IV. Cell Biology | 68. Protein quality control in the endoplasmic reticulum; the fate of misfolded proteins; ERAD Flashcards
- Levels of protein organization
a/ List the levels of protein organization
1/ Primary structure
2/ Secondary structure
3/ Tertiary structure
4/ Quaternary structure
- Levels of protein organization
b/ Characteristics of primary structure?
It is a sequence of a chain of amino acids
- Levels of protein organization
c1/ Characteristics of secondary structure
It is a sequence of amino acids that have hydrogen bridges between carbonyl and amino groups of peptide bonds (α-helix, β-sheet)
- Levels of protein organization
c2/ What are the 2 types of secondary structure
1) α helix: right hand spiral,
H-bonds between amino acids located in three residues distance
- time of formation: ~ 100 ns
2) β pleated sheet:
two or more segments of a polypeptide chain line up next to each other and are held together by hydrogen bonds
- time of formation: ~ 1000 ns
- Levels of protein organization
d/ Characteristics of tertiary structure
when certain attractions are present between α- helices and β-pleated sheets (ex: disulfide bridges, hydrophobic interaction, ionic bonds, H-bonds)
=> forms the overall 3D shape of the protein
- Levels of protein organization
e/ Characteristics of quarternary structure
protein consisting of more than one amino acid chain
- What is Anfinsen’s dogma?
a thermodynamic hypothesis that propose:
- native structure of proteins is determined only by their amino acid sequence
(primary structure)
- What does Levinthal’s paradox state?
There are enormous numbers of possible conformations for protein folding – would take endless amount of years to try all, yet folding is done on a millisecond scale = contradiction.
E.g, a protein composed of 100 amino acids:
- every amino acid has 3 possible conformations
-> 3^100
-> 10^-13 seconds for trying one conformation
-> 10^27 years to try all conformations
- What is Hydrophobic collapse?
Because the main driving force is to find the thermodynamically more favorable position, the cytosol is water base solution (polar) –> try to hide the AA which poses the hydrophobic side chains, the polar AA will face to the cytosol.
=> It is called Molten globule state: secondary strucure is formed but tertiary structure is not finalized, yet.
- Why are proteins driven to their free energy minimum via the folding funnel?
- the folded state of the protein is very stable
- the undesired amino acid interactions along the folding pathway are reduced
- by forming the contacts of key residues the overall topology of the protein is
established
- Success rate of in vitro folding of eukaryotic proteins is only 20-30 %
-> WHY?
1) Macromolecular crowding inside the cells
2) Protein aggregation competes with folding (oligomers, amyloid fibrils and amorphous aggregations)
-> Formation of amyloid fibrils can accompany disease like
Alzheimer’s disease
- How does protein fold in ribosomes?
- α-helices and small tertiary structure elements could be formed inside the exit tunnel
- Prokaryotic proteins are folded post-translationally, while eukaryotic proteins are mainly folded co-translationally
- Protein folding in the cytosol
a/ Which additional proteins are involved in protein folding in cytosol?
Chaperone proteins
- Protein folding in the cytosol
b/ What are chaperone proteins?
They are a functionally related group of proteins assisting protein folding in the cell under physiological and stress condition
- Protein folding in the cytosol
d/ Why are chaperone proteins heat shock proteins? Give an example
1/ Chaperones are heat shock proteins because they are proteins induced in a living cell in response to a rise in temperature (above normal level)
2/ E.g, in the salivary gland chromosome of fly, if put them at high temperature, it presents a specific puffing pattern showing active transcription and translation
- Protein folding in the cytosol
c/ What is the role of chaperone proteins?
- Fold newly made proteins into functional conformations
- Refold misfolded / unfolded proteins into functional conformation
- Disassemble potentially toxic protein aggregates that form due to protein misfolding
- Mediate transformations between active + inactive forms of proteins
- They are ATP binding + use hydrolysis to (1) enhance the binding of target proteins and (2) switch their own conformation
- Protein folding in the cytosol
e/ How can chaperones facilitate proper folding of nascent proteins?
- Prevent aggregation by binding to target polypeptide
- Sequestering it from other unfolded proteins to give nascent protein time to fold
- Protein folding in the cytosol
f/ What are the 2-general families of chaperons?
1) Molecular chaperons: bind to segment of protein substrate and stabilize unfolded or partly folded protein
-> Prevent aggregation/degradation
2) Chaperonins: form folding chambers, where all or parts of unfolded protein can enter
-> Give time to fold properly in appropriate environment
- Protein folding in the cytosol
g/ What is the working principle of Hsp70 (chaperon)?
- ATP binding and hydrolysis drive large conformational
changes - Non-properly folded polypeptide substrate interacts first with Hsp40 and is delivered to ATP-bound Hsp70
- Interaction with Hsp40 triggers ATP hydrolysis on Hsp70 generating the closed state
- ADP-release catalyzed by nucleotide exchange factor (NEF) and rebinding of ATP triggers substrate release for folding or possible transfer to downstream chaperones
- Protein folding in the cytosol
h/ What is the Working principle of GroEL/GroES (chaperonin) nanocage?
- Substrate protein binds as folding intermediate to
the empty GroEL-ring and is encapsulated by GroES
in an ATP-dependent step - The protein is free to fold within the chaperonin
nanocage for the time required to hydrolyze ATP on
each subunit of the heptameric GroEL-ring - ATP binding to the opposite ring then triggers the
release of folded protein and GroES, completing the
cycle - Incompletely folded protein will rebind after release
- Protein folding in ER
a/ How does protein fold in ER?
- The proteins in the ER have a hydrophobic signal
sequence near their N-terminal - A protein-RNA complex, the signal recognition
particle (SRP), binds to the ribosome and stops
translation - The SRP receptor has GTPase activity (SRP as well)
and is located in the ER membrane - The receptor will bind to SRP and open a translocon
channel and the translation of the peptide continues
straight into the lumen of the ER - A signal peptidase in the ER membrane cleaves the
signal sequence and the folded protein will be released into the ER lumen -> Proteins targeted for the ER, are never in contact with the cytosol
- Protein folding in ER
a/ How does protein fold in ER?
- The proteins in the ER have a hydrophobic signal
sequence near their N-terminal - A protein-RNA complex, the signal recognition
particle (SRP), binds to the ribosome and stops
translation - The SRP receptor has GTPase activity (SRP as well)
and is located in the ER membrane - The receptor will bind to SRP and open a translocon
channel and the translation of the peptide continues
straight into the lumen of the ER - A signal peptidase in the ER membrane cleaves the
signal sequence and the folded protein will be released into the ER lumen -> Proteins targeted for the ER, are never in contact with the cytosol
- Protein folding in ER
b/ How do enzymes fold in ER?
- Classical chaperons (Hsp70 and 90) (ex: BIP and GRP94)
- Protein disulfide isomerase (PDI) – produces disulfide bridges (only in ER, since no
possibility of formation of the bridges in the cytosol!) - Carbohydrate binding chaperons = lectins
- Protein folding in ER
c/ Characteristics of lectins
- Carbohydrate binding chaperons
- 2 types of lectin in the ER, both bind to Ca2+ = calnexin + calreticulin
