Midterm #2: Protein Folding Flashcards Preview

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Flashcards in Midterm #2: Protein Folding Deck (31):
1

Protein Folding Overview

  • 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

Four Regimes of Protein Structure

  • 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

Natural proteins contain ___-amino acids almost exclusive

L

4

Positive Amino Acids

​Arg, His, Lys

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Negative Amino Acids

Asp, Glu

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Amino Acids with Polar Uncharged

Ser, Thr, Asn, Gln 

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Special Cases

Cys, Gly, Pro

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Amino Acids with Hydrophobic Side Chain

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

9

Protein Secondary Structure 

  • 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 

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The process of protein folding

  • 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

11

Molecular Chaperones

  • 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

Small Heat Shock Proteins: Ubiquitous Holdases

  • 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 

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13

The Hsp70 Family of Chaperones

  • 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)

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14

GroEL and other Hsp60 Chaperones

  • 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 

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15

GroEl cycle

  • 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 

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16

Chaperones and Protein Translocation

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

17

Hsp90

  • Foldase chaperone that is overexpressed in many forms of cancer

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18

Name this Structure

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Tanespimycin  

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Hsp90 inhibitors  

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

20

Protein Degradation and the Ubiquitin-Proteasome System 

  • 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

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21

The Ubiquitination System 

  • 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 

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22

The Proteasome

  • 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

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23

Name this Structure

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bortezomib 

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bortezomib 

  • protease inhibitor approved for the treatment of relapsed mutliple myeloma 

25

Protein Misfolding Disease: Pathological Protein

  • 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

26

Prion Disease

  • 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 

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 

 

  • 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

28

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

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

29

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

  • 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. 

 

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Name this structure

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

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Name this structure

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  • 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