macromolecular interactions Flashcards
what can an ‘intrinsically disordered protein’ refer to?
regions (between structured domains) or the whole protein
give an example of an intrinsically disordered protein
the carboxy terminus of p53
what are the interactions which cause a protein to fold?
- hydrophobic effect
- hydrogen bonds
- electrostatic interactions
- van der waals
- disulphide bridges
what causes a protein to fold?
interacting with itself in a folded conformation will be enthalpically/entropically favourable, resulting in negative gibbs free energy
give an example of macromolecular interactions
DNA - molecular recognition occurs via hydrogen bonds in complementary base pairing
what is the use of the dissociation constant?
allows us to differentiate between transient and obligate interactions
give examples of interactions from high-low KD
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
what determines specificity?
the difference in KD for one site versus another
why is a large difference in KD required for a drug?
reduce side effects due to decreasing likelihood that drug will bind to off-target protein
what methods can you use to measure KD?
- filter binding
- fluorescence anisotropy
- isothermal calorimetry
- optical interferometry
how does filter binding measure kD?
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
what are the drawbacks of filter binding?
- not the most accurate
- non-equilibrium method
when free ligand is separated from ligand-protein complex, complex begins to dissociate due to disrupted equilibrium
how can fluorescence anisotropy be used to measure KD?
polarised light on a small unbound molecule -> rapid rotation and fluorescence is depolarised
polarised light on a large complex -> slower rotation -> polarised fluorescence
how can isothermal calorimetry be used to meausre KD?
measures the heat released/absorbed upon binding
how can optical interferometry be used to measure KD?
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
why might only weak interactions form between two complementary molecules?
if there is high entropic cost
what increases and decreases entropy?
- freeing bound water molecules increases entropy
- rigidifying molecules decreases entropy
why does the DNA helix have a negative charge?
the negatively charged phosphates are on the outside of the molecule, this charge spreads across the whole molecule
what is a helix-turn-helix motif?
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
why is the Met repressor unusual?
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
