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
What is the Bohr effect?
how the pH and Co2 affect the O2 affinity
tissue: low pH, high Co2, affinity is low so oxygen is released and H+ and Co2 binds
lungs: Co2 is excreted so pH rises and affinity increases. then binds more O2 to be transported back to the peripheral tissues
What is O2 binding to haemoglobin regulated by?
2,3 - bisphosphoglycerate (BPG)
What type of relationship between binding of O2 and binding of BPG?
inverse relationship
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
in what situation is BPG important?
- physiological adaptation to high altitude
- foetal development
Does foetal haemoglobin have lower or higher affinity for BPG than normal adult haemoglobin?
Foetus must extract O2 from its mothers blood so foetal haemoglobin must have greater affinity than the mternal haemoglobin for O2
the foetal haemoglobin have much lower affinity for BPG than normal adult haemoglobin, and a correspondingly higher affinity for O2
BPG and affinity have an inverse relationship
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 are carbon monoxides symptoms w varying %
<10% - symptoms rarely observed
15% - mild headache
20-30% - severe headache with neurological symptoms
30-50% - more severe neurological symptoms
50% - lose consciousness and coma
>60 % - death
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)
what does a immune system do?
- distinguish between molecular “self” from “nonself”
- destroy what is “nonself” i.e. pathogens or molecular threats
What is an immune system?
a sensitive and specific biochemical system built upon reversible binding of ligands to proteins
What are immunoglobulins?
antibodies
How many classes of immunoglobulins and which is the most abundant class?
5 classes
most abundant is IgG
What is an antigen?
any molecule/pathogen capable of eliciting an immune response
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 is ELISA?
enzyme-linked immunosorbent assay
* used to rapidly screen or quantify an antigen in a sample
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 some examples of motor proteins?
- flagellar motion in bacteria involves complex rotational motor at base of flagellum
- helicases, polymerases, other proteins move along DNA as they carry out functions in DNA metabolism
- kinesins and dyneins move along microtubules pulling along other organielles
What are the 2 proteins of skeletal muscles?
Myosin head and actin filament
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 does a vaccine contain? and what does it do?
contains a weakened or killed virus/parts of a viral protein = antigen
when injected, it doesnt cause an infection but teaches the immune system what the viral antigens look like, stimulating production of memory cells so during actual infection, memory cells can spot the virus and trigger an immune response producing antibodies
What strategy could viruse use to evade the immune system?
mutation, change protein structure so it cannot be recognised by exisiting antibodies
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
what antibody would you choose to develop a more effective covid 19 rapid antigen test - X (Kd of 3x10^-7M) or Y (Kd of 5x10^-8)?
Y because lower Kd = higher binding/affinity
Because Y is half saturated at lower ligand concentration. if it needs less ligand to be saturated, means it binds more strongly with ligand.
stronger binding makes covid 19 test more effective and sensitive
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
How does BPG help with high altitude adaptation?
BPG increases
but this BPG adjustment has only a small effect on the binding of oxygen in the lungs, but a considerable effect on the release of oxygen in tissues
affinity of hemoglobin for oxygen decrease = delivery of oxygen to tissues is restored, significantly enables the release of oxygen to the tissues in an environment where the pO2 is considerably lower
What happens after high altitude adaptation when they go back to sea level conditions?
situation is reversed
BPG decreases and affinity of haemoglobin for oxygen increases
What cant be generated from oxidative phosphorylation without oxygen?
ATP
What is rigor mortis?
- post-death stiffening of muscles
- starts immediately after death and is usally seen in a sequence
- smaller muscles over the face first
- followed by muscles in the hands and upper limbs, and finally appears in the large muscles of the lower limbs
- rigor mortis appears ~2 hours after death in the muscles of the face, progresses to the limbs over the next few hours
- completes between 6-8 hours of death
- 24 hours death rigor mortis dissapears because protein starts to degrade
- need ATP to detach myosin and actin
