13 Protein function and molecular recognition Flashcards
What is a ligand?
A molecule that binds reversibly to a protein
Where does a ligand bind?
a binding site on a protein
What are the characteristics of a bidning site?
it is complementary to the ligand in size, shape, charge, and hydrophilic/phobic character
Why are protein-ligand interactions critical to life?
they allow rapid and reversible response to changing environmental and metabolic circumstances
How is the binding of a protein and ligand like an induced fit?
the binding is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand
How can protein-ligand interactions be described in letters?
What is Kd?
dissociation constant = used to express the addinity of a protein for a ligand (how strong they’re binding)
smaller kd = higher affinity
What is theta θ?
fraction of binding sites on the protein that are occupied by ligand
[PL]/ ([PL] + [P]
What is theta and L representing?
theta = fraction of binding sites on the protein that are occcupied by ligand
L= Kd, where half of the lingand binding sites are occupied, can be used to express the affinity of a protein for a ligand
which binds with higher affinity (i.e. binds tighter)
Avidin
smaller kd = higher affinity
the earlier half of ligand binding sites are occupied = stronger binding
which binds with higher affinity (i.e. binds tighter)?
X because it reaches theta (when half the binding sites are occupied) faster = kd is lower
X is half-saturated at a lower ligand concentration
if it needs less ligand to be saturated = it binds more strongly with ligand
when saturated at a lower kd = needs very little to be 50% saturated
if need a lot to be saturated = the ligand is not binding very strongly (binding then coming off) - low affinity
what is an oxygen-binding protein?
haemoglobin
What is the haemoglobin composed of?
4 subunits, 4 heme with iron atom (one each)
When does a haemoglobin undergo structural change?
when binding to O2
What is the T and R state of haemoglobin?
T and R state - 2 main changes
T state - bind with lower affinity w oxygen
R state - bind with higher affinity to oxygen
Why do we have 2 different states of haemoglobin?
High affinity is good for in the lungs to bind efficiently to O2, and Low affinity is good for tissues where O2 can be released
But low affinity in lungs is bad because it cant pick up oxygen, and high affinity in tissues is bad because it wouldnt release them
so haemoglobin transitions between T state (low affinity) to R state (high affinity) as more and more oxygen molecules are bound
What is happening here?
first O2 molecule binds weakly to a subunit in T state, its binding, leads to other subunits transition to R state and further O2 molecules binds tighter
what is positive cooperativity in haemoglobin?
the last O2 binds more strongly than the first
what is the inverse relationship that causes haemoglobin to release O2?
inverse relationship between binding of O2 and the binding of H+ and CO2
How does very low pH and high CO2 conc of peripheral tissues affect haemoglobin?
low Ph = High H+
the affinity of haemoglobin for O2 decreases and are released, then H+ and CO2 can be bound to haemoglobin
What happens to haemoglobin when the environment becomes less acidic?
haemoglobin have higher affinity for oxygen again
Where does BPG bind and what does it do?
binds at a different site from O2 binding site and regulates O2 binding affinity
BPG bind to middle pocket and O2 bind to heme area
How does carbon monoxide affect affinity of haemoglobin?
it affects affinity of remaining haemoglobin subunits for O2
CO makes subsequent binding of oxygen very strong - cannot be released to tissues
what is a molecular disease of haemoglobin?
sickle cell anemia
* single AA substitution, glu -> val at position 6 in the 2 beta chains
* Glu->val creates a sticky hydrophobic contact point at position 6 of the beta chain
* these sticky spots cause the protein to associate abnormally with each other, forming long, fibrous aggregates
* the fibre formation give rise to the sickle shape of RBC
* sickle cells are fragile and rupture easily = anemia (lack of blood)
How does the antigen bind to antibody?
- antibody has a specific antigen-binding site. the antibodies bind tightly and specifically to antigen.
- binding is an induced fit
What are the steps of ELISA?
- coat surface with sample (antigens)
- block unoccupied sites with nonspecific proteins
- incubate with primary antibody against specific antigen
- incubate with secondary antibody-enzyme complex that binds primary antibody
- add substrate
- formation of colored product indicates presence of specific antigen
What is an immunoblot?
- proteins that have been seperated by gel electrophoresis (SDS gel) are transferred to a nitrocellulose membrane
- membrane is treated with primary antibody, secondary antibody linked to enzyme and substrate
- coloured precipitate only forms on the band containing protein of interest
What are the components here
- troponin (a regulatory protein complex, green) is attached to tropomyosin (brown)
- myosin (red)
- actin (blue)
What is the muscle like when relaxed?
tropomyosin blocks the attachment sites for myosin, preventing the myosin filament from binding to actin
what is the process of muscle contraction?
- calcium enters and attaches to troponin which causes troponin to change in shape
- leads to tropomyosin to move away from the myosin attachment sites on the actin filament, exposing the binding sites for myosin to attach
- Myosin ATPase, which is located at the myosin heads, is responsible for hydrolysing ATP into ADP and Pi, and energising the myosin
- energised, the myosin can now attach onto the binding sites on actin, and now the phosphate group is released
- the myosin then bends and slides the actin across. this process is called the power stroke and it creates the actual muslce contraction movement. The ADP is released during this process
- new ATP must bind to the myosin heads to detach them from the actin and the muscle can then go back to the relaxed state
- process repeats
What is the general principle of antigen-antibody interaction in this?
- sample from sample pad go to conjugate pad where there are gold conjugated antibody - gives off the brown purple color
- then there are 2 lines, test and control line with same antibody
- test line for the antigen-antibody and control line with secondary antibody for the gold antibody - to test if kit is working properly
Which is the T state and R state? Where can you find BPG?
Left is T, theres space in the middle and R is more contracted
BPG supports the hemoglobin in a T state
when oxygen starts filling up the binding sites, it starts to compress the structure = no more pocket
What makes the properties of BPG and BPG binding site on haemoglobin complementary to each other?
charge/ionic charge
