Lecture 21 - Protein folding, misfolding and degradation Flashcards
Important component of tertiary structures and its definition
Domains. 100-150 a.a long regions, COMPACTLY FOLDED an that can be made of various motifs. Doesn’t depend on other parts of protein to fold itself
Possible shapes of domains (2 ex,)
fibrous or globular
Exemple of protein with fibrous and globular domain
HA2 protein in influenza virus hemagglutinin (interacts with HA1)
Meaning of modular nature of protein domains
Can be found in diverse proteins or in multiple similar copies within a given protein. Domains can be shuffled because they fold independently of the rest of the protein
What is EGF and where does it come from
epidemical growth factor (1 domain). Comes from EGF precursor which is a protein with many EGF domains
Domains can be found in multiple copies in same prots or in different prots. what is conserved and what is not
a.a sequence is not the same but 3D shape is the same
multimeric proteins is an aspect of _________ structure.
quaternary
multimers covalent or non covalent bonds
non covalent
Supramolecular complexes is an aspect of _________ structure. Can have a MW of more than _______ and can also contain _______
quaternary. more than 1 MDa. nucleic acids
Supramolecular complex def
Molecular machines mad of multiple distinct proteins that have multiple subunits.
Supramolecular complex 2 exemples
transcription initiation complex, nuclear pore complex
Main forces that hold tertiary quaternary structure vs main forces that hold secondary structure
tertiary quaternary -> H bonds, hydrophobic interactions and +/+ +/- interactions
secondary -> ONLY H bonds
Hemoglobin is a ____mer. What’s its structure ? Myoglobin is a ____mer. Leghemoglobin is a _____mer.
Hb tetramer. 2 alpha chains and 2 beta chains.
Myoglobin and leghemoglobin are monomers
Where hemoglobin, myoglobin and leghemoglobin are found
hemoglobin -> vertebrates, myoglobin -> unvertebrates
leghemoglobin -> plants
differences and similarities between beta subunit of Hb, myoglobin and leghemoglobin and why the similarity
myoglobin and beta subunit of Hb -> 30% a.a similarity.
leghemoglobin and beta subunit of Hb -> much less than 30% a.a similarity
similarities -> same 3D structures (look alike) -> 3D structure related to its function
what 3D structure ressemblence between beta subunit of Hb, myoglobin and leghemoglobin shows
all come from an ancestral oxygen-binding heme-carrying protein
2 forms of a protein that can be obtained in vitro
Native and denatured conformations
What can lead to protein denaturation (2 exemples)
Urea, heat
What happens when denaturation conditions are removed/reversed ?
Protein folds back into native form (right conformation) or could possibly misfold
Main characteristic of misfolded proteins and what than can lead to
Hydrophobic residues on the surface (oil drop model not respected) –> Can lead to aggregation of misfolded protein by interactions between the hydrophobic patches on their surfaces
How refolding and nascent protein folding is thought to happen and how is misfolding related to that
Through a folding pathway with multiple steps. Incorrect folding can happen in one of these steps.
What are chaperones
Proteins that help protein fold along the right pathway.
Will put back into an on-pathway folding the proteins that have an off-pathway folding
What is the pathway that proteins that can’t refold go through ?
Protease pathway (gives the ‘‘irreversible accidents’’ category)
How chaperones recognize misfolded proteins
Recognize exposed hydrophobic patches
Chaperones regulation (what can lead to them being in higher/lower number)
Upregulated under conditions that lead to misfolded proteins accumulation, such as heat shocks.
What are HSPs and important side note
Heat-shock proteins -> synonym for chaperones. Chaperones aren’t only upregulated by heat
5 things chaperones can do
1) Fold newly made proteins
2) Refold misfolded or unfolded proteins
3) Disassemble potentially toxic protein aggregates (that form due to misfolding)
4) Assemble/dismantle large multiprotein complexes
5) Mediate transformations between inactive and active forms of some proteins
2 major classes of chaperones and their difference
1) Molecular chaperones (operate as single molecules)
2) Chaperonins (multisubunit refolding chamber)
How chaperones work in general
ATP-dependent cycle of binding and release of misfolded client. Covers hydrophobic patches while torsions facilitate correct refolding
Major MOLECULAR chaperone (+ most studied) and what it does
Hsp70. Helps newly-synthesized proteins follow the correct folding pathway
2 important subunits (it has 3 subunits) on Hsp70
Substrate-binding site and nucleotide-binding site (for ATP)
First step of Hsp70 mode of action + something particular about this step
Rapid protein binding (reversible, probably because low enthalpy/entropy step)
Second step of Hsp70 mode of action
ATP hydrolysis (ADP stays and Pi leaves) and conformational change –> closed substrate-binding site (substrate is locked)
Third step of Hsp70 mode of action
ADP release and ATP binding + end of correct folding
Fourth (last) step of Hsp70 mode of action
Protein release (after proper folding finished)
T/F : Hsp70 acts on a newly made protein when it’s out of the ribosome
F : Can also start helping it fold as elongation of the peptide is happening.
Why chaperones bind to hydrophobic patches specifically
to prevent protein aggregation
Other name for chaperonins and what they do
Hsp60’s : Form a chamber made of inward-facing protein-binding subunits that undergo ATP binding and hydrolysis and conformation change
Structure of chaperonins
2 barrels and a cap.
What is ubiquitin
76 residues protein that can be covalently linked to lysine residues on target proteins
First step of ubiquitin/proteasome system for protein degradation
Poly-ubiquitin tags damaged or misfolded proteins for degradation
Second step of ubiquitin/proteasome system for protein degradation
Ub tagged proteins are fed into a multisubunit chamber in which each subunit is an inward-facing protease
what is E1 and what it does - what drives its reaction
Ubiquitin activating enzyme, binds to Ub on carboxyl group (carbonyl group C=O between the two) to activate it. Driven by ATP hydrolysis to AMP (+PPi pyrophosphate release)
what is E2 and what it does
Ubiquitin-conjugating enzyme. Replaces E1
what is E3, what it does and what follows its action
Ubiquitin ligase. Helps Ub find misfolded or damaged proteins. After that, poly-ubiquitination can happen (adding of Ubs on the previous Ub to form a chain of Ubs)
4 things that happen to Ub tagged protein
Recognition by the cap of the proteasome (has 2 caps), deubiquitination, unfolding and degradation under ATP hydrolysis dependent process. (release of peptides)
How many E3 genes in human genome and what E3 does specifically
100 E3 genes, E3 Ub ligases recognize a variety of chemical side reactions that can occur on amino acids and that aren’t good.
2 Exemple of structures that can be recognized by E3 Ub ligases
Hydrophobic patches
Oxydized methionine
What happens during polyubiquitination
Steps 1 (ATP and E1 dependent Ub activation), 2 (E2 replaces E1) and 3 (E3 helps Ub recognize damaged/misfolded protein) are repeated for each Ub that is added
Accumulation of misfolded proteins = important aspect of what kind of diseases + 3 ex
Neurodegenerative diseases. Parkinson’s, Alzheimer’s and ‘‘mad cow’’ diseases
Example of protein responsible for neurodegenerative diseases due to protein misfolding
Amyloid precursor that becomes amyloid (what was supposed to become and alpha helix becomes beta-sheet)
Why beta-amyloid is bad (2 reasons)
Insoluble
Aggregates into filaments that are resistant to proteolysis
In what form are amyloid deposits in brain tissue visible in the microscope (2 forms)
Plaques
Tangles
