Lecture 21 - Protein folding, misfolding and degradation

Question 1

Important component of tertiary structures and its definition

Answer 1

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

Question 2

Possible shapes of domains (2 ex,)

Answer 2

fibrous or globular

Question 3

Exemple of protein with fibrous and globular domain

Answer 3

HA2 protein in influenza virus hemagglutinin (interacts with HA1)

Question 4

Meaning of modular nature of protein domains

Answer 4

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

Question 5

What is EGF and where does it come from

Answer 5

epidemical growth factor (1 domain). Comes from EGF precursor which is a protein with many EGF domains

Question 6

Domains can be found in multiple copies in same prots or in different prots. what is conserved and what is not

Answer 6

a.a sequence is not the same but 3D shape is the same

Question 7

multimeric proteins is an aspect of _________ structure.

Answer 7

quaternary

Question 8

multimers covalent or non covalent bonds

Answer 8

non covalent

Question 9

Supramolecular complexes is an aspect of _________ structure. Can have a MW of more than _______ and can also contain _______

Answer 9

quaternary. more than 1 MDa. nucleic acids

Question 10

Supramolecular complex def

Answer 10

Molecular machines mad of multiple distinct proteins that have multiple subunits.

Question 11

Supramolecular complex 2 exemples

Answer 11

transcription initiation complex, nuclear pore complex

Question 12

Main forces that hold tertiary quaternary structure vs main forces that hold secondary structure

Answer 12

tertiary quaternary -> H bonds, hydrophobic interactions and +/+ +/- interactions
secondary -> ONLY H bonds

Question 13

Hemoglobin is a ____mer. What’s its structure ? Myoglobin is a ____mer. Leghemoglobin is a _____mer.

Answer 13

Hb tetramer. 2 alpha chains and 2 beta chains.

Myoglobin and leghemoglobin are monomers

Question 14

Where hemoglobin, myoglobin and leghemoglobin are found

Answer 14

hemoglobin -> vertebrates, myoglobin -> unvertebrates

leghemoglobin -> plants

Question 15

differences and similarities between beta subunit of Hb, myoglobin and leghemoglobin and why the similarity

Answer 15

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

Question 16

what 3D structure ressemblence between beta subunit of Hb, myoglobin and leghemoglobin shows

Answer 16

all come from an ancestral oxygen-binding heme-carrying protein

Question 17

2 forms of a protein that can be obtained in vitro

Answer 17

Native and denatured conformations

Question 18

What can lead to protein denaturation (2 exemples)

Answer 18

Urea, heat

Question 19

What happens when denaturation conditions are removed/reversed ?

Answer 19

Protein folds back into native form (right conformation) or could possibly misfold

Question 20

Main characteristic of misfolded proteins and what than can lead to

Answer 20

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

Question 21

How refolding and nascent protein folding is thought to happen and how is misfolding related to that

Answer 21

Through a folding pathway with multiple steps. Incorrect folding can happen in one of these steps.

Question 22

What are chaperones

Answer 22

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

Question 23

What is the pathway that proteins that can’t refold go through ?

Answer 23

Protease pathway (gives the ‘‘irreversible accidents’’ category)

Question 24

How chaperones recognize misfolded proteins

Answer 24

Recognize exposed hydrophobic patches

Question 25

Chaperones regulation (what can lead to them being in higher/lower number)

Answer 25

Upregulated under conditions that lead to misfolded proteins accumulation, such as heat shocks.

Question 26

What are HSPs and important side note

Answer 26

Heat-shock proteins -> synonym for chaperones. Chaperones aren't only upregulated by heat

Question 27

5 things chaperones can do

Answer 27

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

Question 28

2 major classes of chaperones and their difference

Answer 28

1) Molecular chaperones (operate as single molecules) | 2) Chaperonins (multisubunit refolding chamber)

Question 29

How chaperones work in general

Answer 29

ATP-dependent cycle of binding and release of misfolded client. Covers hydrophobic patches while torsions facilitate correct refolding

Question 30

Major MOLECULAR chaperone (+ most studied) and what it does

Answer 30

Hsp70. Helps newly-synthesized proteins follow the correct folding pathway

Question 31

2 important subunits (it has 3 subunits) on Hsp70

Answer 31

Substrate-binding site and nucleotide-binding site (for ATP)

Question 32

First step of Hsp70 mode of action + something particular about this step

Answer 32

Rapid protein binding (reversible, probably because low enthalpy/entropy step)

Question 33

Second step of Hsp70 mode of action

Answer 33

ATP hydrolysis (ADP stays and Pi leaves) and conformational change --> closed substrate-binding site (substrate is locked)

Question 34

Third step of Hsp70 mode of action

Answer 34

ADP release and ATP binding + end of correct folding

Question 35

Fourth (last) step of Hsp70 mode of action

Answer 35

Protein release (after proper folding finished)

Question 36

T/F : Hsp70 acts on a newly made protein when it's out of the ribosome

Answer 36

F : Can also start helping it fold as elongation of the peptide is happening.

Question 37

Why chaperones bind to hydrophobic patches specifically

Answer 37

to prevent protein aggregation

Question 38

Other name for chaperonins and what they do

Answer 38

Hsp60's : Form a chamber made of inward-facing protein-binding subunits that undergo ATP binding and hydrolysis and conformation change

Question 39

Structure of chaperonins

Answer 39

2 barrels and a cap.

Question 40

What is ubiquitin

Answer 40

76 residues protein that can be covalently linked to lysine residues on target proteins

Question 41

First step of ubiquitin/proteasome system for protein degradation

Answer 41

Poly-ubiquitin tags damaged or misfolded proteins for degradation

Question 42

Second step of ubiquitin/proteasome system for protein degradation

Answer 42

Ub tagged proteins are fed into a multisubunit chamber in which each subunit is an inward-facing protease

Question 43

what is E1 and what it does - what drives its reaction

Answer 43

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)

Question 44

what is E2 and what it does

Answer 44

Ubiquitin-conjugating enzyme. Replaces E1

Question 45

what is E3, what it does and what follows its action

Answer 45

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)

Question 46

4 things that happen to Ub tagged protein

Answer 46

Recognition by the cap of the proteasome (has 2 caps), deubiquitination, unfolding and degradation under ATP hydrolysis dependent process. (release of peptides)

Question 47

How many E3 genes in human genome and what E3 does specifically

Answer 47

100 E3 genes, E3 Ub ligases recognize a variety of chemical side reactions that can occur on amino acids and that aren’t good.

Question 48

2 Exemple of structures that can be recognized by E3 Ub ligases

Answer 48

Hydrophobic patches | Oxydized methionine

Question 49

What happens during polyubiquitination

Answer 49

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

Question 50

Accumulation of misfolded proteins = important aspect of what kind of diseases + 3 ex

Answer 50

Neurodegenerative diseases. Parkinson's, Alzheimer's and ''mad cow'' diseases

Question 51

Example of protein responsible for neurodegenerative diseases due to protein misfolding

Answer 51

Amyloid precursor that becomes amyloid (what was supposed to become and alpha helix becomes beta-sheet)

Question 52

Why beta-amyloid is bad (2 reasons)

Answer 52

Insoluble | Aggregates into filaments that are resistant to proteolysis

Question 53

In what form are amyloid deposits in brain tissue visible in the microscope (2 forms)

Answer 53

Plaques | Tangles