CHE316 3/18

"Describe the role of enthalpic contributions in binding events."

"Enthalpic contributions involve interactions such as hydrogen bonds, salt bridging, and Van Der Waals interactions that can release heat (negative delta H) during bond formation. Unfavorable enthalpy indicates that net bonds have been lost."

"Explain the concept of entropic contributions in the context of hydrophobic effects."

"Entropic contributions relate to the entropy of water, where favorable entropy (negative TdeltaS) occurs as hydrophobic surfaces come together, increasing disorder by losing water at the interface."

"How do unfavorable entropies affect ligand binding in proteins?"

"Unfavorable entropies can arise when a flexible ligand becomes restricted in a protein's binding site, making it less favorable for binding, especially if the binding site has many rotatable bonds."

"What is the significance of a binding event with negative delta G?"

"A binding event with negative delta G is spontaneous, indicating that it is entropically driven despite having unfavorable enthalpy, where a large -TdeltaS compensates for the unfavorable enthalpy."

"Define the relationship between enthalpy and binding interactions involving polar groups."

"Unfavorable binding enthalpies are often associated with polar groups that become desolvated and fail to establish strong interactions with the protein, leading to less favorable binding."

"How does a large -TdeltaS value influence binding events?"

"A large -TdeltaS value indicates that the binding event is primarily driven by entropy, suggesting that the increase in disorder is significant enough to favor the binding despite unfavorable enthalpic contributions."

"Explain the importance of TIC in thermodynamic characterization."

"TIC (Isothermal Titration Calorimetry) is crucial for complete thermodynamic characterization, providing stoichiometry, association constants, and binding enthalpy/entropy in a single experiment."

"What are the advantages of using TIC for studying ligand binding?"

"Advantages of TIC include complete thermodynamic characterization, the use of heat as a universal signal without the need for reporter labels, direct determination of binding enthalpy, non-destructive analysis, and the ability to work with optically dense solutions."

"Identify some disadvantages of TIC in ligand binding studies."

"Disadvantages of TIC include the proportionality of the signal to binding enthalpy, complications from dilution heat, the need for a large amount of concentrated protein sample, and its slow throughput, making it unsuitable for high-throughput methods."

"Describe how enthalpy and entropy contribute to the overall binding process."

"The overall binding process is influenced by both enthalpy and entropy, where favorable enthalpic interactions can drive binding, while unfavorable enthalpy can be compensated by favorable entropy, leading to a successful binding event."

"Describe the process of surface plasmon resonance (SPR) in measuring binding interactions."

"SPR involves immobilizing a ligand or protein on a chip surface. When a binding event occurs, it causes a change in surface plasmon resonance, which is detected as a deflection in relative units."

"Explain the typical experimental procedure for conducting an SPR experiment."

"The procedure includes establishing a baseline, flowing in the ligand to measure the on-rate, then flowing in a buffer to measure the off-rate, allowing for the calculation of the association constant (k_a)."

"Define the term K_D in the context of ligand binding."

"K_D is the concentration at which 50% of the ligand is bound to its receptor, indicating the affinity of the ligand for the receptor."

"What are the advantages of using surface plasmon resonance (SPR) in experiments?"

"SPR allows for fast experiments and provides actual on and off rates of binding interactions."

"Identify the disadvantages associated with surface plasmon resonance (SPR)."

"Disadvantages include the high cost of the instrument and potential effects caused by immobilizing ligands or proteins on the surface."

"How can one prevent the destabilization of complexes in SPR experiments?"

"To prevent destabilization, complexes can be chemically cross-linked to inhibit dissociation."

"Differentiate between Kd and Ki in terms of their definitions."

"Kd is the dissociation constant that measures the affinity between a receptor and ligand, while Ki is the inhibition constant that measures the concentration of an inhibitor needed to occupy 50% of an enzyme's binding sites at equilibrium."

"Explain the significance of the Michaelis constant (K_m)."

"K_m represents the apparent affinity of a substrate for an enzyme, indicating the substrate concentration at which the reaction rate is half of its maximum."

"What does IC_50 represent in pharmacology?"

"IC_50 is the concentration of a substance needed to inhibit a specific biological process or component by 50%, often used to describe the effectiveness of drugs."

"Describe the difference between IC50 and EC50."

"IC50 refers to the concentration needed to inhibit a process by 50%, while EC50 refers to the concentration needed to activate a process by 50%."

"What is the principle behind fluorescence resonance energy transfer (FRET)?"

"FRET involves the transfer of energy from an excited donor fluorophore to an acceptor fluorophore when they are in close proximity, allowing for the study of molecular interactions."

