Biochemical Energetics Flashcards
Learning Objectives - Describe the characteristics of protein binding sites - Understand the law of mass action kinetics - Review techniques to determine protein-ligand binding affinity
1
Q
What is a ligand?
A
Any molecule that binds to another molecule
2
Q
Give some examples of ligands.
A
- Substrates
- Ligands that bind to enzymes and membrane transporters - Protein signal molecules + protein transcription factors
3
Q
List 3 protein-ligand binding models.
A
- Lock-and-key
- Induced fit
- Conformational selection
4
Q
Explain the lock-and-key model.
A
- The enzyme and substrate will already fit together
- Active site of an enzyme will fit a specifically shaped substrate
- They are rigid and do not change shapes
5
Q
Explain the induced fit model.
A
- Binding site of the enzyme is adjustable to allow for a good fit
- Binding induces this change
- Hand fitting into a glove (substrate is the hand and the enzyme’s (glove) shape changes to allow the hand to fit)
6
Q
Explain the conformational selection model.
A
- There is no single rigid conformation
- The complex can exist in various states and change depending on the LIGAND
- All states naturally exist
- Ligand chooses the best state
- Binding site on enzyme has already changed shaped before the ligand binds to it
7
Q
What are the characteristics of binding sites? (4)
A
- Specificity
- Affinity
- Competition
- Saturation
8
Q
What is specificity in terms of binding sites?
A
- The ability of a protein to bind to a certain ligand or group of related ligands
- Some proteins are very specific about ligands they bind to while others binds to the whole groups of molecules
9
Q
What is affinity in terms of binding sites?
A
- The degree to which a protein is attracted to its ligand
- If a protein has a high affinity for its given ligand, the protein is more likely to bind to that ligand
- When explaining this on exams, mention the formula and kinetic rate constants (P+L –>(Kon, Koff)<– PL)
10
Q
What is the equilibrium constant?
A
- The ratio of the product concentration to the concentrations of the reactants
- Always the same at equilibrium
11
Q
What happens to equilibrium when the concentration of P or L changes (assume the reaction is P+L–>PL)
A
- If more P or L is added to the system, more PL will be produced
- Equilibrium is shifted right (forward reaction is favored)
- Ratio of products to reactants will be different from Keq
12
Q
What is the law of mass action?
A
The rate of a chemical reaction (# of collisions per unit time) is directly proportional to the product of the activities or concentration of the reactants
13
Q
What does a higher affinity mean for your Keq?
A
- You have a large Keq
- More product in comparison to your reactants
- Enzyme WANTS to bind and react with substrates
14
Q
What is the dissociation constant?
A
- The reciprocal of the equilibrium constant
- A large Kd indicates low binding affinity of the protein for the ligand
- More P and L remains in an unbound state
Kd = [P][L]/[PL]
15
Q
How do absolute concentrations of the reactants and products affect Kd?
A
They do not, only the relative proportions affect it
16
Q
What is competition in terms of binding sites?
A
- Comparing Kd values can tell us which ligand is more likely to bind to which protein
- Competitors are ligands that compete for binding sites
17
Q
What is saturation in terms of binding sites?
A
- Conditions where all available binding sites on the proteins are occupied by the ligand
- Adding more ligand will not significantly increase the amount of PL formed
- Each protein has a finite (one or more) number of binding sites
- Once all sites are occupied, the protein is fully saturated
18
Q
What is the purpose of isothermal titration calorimetry (ITC)?
A
- Used to measure the thermodynamics of molecular interactions (binding affinity, enthalpy, and entropy changes)
- Determines the heat released or absorbed from reaction
- Helpful to understand how 2 molecules will interact with one another and the stability of their interaction
19
Q
Outline the steps of ITC.
A
- Macromolecule (protein) of interest is placed in a sample cell
- Titrate (ligand or substrate) is slowly injected into the cell
- As reaction occurs, heat is released or absorbed is measured and plotted against the ligand concentration
20
Q
What is the twin cell design and its purpose in ITC?
A
- A sample cell contains the analyte and is injected with the titrant
- A reference cell has a buffer solution for baseline comparisons
- Both cells placed in an adiabatic (no heat in/out) jacket
21
Q
How are heat changes detected in ITC?
A
- Both cells are maintained at a constant temperature
- Any heat change from reaction creates a temperature difference between the two cells
- Sensitive thermopile sensors are used to monitor temperature
- Feedback heaters kept a constant temperature in the cell as heat is gained or lost
22
Q
Why is maintained a constant temperature important during ITC?
A
- Constant temperature allows for detection of heat changes caused by the binding reacrtion
- Ensures accuracy and consistency of readings
23
Q
Describe the output of ITC experiments?
A
- Heat changes are recorded as a series of peaks after each titration step
- Peaks represent the heat released or absorbed during each injection (integrate them to find total heat)
24
Q
How does the ITC instrument react to an exothermic reaction?
A
- Less heat per unit time is needed by the sample cell to keep the twin cells in thermal equilibrium
- Remember, the reference cell receives a constant power supply
25
What is surface plasmon resonance (SPR) used for?
- Used to analyze affinity and selectivity of biomolecular interactions like protein-ligand, antigen-antibody, and receptor-ligand reactions
- Determines kinetic rate constants and the equilibrium constant
26
What does the data curves from SPR tell you?
How the formation of complexes on the sensor surface increased the device's signal over time
27
How is SPR done?
1. A protein is immobilized on a gold sensor surface
2. Ligands flows over the sensor at a controlled flow rate
3. Changes in refractive index at the sensor surface caused by binding events are detected
4. Association and dissociation phases are recorded
28
What is the dissociation phase of SPR?
- Stopping the flow of the ligand over the sensor and replacing it with a buffer
- Observe the decrease in signal as the ligand dissociates from protein
29
What does fluorescence polarization (FP) measure?
How much light emitted from a fluorescently tagged molecule is polarized
30
What are the steps of FP?
1. Ligand is labeled with a fluorescent tag
2. Tagged ligand is mixed with target protein in a buffer
3. Sample is exposed to polarized light (exciting the fluorophore)
4. As fluorophore relaxes, light is emitted
31
How do unbound (free) ligands appear on FP results?
- Rapid rotation and tumbling
- Emitted light is depolarized
- Low fluorescence polarization
32
How does a ligand bound to the larger molecule appear on FP?
- Slower rotation and tumbling
- Higher polarization of emitted light
