Highly sensitive
What are the cons of SPR?
-Immobilisation effects possible
-Steric hindrance with binding events
-Non-specific binding to surfaces
-Some other caveats, like mass transport limitations
-Control experiments must be meticulously designed
-Expense of sensor chips and instrumentation
