11 - Allostery Flashcards
What does allostery do?
It alters affinity response
What does a 100 fold change in ligand concentration lead to?
9 fold change in fractional occupancy
What is a graded response?
Continuum
What is a linear response?
Stepwise
What does an ultrasensitive response look like?
It is steeper (10 fold change in input for 9 fold change in fractional occupancy)
On a linear scale, what do graded and ultrasensitive responses look like?
Graded response is hyperbolic, while ultrasensitive response is sigmoidal
What is a graded response similar to?
A dimer
What is an ultrasensitive response similar to?
A switch
What is cooperativity?
Binding of one ligand molecule changes the affinity for the second ligand binding
What is allostery?
Binding occurs at a different site (not the active site)
What is needed for allostery?
More than one binding site
True or false: all cooperative binding is allosteric
False: not all cooperative binding is allosteric
What is an example of cooperative binding that is not allosteric?
2 binding sites on DNA for transcription factors, can increase or decrease binding
Why is allosteric feedback common at the beginning of biochemical pathways?
Can regulate whether the pathway proceeds or not (it has the proper substrates)
What is the MWC model of allostery?
Induced fit model, change conformation of active site
In a plot of E vs conformation, what would the MWC model predict?
The graph would shift to the right (conformational shift)
What is the KNF model of allostery?
Transient formation of low and high state, binding shifts equilibrium
In a plot of E vs conformation, what would the KNF model predict?
A narrower curve (population shift)
True or false: ligand binding changes protein dynamics
True: this affects the jiggles and wiggles of the protein
How does bacterial chemotaxis work?
When bound to a repellant, CheY is phosphorylated, causing clockwise rotation and tumbling
What type of response is pCheY?
Ultrasensitive response
What is the general structure of a protein kinase?
N lobe, C lobe, and ATP pocket
What is found in the N lobe of a protein kinase?
DFG motif (activation loop)
What regulates the DFG motif in a kinase?
Helix alpha C
What does the P loop do in a kinase?
Covers ATP product
What does a kinase cycle between?
Active and inactive states
What happens when the activation loop is phosphorylated?
It locks the kinase into the active configuration
How does helix alpha C regulate the kinase?
It can either break salt bridge, or flip DFG motif
What are kinase cascades an example of?
An ultrasensitive switch (multiply system output)
Is the response of MAPK3 shallow or steep and why?
Shallow (initial protein in cascade)
Is the response of MAPK shallow or steep and why?
Steep (amplification by enzyme activity)
What affects the steepness of the response?
Cooperativity
What is positive cooperativty?
Increases affinity (Kd1 > Kd2)
What is negative cooperativity?
Decreases affinity (Kd1 < Kd2)
How does positive cooperativity change the slope steepness?
It increases it
How does negative cooperativity change the slope steepness?
It decreases it
What is the significance of negative cooperativity?
Wide range of sensitivity (extend dynamic range, better at detecting high vs. low)
How does oxygen transport work?
Load hemoglobin with high O2 in lungs, release with low O2 in tissues
What is the importance of myoglobin?
It scavenges O2 away from hemoglobin to keep O2 levels low
What is the structure of hemoglobin?
4 subunits
What happens when O2 binds to a hemoglobin subunit?
It causes a conformational change, and increases affinity
How does hemoglobin give rise to cooperativity?
Switch between different states of affinity
What is the high affinity state in hemoglobin?
R
What is the low affinity state in hemoglobin?
T
If hemoglobin goes from R to T, what cooperativity is this?
Negative
If hemoglobin goes from T to R, what cooperativity is this?
Positive
What is the response to switch between 2 affinity states?
A sigmoidal response
What happens at f/(1-f) = 1?
f = 0.5, Kd = L
How can you find Kd from f/(1-f) and L?
Plot f/(1-f) and log(L), and log(Kd) is when f/(1-f) = 1
What happens if two isotherms are far apart?
There is stronger cooperativity?
What quantifies binding cooperativity?
Hill coefficient (slope in log (f/(1-f)) vs log(L) plot at half ligand saturation)
What is the formula for hill coefficient?
nH = 2/(1+sqrt(Kd2/Kd1))
What happens if nH > 1?
Positive cooperativity (slope > 1)
What happens if nH < 1?
Negative cooperativity (slope < 1)
What is the maximum of nH?
2
What is the minimum of nH?
0
What does the maximum value of nH represent?
The minimum number of interacting binding sites
What is the max nH for hemoglobin?
4
What happens if Kd1 = Kd2?
nH = 1, no cooperativity (no change in affinity)
What is the structure of deoxyhemoglobin?
It has a twisted heme group
What happens when deoxyhemoglobin becomes oxygenated?
The heme group is flattened, changing the conformation and changing affinity states (rotation)
What does biphosphoglycerate do?
It stabilizes deoxyhemoglobin
What do BPG and CO2 have in common?
They reduce oxygen affinity binding
What is allosteric regulation?
Stabilize low affinity state
What is the Bohr effect?
Fine tune O2 transport by decreases in pH
Which has the higher pH: lungs or tissues?
Lungs
How does CO2 impair oxygen affinity?
It interacts with K40 and D94 to disrupt salt bridge
