macromolecular interactions

Question 1

what can an ‘intrinsically disordered protein’ refer to?

Answer 1

regions (between structured domains) or the whole protein

Question 2

give an example of an intrinsically disordered protein

Answer 2

the carboxy terminus of p53

Question 3

what are the interactions which cause a protein to fold?

Answer 3

• hydrophobic effect
• hydrogen bonds
• electrostatic interactions
• van der waals
• disulphide bridges

Question 4

what causes a protein to fold?

Answer 4

interacting with itself in a folded conformation will be enthalpically/entropically favourable, resulting in negative gibbs free energy

Question 5

give an example of macromolecular interactions

Answer 5

DNA - molecular recognition occurs via hydrogen bonds in complementary base pairing

Question 6

what is the use of the dissociation constant?

Answer 6

allows us to differentiate between transient and obligate interactions

Question 7

give examples of interactions from high-low KD

Answer 7

highest- Enzyme-ATP: mM-uM KD
signaling protein binding to target uM KD
sequence specific DNA recog. by transcription factor - nM KD
small molecule protein inhibitor - nM-pM KD
biotin binding to avidin - fM KD

Question 8

what determines specificity?

Answer 8

the difference in KD for one site versus another

Question 9

why is a large difference in KD required for a drug?

Answer 9

reduce side effects due to decreasing likelihood that drug will bind to off-target protein

Question 10

what methods can you use to measure KD?

Answer 10

• filter binding
• fluorescence anisotropy
• isothermal calorimetry
• optical interferometry

Question 11

how does filter binding measure kD?

Answer 11

add a radiolabelled ligand to substrate
nitrocellulose filter traps protein and protein-ligand complex
unbound ligand is in solution
cut up filter paper with bound ligand is used to measure radioactivity

Question 12

what are the drawbacks of filter binding?

Answer 12

• not the most accurate
• non-equilibrium method

when free ligand is separated from ligand-protein complex, complex begins to dissociate due to disrupted equilibrium

Question 13

how can fluorescence anisotropy be used to measure KD?

Answer 13

polarised light on a small unbound molecule -> rapid rotation and fluorescence is depolarised

polarised light on a large complex -> slower rotation -> polarised fluorescence

Question 14

how can isothermal calorimetry be used to meausre KD?

Answer 14

measures the heat released/absorbed upon binding

Question 15

how can optical interferometry be used to measure KD?

Answer 15

binding between an immobilised ligand on the biosensor tip and an analyte in solution produces a increase in optical thickness, resulting in a wavelength shift

Question 16

why might only weak interactions form between two complementary molecules?

Answer 16

if there is high entropic cost

Question 17

what increases and decreases entropy?

Answer 17

• freeing bound water molecules increases entropy

- rigidifying molecules decreases entropy

Question 18

why does the DNA helix have a negative charge?

Answer 18

the negatively charged phosphates are on the outside of the molecule, this charge spreads across the whole molecule

Question 19

what is a helix-turn-helix motif?

Answer 19

two a-helices separated by a tight turn. residues of the recognition helix make contacts with bases of the major groove, whilst residues of the first helix interact primarily with the backbone of the DNA

Question 20

why is the Met repressor unusual?

Answer 20

it is a regulatory protein that binds DNA through insertion of a pair of B-strands into the major groove. bacterial regulatory protein that does not bind via H-T-H