Molecular Recognition

Question 1

What kind of interfaces are there in permanent interactions?

Answer 1

Large hydrophobic interfaces

Question 2

What kind of interfaces are there in transient interactions?

Answer 2

Hydrophobic and hydrophilic

Question 3

How does hydrophobic interaction affect affinity?

Answer 3

Increases BUT not specific so use polar or charged groups too

Question 4

What is Ka? What is Kd?

Answer 4

Association constant, dissociation constant

Question 5

What is fraction bound?

Answer 5

[protein:ligand]/[protein]+[protein:ligand]

Question 6

What is the other eqn for fraction bound?

Answer 6

[L]eqm / [L]eqm + Kd

Question 7

If ligand is in excess, what is the equation for fraction bound?

Answer 7

[L]total / Kd + [L]total because L at eqm would be the same as total L

Question 8

What is Kd?

Answer 8

The conc when 50% of protein is bound to ligand

Question 9

What is Kd of efficient drugs?

Answer 9

Lower because they must compete with natural ligand

Question 10

Why is Kd hard to measure in very high affinity interactions?

Answer 10

Need data point below is with very low [protein:ligand] so not possible to measure

Question 11

What is isothermal titration calorimetry?

Answer 11

Measures heat changes when ligand added, keep at constant temp

Question 12

How do you measure kon and koff?

Answer 12

Surface plasmon resonance, immobilise protein and flow ligand over, causes change in refractive index, slopes give on and off rate

Question 13

What is the structure of the epidermal growth factor receptor?

Answer 13

Single alpha helix through the membrane

Question 14

What does ligand do to EGF?

Answer 14

Causes conformational change to expose domain II - two receptors join at domain II (dimerisation state), domain I and III join to form EGF binding site and kinase activated but only one

Question 15

What does the SH2 domain bind to?

Answer 15

Phosphorylated tyrosine and Grb2

Question 16

What is the structure of an SH2 domain?

Answer 16

Antiparallel B sheet and two alpha helices

Question 17

How does phosphopeptide bind in SH2 domain?

Answer 17

As an extended strand

Question 18

What interacts with the negative phosphate group of the SH2 domain?

Answer 18

Arg and Lys

Question 19

What are the two binding pockets of an SH2 domain?

Answer 19

One to phosphotyrosine, one to side chains from binding peptide

Question 20

What does Sos do to Ras?

Answer 20

Changed conformation, bound by large electrophilic interface

Question 21

What does Grb2 do?

Answer 21

Recruits Sos

Question 22

Which domains does Grb2 have?

Answer 22

SH2 to bind to tyrosine and two SH3 groups to bind to proline and Sos

Question 23

How does Grb2 have such strong interactions with Sos?

Answer 23

Binding surface hydrophobic

Question 24

Where does proline from Sos bind to Brb2?

Answer 24

Groove in it

Question 25

What causes Ras to swap GDP for GTP?

Answer 25

Sos

Question 26

What is the structure of an SH3 domain?

Answer 26

5 antiparallel B strands, aromatic residues from hydrophobic patch, binds a lefthanded polyproline helix

Question 27

Why is Ras active with GTP?

Answer 27

Forms H bonds with gamma phosphate, downstream effectors recognise its active conformation

Question 28

How does Ras's "off switch" work?

Answer 28

Uses GTPase activating protein, inserts Arg into active site to stimulate hydrolysis

Question 29

What happens when Ras binds to Raf?

Answer 29

Forms intermolecular antiparallel B sheet, H bonds and ionic interactions, no contact between Raf and GTP

Question 30

What does Raf contact?

Answer 30

One of the main switch regions whose conformation changes when GTP binds to Ras

Question 31

What does Raf phosphorylate?

Answer 31

Ser and Thr

Question 32

Why is Raf inactive and how does it activate?

Answer 32

Because it loops in on itself, when Ras binds to N terminal region it straightens out and can now phosphorylate other proteins

Question 33

Where does DNA binding specificity come from?

Answer 33

H bonds to side chains and base pairs, inserts alpha helix into major groove

Question 34

How do Fos and Jun interact?

Answer 34

Spirals with Leu in the centre which fit into major grooves, non-specific ionic interactions with phosphates, specific H bonds with bases

Question 35

How do kinases become inactive?

Answer 35

When activation loop blocks active site, phosphorylation moves it so any -ve group would mimic phosphate and cause overactive kinase

Question 36

What is the usual Raf mutation in malignant melanomas?

Answer 36

Val > Glu in activation loop so Raf is activated without signal

Question 37

What is fragment based drug discovery?

Answer 37

Small molecule drugs are hard to find because need to screen too many, instead start with fragments that work and add functional groups

Question 38

What does Vemurafinib treat?

Answer 38

Melanoma

Question 39

What are advantages of small molecule drugs?

Answer 39

Designed for good properties, easy and cheap, can enter active sites and transition state mimic, easily go through membrane

Question 40

What are disadvantages of small molecule drugs?

Answer 40

Can't inhibit protein-protein interactions because flat surface and few pockets, high failure rate because of unexpected side effects