10 - Ligand Binding and Equilibria II

Question 1

True or false: binding is a kinetic process

Answer 1

True: it depends on on and off rate

Question 2

True or false: all collisions lead to reactions

Answer 2

False: they need sufficient speed and orientation

Question 3

What is the order of the association reaction?

Answer 3

2nd order (1/Ms)

Question 4

What is the order of the dissociation reaction?

Answer 4

1st order (1/s)

Question 5

For a first order reaction, what is the equation for the half time?

Answer 5

t1/2 = ln(2)/k

Question 6

What does the association rate depend on?

Answer 6

Diffusion

Question 7

What is the fastest possible type of reaction?

Answer 7

A diffusion limited reaction

Question 8

How does stopped flow work?

Answer 8

Two reactants are mixed, and it is measured how solution ages over time

Question 9

On what order does stopped flow work?

Answer 9

Reactions on order of seconds (mixing on order of ms)

Question 10

What is measured in stopped flow?

Answer 10

A change in absorbance or fluorescence

Question 11

What is calculated in stopped flow?

Answer 11

kon

Question 12

How is kon calculated in stopped flow?

Answer 12

Measure the fluorescence over time for different [L] levels, then plot k vs L to find kon and koff.

Question 13

For stopped flow, what line is plotted?

Answer 13

kobs = (kon)[L]T + koff

Question 14

How does SPR work?

Answer 14

A gold membrane is used to bind antibodies to receptor (immobilize protein), and change in density is measured

Question 15

What is SPR used frequently for?

Answer 15

Protein/antibody, not protein/small molecule

Question 16

How come SPR is used for protein/antibody?

Answer 16

Need a large change in molecular weight for detection

Question 17

How does FRET work?

Answer 17

Direct observation by a FRET signal (2 fluorophores close together)

Question 18

What are the advantages of FRET?

Answer 18

Very sensitive, little background, very fast, small concentrations

Question 19

What interactions can PDZ have?

Answer 19

Protein/protein (non-canonical) and protein/peptide (canonical)

Question 20

True or false: if two proteins have different kinetics, they must have different Kds

Answer 20

False: they can have different kinetics, but same Kd

Question 21

What are the kinetics for a protein/protein interaction?

Answer 21

Stay on for longer, slower kinetics

Question 22

What are the kinetics for a protein/peptide interaction?

Answer 22

Pop off very frequently, faster kinetics

Question 23

What are the two major classes of protein/protein interactions?

Answer 23

Forming an interface, or peptide recognition

Question 24

What is the typical protein/protein interface?

Answer 24

700-800 A^2, 10 H-bonds

Question 25

What is the size of a typical protein?

Answer 25

100 A (largest dimension)

Question 26

What percentage of a protein is an interface?

Answer 26

10%

Question 27

True or false: interfaces are based on one interaction

Answer 27

False: they are a mix of multiple interactions

Question 28

What two factors are needed for an interface?

Answer 28

Affinity and specificity

Question 29

What is the role of hydrophobic interactions in interfaces?

Answer 29

High affinity, low specificity

Question 30

What is the role of electrostatics in interfaces?

Answer 30

Give high specificity (positive and negative discrimination)

Question 31

What is the role of water in interfaces?

Answer 31

Can mediate contacts (solvation, or H-bond network)

Question 32

What is required for polar interactions?

Answer 32

Desolvation

Question 33

What is a positive delta G elec?

Answer 33

Repulsive

Question 34

What is a negative delta G elec?

Answer 34

Attractive

Question 35

Are electrostatics usually attractive or repulsive and why?

Answer 35

Repulsive, because desolvation is a big energy cost)

Question 36

Describe how forming a salt bridge is a repulsive electrostatic interaction

Answer 36

The surface is solvated, so there is a large enthalpic cost to desolvate. Not enough enthalpy is formed back when the salt bridge forms

Question 37

What are some examples of peptide recognition domains?

Answer 37

PTB, SH2, SH3, WW

Question 38

What is the purpose of combining modular domains?

Answer 38

Higher specificity and affinity

Question 39

How does the Kd change if two domains are joined?

Answer 39

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)

Question 40

What is the MAGuK family?

Answer 40

Different scaffolds that can be combined together for different recognition

Question 41

What is the energetic cost for peptide recognition?

Answer 41

Entropic loss (less freedom), but enthalpic gain (bonding)

Question 42

Why is concentration required for specificity?

Answer 42

At low concentrations, there will be higher specificity than at higher concentrations

Question 43

What is alpha?

Answer 43

Specificity factor

Question 44

What is the formula for alpha?

Answer 44

alpha = [RoL]/sum([RiL]) (specific/non-specific)

Question 45

What happens to alpha if Kd(R0) < L < Kd (R1)

Answer 45

Alpha is high, only desired target, high specificity

Question 46

What happens to alpha if Kd(R0) < L = Kd(R1)?

Answer 46

Alpha decreases, more off target complexes

Question 47

What happens to alpha if Kd(R0) < Kd (R1) < L?

Answer 47

Alpha approaches one, everything is saturated

Question 48

What balance is needed with specificity?

Answer 48

Want concentration as low as possible for specificity (minimize off target effects), but need a certain amount to actually inhibit (bind to) target

Question 49

What is IC50%?

Answer 49

Inhibitor concentration for 50% inhibition

Question 50

What is Ki?

Answer 50

Dissociation constant for inhibitor

Question 51

What does IC50% depend on?

Answer 51

Concentration

Question 52

What interactions dominate DNA binding?

Answer 52

Electrostatic and polar interactions

Question 53

For DNA binding, what charge is the protein?

Answer 53

Positive (DNA is negative)

Question 54

How is dsDNA distinguished from dsRNA?

Answer 54

Differences in major/minor groove (complementary shape to specific protein in spacing of phosphates)

Question 55

How does DNA bending affect binding?

Answer 55

Contributes to affinity, but lacks specific constraints, and is energetically costly (loss of H bonds and base stacking)

Question 56

How do aromatic residues affect DNA binding?

Answer 56

These can lead to interactions beyond electrostatics

Question 57

What are the key determinants of DNA binding specificity?

Answer 57

H-bonds

Question 58

How can specificity be increased in DNA binding?

Answer 58

Tandem DNA binding domains

Question 59

What is an example of a tandem repeat for DNA binding?

Answer 59

Zinc finger

Question 60

What sequences do tandem domains recognize?

Answer 60

Palindromes (dimers)

Question 61

What cooperativity is present in DNA binding?

Answer 61

Protein complexes that also bind to DNA

Question 62

What is the problem of specificity in DNA binding?

Answer 62

Need to bind 20 bp in 10^9 bp sequence

Question 63

True or false: transcription factors always bind to the right site

Answer 63

False: there are so many sites, so they can bind to the wrong site

Question 64

What can be used to observe DNA binding and target searching?

Answer 64

Fluorescent microscopy (use a chip and fluorescence or FRET), and gel shift (have free DNA, and increase protein concentration leads to changes in mobility)