FOM 1.3.1 Flashcards
What is an apo-protein?
protein not bound to anything
What is a ligand?
Anything that binds a protein.
What is a holo-protein?
A complex of all components (proteins, subunits, and all ligands) needed for function
Give examples of some of the things that proteins bind.
small sugars, nucleotides, RNA, water, organic molecules, O2, large carbs, metal ions, proteins, lipids, CO2, protons, DNA, drugs, photons, NO
What constitutes a protein binding site?
A well-defined region of the protein that contacts a ligand. These contacts are made by specific AA side chains or backbone.
Give an example of a protein binding site interaction with a ligand.
An aromatic tryptophan ring stacking against the hexose ring of the sugar ligand.
How does ATP show chemical complementarity?
ATP has numerous domains that interact with binding sites including: Hydrophobic regions, positive charges, accepting and donating H bonds.
What happens when an ATP tries to bind in an inverted manner?
Chemical repulsion and steric clashes. ATP won’t bind
How can mutations alter binding? (2)
It can affect how tightly the ligand is bound (affinitiy) It can affect which ligand is bound most tightly (specificity, similar to selectivity)
What has occurred in patients who present with Vitamin D resistant rickets?
A mutation has occurred in the receptor that alters binding stability b/t receptor and ligand.
In the example with Vitamin D resistant rickets, how were doctors able to increase vitamin D uptake by cell lines? What’s the limitation or obstacle with this route?
The doctors were able to alter the vitamin D to better fit w/ the cells mutant receptor. The obstacle is that patients may have different mutations.
Can ligands affect the protein structure?
Yes and they most likely will.
How do ligands affect the equilibrium of proteins b/t their folded and unfolded form?
Shift equilibrium in favor of the folded forms.
What type of roles can metal ions play in proteins? Would we use drugs to mimic these effects?
“structural” roles Yes, drugs that mimic these effects are used in unfolding dz’s (e.g. CFTR)
Can different ligands affect the way a protein will fold? If so, how much of a folding difference could be achieved?
Yes 1000-fold difference
P + L PL , give the dissociation and association reaction contants, k
Ka = [PL]/[P][L], units M-1 Kd = [P][L]/[PL], units M
In the dissociation reaction, when [P]free = [PL], what is Kd equal to?
[L]
Where can Kd be found on a sigmoidal curve?
At the 50/50 point, the point where half of the protein is bound.
When looking at differentiation curves, what is important to note about the axises? How could it affect the curve?
Standard unit distribution = hyperbolic shape Logarithmic distribution= sigmoidal curve
What is the slope around the half-way point of an uninfluenced sigmoidal curve?
1
When looking at a single ligand-protein binding sigmoidal dissociation curve, what do shifts to the left (smaller []) and right indicate?
Left: Tighter affinity Right: Weaker affinity
When analyzing the capability of a drug to bind a protein, what type of concentration is desirable?
A drug that can achieve a concentration higher than its midpoint inside a cell is desirable.
What is the midpoint of a drug concentration in vivo called?
EC50 instead of Keq
What type relationship exists when the binding of the first has no effect on the binding of the subsequent?
Independent
What two types of relationships are possible if binding the 1st alters binding of the 2nd?
Cooperative, Allosteric
The thermodynamic cycle is…
pathway independent
Name this type of binding relationship: Same binding site, same ligand
Cooperative
Name this type of binding relationship: Different binding site, different ligand
Allosteric
Name this type of binding relationship: Different binding site, different ligand
Allosteric
Break down the word allostery. Allos: ____ Steric: ____
Allos: other Steric: placement of atoms
Give an example of an allosteric interaction
A protein binding w/ DNA & sugar
In inhibitory allostery, how is the sigmoidal curve altered?
Shifted to the right b/c its weaker
In enhancing allostery, how is the binding sigmoidal curve shifted?
To the left, b/c its stronger
What are some possible benefits/uses of allostery in nature?
Regulation! Modulating function instead of on/off Received signal and transform response
Give 2 examples of how allostery is utilized w/in the body.
Metabolism - change nutrients to allow more or less metabolism DNA Txn - change transcriptome/proteome in response to environment
Give two examples of how cooperativity could be arranged.
Binding repeated subunits on homo-oligomers Multiple identical binding domains on a single polypeptide chain (e.g. calmodulin)
The sigmoidal curves represents what of the multiple ligands
weighted average b/c its impossible to distinguish b/t the 2(+) binding sites
What happens to the curve with positive cooperativity?
The sigmoidal curve becomes steeper
What is different about a linear plot of ligand-protein binding with positive cooperativity?
The positive cooperativity curve shows sigmoidal characteristics
What is binding that one ligand makes subsequent binding more difficult?
Negative cooperativity
With negative cooperativity, will it ever reach 100% bound?
No
What are the 3 ultimate results of positive cooperativity?
Response range to ligand change narrows Dramatic biological effects Closer to on/off
What are the 2 ultimate results of negative cooperativity?
Changes @ higher ligand [] don/t affect protein function Constant protein function across a wide range of ligand []s
Name at least 3 of the possible advantages of allosteric drugs.
-More subtle effects -Slow function/Ramp up function -Expands # of search targets
What are the units for the association reaction? What are the units for the dissciation reaction?
Association M^-1
Dissociation M
What is the difference between a sigmoidal and hyperbolic graph?
The hyperbolic graph “tends” to be a classical representation and the sigmoidal graph is in logarithmic function.
What is the shifting in this graph demonstrating?
Shift to the right is a weaker affinity and a shift left is a stronger affininty
Answer this
B - because it uses the least amount of ligand to bind the most amount of protein. MDV3100 would be the most effective binder.
These two graphs demonstrate an example of what?
Inihibitory allostery
This graph demonstrates what type of allostery?
Enhancing allostery
Explain what each line is and what is happening to the slope of the green line
Pink is pos cooperativity
Orange is no cooperativity
Green is negative cooperativity, and it will never reach 100% of the positive
D - The shift to the right shows that there is a higher amount of Ca2+ needed to bind that same amount of CaM. This shows that it is diminishing the binding of Ca2+. The slope of the line is unchanged, meaning that it is allosterically diminishing the effect and not cooperatively.
