10 - Ligand Binding and Equilibria II Flashcards
True or false: binding is a kinetic process
True: it depends on on and off rate
True or false: all collisions lead to reactions
False: they need sufficient speed and orientation
What is the order of the association reaction?
2nd order (1/Ms)
What is the order of the dissociation reaction?
1st order (1/s)
For a first order reaction, what is the equation for the half time?
t1/2 = ln(2)/k
What does the association rate depend on?
Diffusion
What is the fastest possible type of reaction?
A diffusion limited reaction
How does stopped flow work?
Two reactants are mixed, and it is measured how solution ages over time
On what order does stopped flow work?
Reactions on order of seconds (mixing on order of ms)
What is measured in stopped flow?
A change in absorbance or fluorescence
What is calculated in stopped flow?
kon
How is kon calculated in stopped flow?
Measure the fluorescence over time for different [L] levels, then plot k vs L to find kon and koff.
For stopped flow, what line is plotted?
kobs = (kon)[L]T + koff
How does SPR work?
A gold membrane is used to bind antibodies to receptor (immobilize protein), and change in density is measured
What is SPR used frequently for?
Protein/antibody, not protein/small molecule
How come SPR is used for protein/antibody?
Need a large change in molecular weight for detection
How does FRET work?
Direct observation by a FRET signal (2 fluorophores close together)
What are the advantages of FRET?
Very sensitive, little background, very fast, small concentrations
What interactions can PDZ have?
Protein/protein (non-canonical) and protein/peptide (canonical)
True or false: if two proteins have different kinetics, they must have different Kds
False: they can have different kinetics, but same Kd
What are the kinetics for a protein/protein interaction?
Stay on for longer, slower kinetics
What are the kinetics for a protein/peptide interaction?
Pop off very frequently, faster kinetics
What are the two major classes of protein/protein interactions?
Forming an interface, or peptide recognition
What is the typical protein/protein interface?
700-800 A^2, 10 H-bonds
What is the size of a typical protein?
100 A (largest dimension)
What percentage of a protein is an interface?
10%
True or false: interfaces are based on one interaction
False: they are a mix of multiple interactions
What two factors are needed for an interface?
Affinity and specificity
What is the role of hydrophobic interactions in interfaces?
High affinity, low specificity
What is the role of electrostatics in interfaces?
Give high specificity (positive and negative discrimination)
What is the role of water in interfaces?
Can mediate contacts (solvation, or H-bond network)
What is required for polar interactions?
Desolvation
What is a positive delta G elec?
Repulsive
What is a negative delta G elec?
Attractive
Are electrostatics usually attractive or repulsive and why?
Repulsive, because desolvation is a big energy cost)
Describe how forming a salt bridge is a repulsive electrostatic interaction
The surface is solvated, so there is a large enthalpic cost to desolvate. Not enough enthalpy is formed back when the salt bridge forms
What are some examples of peptide recognition domains?
PTB, SH2, SH3, WW
What is the purpose of combining modular domains?
Higher specificity and affinity
How does the Kd change if two domains are joined?
The sum of the delta G’s can be used to find the overall Kd (Kd joined = product of Kds), and it should be lower than each individually (higher affinity)
What is the MAGuK family?
Different scaffolds that can be combined together for different recognition
What is the energetic cost for peptide recognition?
Entropic loss (less freedom), but enthalpic gain (bonding)
Why is concentration required for specificity?
At low concentrations, there will be higher specificity than at higher concentrations
What is alpha?
Specificity factor
What is the formula for alpha?
alpha = [RoL]/sum([RiL]) (specific/non-specific)
What happens to alpha if Kd(R0) < L < Kd (R1)
Alpha is high, only desired target, high specificity
What happens to alpha if Kd(R0) < L = Kd(R1)?
Alpha decreases, more off target complexes
What happens to alpha if Kd(R0) < Kd (R1) < L?
Alpha approaches one, everything is saturated
What balance is needed with specificity?
Want concentration as low as possible for specificity (minimize off target effects), but need a certain amount to actually inhibit (bind to) target
What is IC50%?
Inhibitor concentration for 50% inhibition
What is Ki?
Dissociation constant for inhibitor
What does IC50% depend on?
Concentration
What interactions dominate DNA binding?
Electrostatic and polar interactions
For DNA binding, what charge is the protein?
Positive (DNA is negative)
How is dsDNA distinguished from dsRNA?
Differences in major/minor groove (complementary shape to specific protein in spacing of phosphates)
How does DNA bending affect binding?
Contributes to affinity, but lacks specific constraints, and is energetically costly (loss of H bonds and base stacking)
How do aromatic residues affect DNA binding?
These can lead to interactions beyond electrostatics
What are the key determinants of DNA binding specificity?
H-bonds
How can specificity be increased in DNA binding?
Tandem DNA binding domains
What is an example of a tandem repeat for DNA binding?
Zinc finger
What sequences do tandem domains recognize?
Palindromes (dimers)
What cooperativity is present in DNA binding?
Protein complexes that also bind to DNA
What is the problem of specificity in DNA binding?
Need to bind 20 bp in 10^9 bp sequence
True or false: transcription factors always bind to the right site
False: there are so many sites, so they can bind to the wrong site
What can be used to observe DNA binding and target searching?
Fluorescent microscopy (use a chip and fluorescence or FRET), and gel shift (have free DNA, and increase protein concentration leads to changes in mobility)