"Explain the basic concept of fluorescence in biological experiments."

"Fluorescence occurs when a substance absorbs light and re-emits it at a longer wavelength after thermal relaxation or internal conversion."

"What is photobleaching in the context of fluorescence?"

"Photobleaching is the process where a fluorophore is destroyed due to excessive internal relaxation and oxidation, leading to loss of fluorescence."

"How should one design experiments to avoid eliminating fluorophores?"

"Care should be taken to avoid excessive measurements that could lead to photobleaching and destruction of the fluorophore."

"What is the role of resonance energy transfer in fluorescence techniques?"

"Resonance energy transfer is used in techniques like FRET and BRET to study interactions between proteins by measuring energy transfer between fluorophores."

"Describe the basic mechanism of energy transfer in FRET."

"The basic mechanism involves exposing a donor fluorophore to a specific wavelength and measuring the output of a secondary wavelength associated with an acceptor fluorophore. For example, a donor emitting green light transfers energy to an acceptor, resulting in red light emission if the acceptor is close."

"Explain why energy transfer in FRET is proportional to 1/R^6."

"Quantum mechanics indicates that the electric field created by an excited donor decreases at a rate of 1/r^3. Therefore, the rate of energy transfer, which is the square of this decline, results in a proportionality of 1/R^6."

"Define the significance of the distance R_o in FRET."

"R_o, typically ranging from 2-8nm, is significant because it represents the ideal distance for energy transfer, often corresponding to the scale of protein-protein interactions or conformational changes."

"How do intramolecular and intermolecular FRET experiments differ?"

"Intramolecular FRET experiments involve fluorophores that are distant in one conformation and brought together after a conformational change, while intermolecular FRET experiments detect dimerization through the interaction of donor and acceptor fluorophores."

"List some applications of FRET."

"Applications of FRET include studying protein-protein interactions, screening for inhibitors of these interactions, and conducting enzyme assays, particularly in cases where detecting products is challenging."

"Explain the concept of quenching in the context of FRET."

"Quenching occurs when a donor fluorophore transfers energy to a non-fluorescent quencher, which then radiates energy as heat. This results in no fluorescence when the donor and quencher are close, but fluorescence is observed when the quencher is cleaved off, allowing the donor to emit light."

"Describe how FRET can be utilized in RT-PCR."

"In RT-PCR, the use of a quencher can enhance the detection of specific sequences by preventing fluorescence until the quencher is removed, allowing for real-time monitoring of the reaction."

"What is the role of fluorophores in FRET?"

"Fluorophores serve as the donor and acceptor in FRET, where the donor emits light that can be transferred to the acceptor, resulting in a measurable change in fluorescence."

"How does the proximity of donor and acceptor fluorophores affect FRET efficiency?"

"The efficiency of FRET increases as the distance between the donor and acceptor fluorophores decreases, with optimal energy transfer occurring at distances typically within 2-8nm."

"Explain the importance of measuring the output of a secondary wavelength in FRET experiments."

"Measuring the output of a secondary wavelength allows researchers to detect the energy transfer from the donor to the acceptor, providing insights into molecular interactions and conformational changes."

"Describe the relationship between quencher distance and DNA growth."

"The quencher becomes increasingly distant in proportion to the extent of DNA growth."

"Explain the BRET assay and its components."

"The BRET assay uses bioluminescence instead of laser illumination, involving a bioluminescent protein like luciferase that emits light at 480nm in the presence of certain chemicals and ATP."

"How does resonance energy transfer work in the context of BRET?"

"If the bioluminescent protein is not near an acceptor that overlaps with its emission, there is no resonance energy transfer, resulting in no emission from the acceptor."

"Define the FRET/BRET issue related to fluorescent proteins."

"The FRET/BRET issue arises because most fluorescent proteins do not have very sharp emission spectra, leading to potential bleedthrough where the donor emits light that overlaps with the acceptor's emission."

"What complications arise when working with living cells in fluorescence assays?"

"In living cells or complex systems, light can shatter or other species can fluoresce, complicating the results of fluorescence assays."

"Explain the concept of time-resolved FRET (HTRF)."

"Time-resolved FRET introduces a delay before measuring emission, requiring a fluorophore with a long-lived excited state to improve specificity."

"How does the donor-acceptor pair function in time-resolved FRET?"

"In time-resolved FRET, the donor is excited, a delay is introduced, and then measurement is made; if there is no interaction, only the donor emits, but if there is interaction, the acceptor emits as well."

"What is the benefit of using time-resolved FRET in experiments?"

"The benefit of time-resolved FRET is that it eliminates non-specific short-lived background emissions, allowing for a clearer specific signal."