Flashcards
BIOC222 - Mod3
Cards in this deck (23)
- 1What may proteins interact with in order to do their work? What are these known as?Other small molecules known as ligands
- 2How may protein interactions be measured?Both qualitatively (if they bind) & quantitatively (how tightly they bind)
- 3Qualitative methods of measuring protein interactions tend to be ____, but are more ____faster, error prone
- 4Quantitative methods of measuring protein interactions tend to be ____, but are more ____More precise and accurate, expensive and slow
- 5What are two quantitative techniques used to measure protein interactions?Differential scanning fluorimetry
Equilibrium dialysis - 6What are some advantages of differential scanning fluorimetry?Fast, inexpensive, easy to run, tends to work well. Semi-quantitative
- 7How does differential scanning fluorimetry work?Folded protein mixed with dye that fluoresces upon binding hydrophobic pockets (exposed when unfolded). Heat mixture, observe & measure fluorescence.
- 8What is EC50? What is its relevance?Effector concentration at which it has 50% of its maximum effect. Gives an idea of saturation concentrations & affinity of effector for protein
- 9What does equilibrium dialysis allow the calculation of? Why is this important?The Kd of the receptor-ligand pair. This is a precise measure of affinity
- 10How does equilibrium dialysis work?Known concs of ligand and protein are loaded into 2 chamber system, separated by membrane. Some ligand will move across to bind protein, equilibrium will be reached. Measure conc of ligand once equilibrium reached.
- 11What values of Kd correspond to what binding tightness?mM: weak bind
µM: medium bind
nM: good strength
pM: outstanding strength - 12In equilibrium dialysis, what is fractional occupancy?The fraction of receptor/protein with bound ligand as a function of [L] & Kd
- 13What is the formula for fractional occupancy (Y)?= [L] / [L] + Kd
- 14In order to calculate Kd (Kl) from an equilibrium dialysis experiment, what data must be have?Free ligand
Bound ligand
Total ligand - 15What data can be ascertained from equilibrium dialysis?Kd or Ka (reciprocals)
Stoichiometry of binding - 16What can be found using isothermal calorimetry to measure protein interaction?Everything you find from equilibrium dialysis + enthalpy & entropy of binding, & heat capacity
- 17What energy unit is used in isothermal calorimetryMicro calories (µcal)
- 18How does isothermal calorimetry function?Syringe w/ free ligand injects into protein sample at intervals, freeing some ligand to bind protein. Heat evolved from this binding is measured & plotted. As protein becomes saturated, less heat eventuates from each injection until full saturation is reached
- 19On a plotted curve of data from an isothermal calorimetry experiment, what represents what?
- 20What is surface plasmon resonance?A kinetically-oriented method that involves protein attachment to a gold chip, where ligand binding events are measured
- 21What are the two constants of significance in surface plasmon resonance, and what does each one represent?Kon: how fast the receptor + ligand form into the receptor-ligand complex
Koff: how fast the receptor-ligand complex breaks down - 22How is Kd ascertained from surface plasmon resonance?Koff/Kon
- 23Discuss how surface plasmon resonance functionsLight is reflected off the gold film on which the protein resides. Ligand flows past in a flow channel, and as a binding event occurs, the angle of reflection will change. The angle change is proportional to the amount of ligand bound
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- Flashcards on BIOC222 - Mod3, created by Guy Turner on 06/10/2018.
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- Guy Turner
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