Final 18 Flashcards
What is puromycin and what does it do?
It is a toxin that inhibits the peptidyl-transferase of a ribosome, stopping protein synthesis.
What is the other toxin that stops protein synthesis by inhibiting the peptidyl-tranferase?
Choramphenicol/Linezolid
What does cyclohexamide do?
It inhibits peptidyl-transferase on the 80S ribosomal subunit instead of the 70S.
What does Diptheria Toxin do?
It glycosylases eEF-2 factor.
This will inhibit eEF-2’s function, restricting GTP hydrolysis, inhibiting the dissociation of the tRNA.
What does Ricin do?
It inactivates the large ribosomal subunit via glycosylation.
It removes an A from DNA without breaking the backbone, this causes tRNA from functioning and it inhibits GTP hydrolysis.
It kills the ribosome.
What is the main driving force of protein folding?
Hydrophobic interactions.
What are Hsp70 Chaperones?
They are proteins that have a hydrophobic domain and they work by binding to a protein and preventing it from annealing.
Also known as DnaK.
What are Hsp60 Chaperones?
They are a barrel-like structure that allows a protein to go inside and bind to the walls of the barrel.
The barrel is hydrophobic in nature, and once the cap is put on top, the chaperone induces a conformation change, making the hydrophobic AA, hydrophilic, and forcing the protein to refold in its native, correct state. Then it is released.
How does a cell know where to go?
Sorting Motif.
Typically located at an end of a protein, on either N-terminal or C-terminal ends.
What is the general protein export system? (5)
- Protein product + Softing Motif
- SecB binds to the protein and brings it to the membrane.
- ATP hydrolysis causes SecB to dissocaite, transfering the protein onto SecA.
- SecA uses ATP to feed the protein through the transporter.
- Once the protein is on the other side, the motif is cleaved.
What is the signaling motif for the general protein export consists of? Start from the cleavage site and go out.
Cleavage Site -> Polar Residues -> Hydrophobic Core -> Positively Charged AA
What is the goal of SRP and explain the 5 steps associated with it.
SRP will target proteins into the ER.
- Protein synthesis + protein motif.
- SRP will bind to the motif and the A site of the ribosome, stopping synthesis.
- SRP finds the docking site/SRP receptor on the ER.
- SRP ensures that the protein fits in the peptide translocation complex, and then dissociates so that protein synthesis can occur again.
- Once inside the ER, the motif is cleaved.
What is the role of the SRP protein?
- Recognition of the motif.
- Stops protein synthesis.
- Finds the docking site.
What is the motif for SRP targeting to the ER?
The same as in bacteria.
Cleavage -> Polar Residues -> Hydrophobic Core -> Positively Charged AA
What is glycosylation? Where does it happen?
Glycosylation is the addition of a carbohydrate onto a protein and this happens in the ER.
What are the four steps of glycosylation?
- Carbohydrates form on the exterior of the ER.
- The carbohydrates get translocated into the ER where they will be modified and altered.
- The carbohydrates then get added onto an asparagine o the protein that is being transported.
- Once the protein is fully made, the glycosyl group can add as a binding site, signal, or it can get cleaved off.
Where does a carbohydrate get added onto a protein?
On the asparagine close to a terminal end.
Translocation from the ER into the golgi requires what kind of modfication?
O-linked glycoslyation on a serine.
N-linked oligosaccharide on a asparagine.
What is NLS?
It is a motif on a protein that signals it to go into the cell?
What binds to the NLS signal to bring it into the cell?
Importins -> alpha and beta
What molecule allows for the dissociated of the beta-subunit in NLS from the protein?
Ran-GTP
How does Ran-GDP get back into the nucleus?
Through a protein called NTF2.
What does CAS do?
CAS allows for the dissociation of the alpha-subunit from the protein with the NLS signal.
The tendrils of the nuclear poor have what type of AA residues?
Threonine and Leucine.
What are four unique features of NLS?
