Molecular Recognition Flashcards
What kind of interfaces are there in permanent interactions?
Large hydrophobic interfaces
What kind of interfaces are there in transient interactions?
Hydrophobic and hydrophilic
How does hydrophobic interaction affect affinity?
Increases BUT not specific so use polar or charged groups too
What is Ka? What is Kd?
Association constant, dissociation constant
What is fraction bound?
[protein:ligand]/[protein]+[protein:ligand]
What is the other eqn for fraction bound?
[L]eqm / [L]eqm + Kd
If ligand is in excess, what is the equation for fraction bound?
[L]total / Kd + [L]total because L at eqm would be the same as total L
What is Kd?
The conc when 50% of protein is bound to ligand
What is Kd of efficient drugs?
Lower because they must compete with natural ligand
Why is Kd hard to measure in very high affinity interactions?
Need data point below is with very low [protein:ligand] so not possible to measure
What is isothermal titration calorimetry?
Measures heat changes when ligand added, keep at constant temp
How do you measure kon and koff?
Surface plasmon resonance, immobilise protein and flow ligand over, causes change in refractive index, slopes give on and off rate
What is the structure of the epidermal growth factor receptor?
Single alpha helix through the membrane
What does ligand do to EGF?
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
What does the SH2 domain bind to?
Phosphorylated tyrosine and Grb2
What is the structure of an SH2 domain?
Antiparallel B sheet and two alpha helices
How does phosphopeptide bind in SH2 domain?
As an extended strand
What interacts with the negative phosphate group of the SH2 domain?
Arg and Lys
What are the two binding pockets of an SH2 domain?
One to phosphotyrosine, one to side chains from binding peptide
What does Sos do to Ras?
Changed conformation, bound by large electrophilic interface
What does Grb2 do?
Recruits Sos
Which domains does Grb2 have?
SH2 to bind to tyrosine and two SH3 groups to bind to proline and Sos
How does Grb2 have such strong interactions with Sos?
Binding surface hydrophobic
Where does proline from Sos bind to Brb2?
Groove in it
What causes Ras to swap GDP for GTP?
Sos
What is the structure of an SH3 domain?
5 antiparallel B strands, aromatic residues from hydrophobic patch, binds a lefthanded polyproline helix
Why is Ras active with GTP?
Forms H bonds with gamma phosphate, downstream effectors recognise its active conformation
How does Ras’s “off switch” work?
Uses GTPase activating protein, inserts Arg into active site to stimulate hydrolysis
What happens when Ras binds to Raf?
Forms intermolecular antiparallel B sheet, H bonds and ionic interactions, no contact between Raf and GTP
What does Raf contact?
One of the main switch regions whose conformation changes when GTP binds to Ras
What does Raf phosphorylate?
Ser and Thr
Why is Raf inactive and how does it activate?
Because it loops in on itself, when Ras binds to N terminal region it straightens out and can now phosphorylate other proteins
Where does DNA binding specificity come from?
H bonds to side chains and base pairs, inserts alpha helix into major groove
How do Fos and Jun interact?
Spirals with Leu in the centre which fit into major grooves, non-specific ionic interactions with phosphates, specific H bonds with bases
How do kinases become inactive?
When activation loop blocks active site, phosphorylation moves it so any -ve group would mimic phosphate and cause overactive kinase
What is the usual Raf mutation in malignant melanomas?
Val > Glu in activation loop so Raf is activated without signal
What is fragment based drug discovery?
Small molecule drugs are hard to find because need to screen too many, instead start with fragments that work and add functional groups
What does Vemurafinib treat?
Melanoma
What are advantages of small molecule drugs?
Designed for good properties, easy and cheap, can enter active sites and transition state mimic, easily go through membrane
What are disadvantages of small molecule drugs?
Can’t inhibit protein-protein interactions because flat surface and few pockets, high failure rate because of unexpected side effects
