molecular recognition

Question 1

Measuring KD…

Answer 1

any method that allows you to measure the concentration of the complex and one free component

e.g. filter binding, fluorescent anisotropy, isothermal calorimetry, eqbm dialysis

Question 2

filter binding =

Answer 2

• bind a radiolabelled ligand to the protein
• pass through a nitrocellulose filter
• nitrocellulose filter is negatively charged
• as proteins have a net positive charge they are immobilised (traps proteins and protein-ligand complex), whilst unbound ligand passes through
• measure radioactivity to determine how much ligand bound to protein
• flawed method as when bound complex is separated from free ligand the complex can start to dissociate as ligand conc. has decresed (non-eqbm method of measurement)

Question 3

fluorescent polarisation =

Answer 3

• small unbound molecule with fluorophore on it it will rotate rapidly in the presence of polarised light (fluorescence de-polarised as molecule moves)
• if the small molecule with fluorophore on it binds to a large complex molecule it will rotate much slower in the presence of polarised light (fluoresence polarised)
• carry out titrations at different conc. to work out KD

Question 4

isothermal calorimetry =

Answer 4

• determines the thermodynamics of interactions in solution
• temperature differences are detected between a reference cell filled with water and the sample cell containing the macromolecule
• ligand is titrated into the sample cell
• time dependent measurements of power required to maintain equal temperature in both cells is taken

Question 5

eqbm dialysis =

Answer 5

• measures amount of ligand bound to macromolecule
• free ligand is passed through a membrane until its concentration across the membrane is at eqbm, in order for free ligand conc to be measured
• if no ligand is bound = ligand is free to cross the membrane so at eqbm the conc of ligand in the assay will be half
• if ligand is bound = ligand is unable to cross the membrane so at eqbm the conc of ligand in the assay will be reduced by the the total amount of ligand bound to the protein divided by two

Question 6

two ordered molecules coming together =

Answer 6

e.g. lock and key (enzyme and substrate)

enthalpically favorable

Question 7

one order molecule binding to a disorder molecule =

Answer 7

enthalpically favourable

entropically disfavourable on forming interaction

Question 8

two flexible molecules interacting with each other =

Answer 8

become ordered

strongly entropically disfavourable

Question 9

two ordered molecules coming together and displace water

Answer 9

entropically favorable as release of water creates disorder

Question 10

measuring entropy and enthalpy…

Answer 10

measure KD at different temps using isothermal calorimetry

Question 11

all DNA binding proteins are…

Answer 11

positively charged = can bind to negative phosphodiester backbone (attracted to DNA via electrostatic steering)

Question 12

most common way proteins bind to DNA =

Answer 12

434 repressor

• has a helical reading head that sits in the major groove and can recognise 4-6 base pairs

= not specific enough = acts as a dimer to read 8-12 base pairs