Flashcards in Understanding interactions between macromolecules Deck (47):
What do 2 single-stranded DNA molecules make?
What do DNA and a transcription factor make?
DNA-transcription factor complex
What does an enzyme and a substrate make?
An enzyme-substrate complex
What does an enzyme and a drug make?
An enzyme-drug complex
What does a cell surface signalling receptor and a hormone make?
A hormone-receptor complex
What does an antibody and an antigen protein make?
An antibody-antigen complex
What is the equation for the dissociation constant?
A + B AB
Kd = ([A] [B]) / ([AB])
Units are concentration
What is specificity?
The difference in KD for one site versus another
Why is specificity important?
There are many families of similar proteins
Binding sites in a sea of other DNA
On- and off- target activities
In reality there is a continuum of different binding affinities..
And a particular protein will be distributed among binding sites according to the different KD's.
A high degree of specificity does not necessarily mean..
High affinity, and vice versa
What methods can you use to measure KD?
Surface plasmon resonance - BiaCore
Optical interferometry - Octet
Two molecules will interact if there is a loss of..
Gibbs free energy on the formation of a complex
What is the equation for Gibbs free energy?
deltaG = deltaH - (T deltaS)
where deltaH is enthalpy (heat), and deltaS is entropy (disorder)
Both require intimate contact between two molecules
H-bonds, electrostatic interactions, van der Waals interactions
Does freeing bound water increase or decrease entropy?
Does rigidifying macromolecules increase or decrease entropy?
What types of interactions can you get in terms of rigid and flexible?
(Don't forget the solvent)
How can you measure the change in enthalpy and the change in entropy?
Measure KD's at several different temperatures
Using isothermal calorimetry
delta G = delta H - (T delta S) = RT ln Kd
What part of the DNA helix can help to achieve specificity?
The major (wide) and minor (narrow) grooves
What charges do the blue, red and white parts of the chemical space-fill models represent?
Blue - positive potential
Red - negative potential
White - neutral
What properties does the 434 repressor binding site have?
4-6 base pairs
Do hydrogen bonds have a strong or weak dipole? And how does this affect alpha helices?
Hydrogen bonds have a weak dipole
Alpha helices 'add up' weak dipoles
Is a 6 base pair binding site specific enough? And explain why
The human genome is ~3 billion base pairs
If sequence is random then a 6 base pair binding site will occur >500000 times
Therefore 6 base pairs is not enough to be specific
How many times does a 12 base pair binding site occur in the human genome?
Around 200 times
What is the KD value for the 434 repressor?
What properties does the CAP activator binding site have?
What properties does the engrailed homeodomain binding domain have?
Not a dimer
Arginines in the minor groove
What properties does the met repressor binding domain have?
Beta-strands in the major groove
Give examples of zinc-binding DNA recognition motifs
Classical zinc finger (tramtrack), e.g. zif 268 - zinc finger contains tandem repeats to read more sequence
Hormone receptor, e.g. oestrogen receptor DNA binding domain (GATA-I) contains half a NR DBD and a long tail in the minor groove
TBP-TATA binding protein contains a protein saddle in the minor groove which is splayed open
Are cartoon chemistry figures accurate or misleading?
What does the CAP activator help to do?
Name a protein that can cause extraordinary deformation of the DNA
TBP-TATA binding protein
What are the lifetimes of protein:protein interactions?
Obligate (often co-folding)
Transient (often weak but highly specific)
What types of interaction can be protein:protein interactions?
What drives the interaction of protein:protein interactions?
Role of enthalpy and entropy
What is a rigid:rigid interaction also known as?
Lock and key
What is a rigid:flexibility interaction also known as?
What is a flexible:flexible interaction also known as?
What is the KD value for the barnase:barstar interaction?
10^-14 M (= Koff/Kon)
Describe the barnase:barstar interaction
Buried residues at the interface
Importance of charge steering on on rate
Can transient interactions also be co-folding?
What do charged surfaces cause?
Are affinity, specificity and discrimination the same or different?
What is the additive effect of interactions also called?
In terms of protein:drug interactions, the affinity of interaction is often driven by entropy, hence often largely..