Midterm #2: Protein Folding

Question 1

Protein Folding Overview

Answer 1

• 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

Question 2

Four Regimes of Protein Structure

Answer 2

• 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

Question 3

Natural proteins contain ___-amino acids almost exclusive

Answer 3

L

Question 4

Positive Amino Acids

Answer 4

​Arg, His, Lys

Question 5

Negative Amino Acids

Answer 5

Asp, Glu

Question 6

Amino Acids with Polar Uncharged

Answer 6

Ser, Thr, Asn, Gln

Question 7

Special Cases

Answer 7

Cys, Gly, Pro

Question 8

Amino Acids with Hydrophobic Side Chain

Answer 8

Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp

Question 9

Protein Secondary Structure

Answer 9

• 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

Question 10

The process of protein folding

Answer 10

• Driven by attractive and replusive interactions between amino acids

1. H-bonding
2. Charge-Charge (electrostatic) interactions
3. The hydrophobic effect
4. van der Waals forces

• Govern the intermediate confomations adopted by the polypeptide chain as it searches for the native structure

Question 11

Molecular Chaperones

Answer 11

• 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.

Question 12

Small Heat Shock Proteins: Ubiquitous Holdases

Answer 12

• 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

Question 13

The Hsp70 Family of Chaperones

Answer 13

• 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)

Question 14

GroEL and other Hsp60 Chaperones

Answer 14

• 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

Question 15

GroEl cycle

Answer 15

• 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

Question 16

Chaperones and Protein Translocation

Answer 16

Chaperones also play key roles in protein translocation through a membrane, such as translocation from the cytosol into the mitochondria

Question 17

Hsp90

Answer 17

• Foldase chaperone that is overexpressed in many forms of cancer

Question 18

Name this Structure

Answer 18

Tanespimycin

Question 19

Hsp90 inhibitors

Answer 19

• Tanespimycin (17-AAG) are under investigation as novel cancer therapies

Question 20

Protein Degradation and the Ubiquitin-Proteasome System

Answer 20

• 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

Question 21

The Ubiquitination System

Answer 21

• 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:
  
  1. ​Ub-activating enzymes (E1)
  2. Ub-conjugating enzymes (E2)
  3. Ubiquitin ligases (E3)
• ​Different combinations of E1, 2 and 3 working in concert enable the tight regulation of the ubiquitnation system

Question 22

The Proteasome

Answer 22

• 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:

1. recognition: of polyubiquinated substrates (requires ATP binding)
2. deubiquitination & release of free Ub
3. substrate unfolding (powered by ATP hydrolysis)
4. translocation of substrate to core particles

• The core particles contain multiple protease sites and are the sites of proteolysis

Question 23

Name this Structure

Answer 23

bortezomib

Question 24

bortezomib

Answer 24

• protease inhibitor approved for the treatment of relapsed mutliple myeloma

Question 25

Protein Misfolding Disease: Pathological Protein

Answer 25

• Pancreatic amyloid deposits with type 2 diabetes
• Pathological protein aggregates are typically insoluable, fibrillar and rich in B-sheet structure and referred to as amyloid
• Amyloid-forming proteins generally display “templated” or “seeded” aggregation
  
  • a misfolded protein can induce the conversion of native protein into a misfolded form

Question 26

Prion Disease

Answer 26

• Templated protein misfolding is the basis for prion disease
  
  • particular conformations of a protein called PrP can act as infectious particles
• Toxicity in amyloid disease is a result of small intermediates along the aggregation pathway
• Even without aggregation, it can cause degradation of a protein and consequent loss of function

Question 27

Disease:Aggregating Protein

• Alzheimer’s Disease
• Parkinson’s Disease/Dementia with Lewy bodies
• Type 2 Diabetes
• Huntington’s Disease
• Creutzfeldt-Jakob disease/Bovine Spongiform Encephalopathy/Kuru
• Amyotrophic lateral sclerosis
• Familial Amyloid Polyneuropathy/Senile systemic amyloidosis
• Dialysis-related amyloidosis

Answer 27

• Alzheimer’s Disease
  
  • amyloid-B
  • tau
• Parkinson’s Disease/Dementia with Lewy bodies
  
  • alpha-synuclein
• Type 2 Diabetes
  
  • islet amyloid polypeptide
• Huntington’s Disease
  
  • huntingtin
• Creutzfeldt-Jakob disease/Bovine Spongiform Encephalopathy/Kuru
  
  • PrP
• Amyotrophic lateral sclerosis
  
  • SOD1
• Familial Amyloid Polyneuropathy/Senile systemic amyloidosis
  
  • Transthyretin
• Dialysis-related amyloidosis
  
  • Beta2-microglobulin

Question 28

Progression of Huntington’s Disease is Directly Linked to Aggregation Propensity

Answer 28

• aggregation of the huntingtin protein in striatal neurons
• chorea, depression, agression, hypokinesia and rigidity
• CAG repeats in huntingtin
  
  • polyglutamine
  • more CAG, earlier onset

Question 29

Aggregation of what is implicated in familial amyloid polyneuropathy and senile systemic amyloidosis

Answer 29

• aggregation of transthyretin (TTR) is implicated in familial amyloid polyneuropathy and senile systemic amyloidosis
• Aggregation requires the dissociation of the native TTR tetramer into monomers.

Question 30

Name this structure

Answer 30

• Tafamidis binds to and stabilizes the tetramer, potently inhibits aggregation, and arrests disease progression. (TTR and familial amyloid polyneuropathy)

Question 31

Name this structure

Answer 31

• 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