- The motif isn’t always N-terminal (can be C-terminal)
- The motif is NOT cleaved.
- The use of importins and Ran-GTPase
- Proteins can enter into the nucleus in their 3D structure.
What two protein are used during endocytosis?
Clathrin and Dynamin
Phosphorylation on post-transcriptional modifications can happen on which AA?
Serine, Threonine, and Tyrosine.
What is the JAK-STAT pathway?
It is an example displaying the important of phosphorylation.
What are the four steps of the JAK-STAT pathway?
- Alpha-interferon (a cytokine) is secreted in response to a viral response.
- Alpha-interferon binds to the cell receptor, causing JAK to auto-phosphorylate itself.
- This auto-phosphorylation recruits STAT, which also gets phosphorylated, causing it dimerize and become active.
- STAT then leaves and enters the nucleus where it can activate transcription in response to the viral infection.
What is methylation? What is its effect?
It is the addition of a methyl group onto a protein.
It can serve as a binding site for other molecules or it can change the proteins conformation.
What is the net effect of lipid attachment to proteins?
It targets the protein to the membrane.
What are the two main FA that are added onto proteins?
- Myristoyl
2. Palmitoyl.
Explain the addition of Myristoyl and Palmitoyl onto a protein.
- Myristoyl is bonded onto a protein via a glycine through an amide bond. (strong)
- Palmitoyl is bonded to a protein through an ester linkage. (weak)
Explain how different types of lipid attachment will effect its affinity to the membrane.
- Myristoyl is connected to the protein through a strong bond so it will associated with the membrane really well.
- Palmitoyl is connected to the protein through a weak bond so it is easily cleaved. It will not associate with the membrane well.
What are the other two FA talked about in lipid attachment? Are they strong? Weak?
Isoprenoid -> strong
Phospholipid -> weak
Explain how proteins can have more than one modification and the effect of it using RAS.
RAS (a GTPase) can have more than one modification.
If it is farnesylated and palmitoylated it will be targeted to the membrane.
If it is ONLY farnesylated, it will be targeted to the Golgi.
What are 5 effects from glycosylation?
- Increase solubility
- Prevent aggregation
- Signaling
- Protection from proteases
- Binding sites on cell surface for recognition
N-linked glycosylation is through what AA?
Asparagine
O-linked glycosylation is through what AA?
Serine
What is the specific degradation signal?
Ubiquitin- Mediated Pathway
What are the three enzymes that catalyze the addition of a ubiquitin molecule?
E1: Activating Enzyme
E2: Carrier Enzyme
E3: Ligase (activity of addition)
Explain the three step process of ubiquitin-mediated pathway.
- Activation of ubiquitin requires ATP. It forms thioester bond on the C-terminal end of a Glycine with ATP.
- Transfer of the ubiquitin onto a cysteine-thiol of E2, which is the carrier protein.
- E2 brings ubiquitin to E3 where it will add it to the target via Lysine residue.
What kind of bond is formed between ubiquitin and the E1 enzyme when activating it and on what AA?
Thioester bond on the C-terminal end of a Glycine.
During the transfer from E1 to E2 in the ubiquitin-mediated pathway, that is it transfered onto?
A cysteine-thiol on E2.
What is ubiquitin added onto on the target protein?
A lysine residue.
What can happen after a target protein has been ubiquitinated?
The ubiquitin itself can be ubiquitinated.
What will signal for degradation?
Ubiquitination of Ubiquitin on K11 or K48.
What is a ubiquitin-like molecule?
SUMO vis sumolyation.
How is the protein degraded after it has been ubiquitinated?
It will be targeted to the degradation machinery, the proteosome.
The machinery has ATPase activity on the end and will unravel and feel the protein through the barrel. Within the barrel, there will be proteolytic activity where it will chop up the protein into smaller pieces.
What is the protein half-life?
The life of a protein is dictated by the AA present on the N-terminal end.
Ala, Gly, Met > 20 Hours
Glu
