Midterm #2: Protein Folding Flashcards
1
Q
Protein Folding Overview
A
- Most (but not all) polypeptides mus fold into a unique and stable 3D arrangement called the native state in order to carry out their biological function.
- Some fold spontaneous; others require involvement of chaperones
- Misfolding into incorrect and potentially toxic confirmations is a hallmark of disease
2
Q
Four Regimes of Protein Structure
A
- Primary: linear AA sequence
-
Secondary: local, regular arrangements of amino acids stabalized by hydrogen bonding
- alpha helix and beta sheets
- Tertiary: compact 3D structure of a single polypeptide chain
- Quarternary: the arrangement of multiple polypeptide molecules in a multi-subunit complex
3
Q
Natural proteins contain ___-amino acids almost exclusive
A
L
4
Q
Positive Amino Acids
A
Arg, His, Lys
5
Q
Negative Amino Acids
A
Asp, Glu
6
Q
Amino Acids with Polar Uncharged
A
Ser, Thr, Asn, Gln
7
Q
Special Cases
A
Cys, Gly, Pro
8
Q
Amino Acids with Hydrophobic Side Chain
A
Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp
9
Q
Protein Secondary Structure
A
- motiffs are determined primarily by backbone hydrogen-bonding patterns and by side-chain steric bulk
- Alpha helices are right handed helices because of L-amino acids
- 3.6 residues/turn (or) 5.4 angstrom/turn
10
Q
The process of protein folding
A
- Driven by attractive and replusive interactions between amino acids
- H-bonding
- Charge-Charge (electrostatic) interactions
- The hydrophobic effect
- van der Waals forces
- Govern the intermediate confomations adopted by the polypeptide chain as it searches for the native structure
11
Q
Molecular Chaperones
A
-
Holdases: bind misfolded of completely unfolded proteins and keep them soluable until they can spontaneously assume their correct fold
- prevent hydrophobic patches from aggregating
- Foldases: actively (with ATP) force misfolded proteins into the correct conformation
- First chapeerones were discovered were *heat-shock proteins. *They are induced by a variety of cellular stresses including heat, infection, inflammation, exposure to toxins (ethanol, trace metals, UV light), starvation, hypoxia, etc.
- 70s housekeeping sigma, 32s turned on when cell is stressed.
12
Q
Small Heat Shock Proteins: Ubiquitous Holdases
A
- sHSPs (such as HSP27 and alphaB-crystalline) exist as a mixture of dimers and larger oligomers in the cell, and bind to unfolded or misfolded proteins in the cell so as to prevent their aggregation
- alpha-crystallines are also extreamly abundant in the eye, where they defend against cataract formation
- Form protective bubble aroundn misfolded
13
Q
The Hsp70 Family of Chaperones
A
- Ex: DnaK (prokaryotes) and Hsp70 & Hsc70 (eukaryotes)
- Resting state is ATP-bound
- Cochaperones (DnaJ or Hsp40) facilitate recognition and binding of misfolded client proteins, or hydrophobic sequences in nascent proteins exiting the ribosome
- This triggers ATP hydrolysis and tight binding to the protein, aiding folding
- Nucleotide exchange factor (NEF) proteins stimulate ADP-ATP exchange and the release of substrate proteins
- Client proteins that are still misfolded may be passed on to other chaperones (Hsp60, Hsp90)
14
Q
GroEL and other Hsp60 Chaperones
A
- multi-subunit cages that physically sequester client proteins, providing a “safe” environment for refolding
- 7 subunits/ring (14 total) ~60kDa each
- hydrophobic patch in the middle
15
Q
GroEl cycle
A
- Misfolded client proteins bind to hydrophobic patches in the ATP-bound Gro-EL barrel
- GroES binding traps the client in the cis chamber and blocks the hydrophobic patches, promoting folding
- This promotes release of ADP and GroES from the Trans chamber
- Hydrolysis of the 7 ATPs in the cis allows ATP binding in the tran chamber .
- Trans client binding displaces cis ATPs, GroES and client and the cycle continues
17
Q
Hsp90
A
- Foldase chaperone that is overexpressed in many forms of cancer
18
Q
Name this Structure
A
Tanespimycin
20
Q
Protein Degradation and the Ubiquitin-Proteasome System
A
- Protein synthesis has a failure rate of 30% due to inaccurate translation or post-translational folding
- needs to be degraded and the amino acids recycled
- Normal proteins also need to be turned over from “wear and tear” or because their biological function is transient
- Proteins are tagged for degradation by the ubiquitination system and are then degraded by the proteasome
21
Q
The Ubiquitination System
A
- Ubiquitin is a small 76aa protein
-
Polyubiquitination typically tags a protein for degradation
- Ub can also alter the stability, function and intracellular localization of a wide variety of target proteins (ex: histones)
- Ubiquitination involves 3 enzymes:
- Ub-activating enzymes (E1)
- Ub-conjugating enzymes (E2)
- Ubiquitin ligases (E3)
- Different combinations of E1, 2 and 3 working in concert enable the tight regulation of the ubiquitnation system
22
Q
The Proteasome
A
- 26S proteasome is a large (~2,000 kDa) multisubunit complex and consists of a core (20S) and two regulatory (19S) particles
- Each 19S particles in turn contains base and lid subunits
- The regulatory particle has 4 activites:
- recognition: of polyubiquinated substrates (requires ATP binding)
- deubiquitination & release of free Ub
- substrate unfolding (powered by ATP hydrolysis)
- translocation of substrate to core particles
- The core particles contain multiple protease sites and are the sites of proteolysis
23
Q
Name this Structure
A
bortezomib
30
Q
Name this structure
A
- Tafamidis binds to and stabilizes the tetramer, potently inhibits aggregation, and arrests disease progression. (TTR and familial amyloid polyneuropathy)
31
Q
Name this structure
A
- Ivacaftor can treat some of the latter mutations by binding to misfolded CFTR and forcing it into a native (functional) state.
- Mutations in CFTR ion channel lead to either premature degration or insertion of misfolded protein into cell membrane
