Midterm #2: Protein Folding Flashcards Preview

Medchem 570 > Midterm #2: Protein Folding > Flashcards

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

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


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


Natural proteins contain ___-amino acids almost exclusive



Positive Amino Acids

​Arg, His, Lys


Negative Amino Acids

Asp, Glu


Amino Acids with Polar Uncharged

Ser, Thr, Asn, Gln 


Special Cases

Cys, Gly, Pro


Amino Acids with Hydrophobic Side Chain

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


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


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. 


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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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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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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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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Chaperones and Protein Translocation

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



  • Foldase chaperone that is overexpressed in many forms of cancer

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Name this Structure

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Hsp90 inhibitors  

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


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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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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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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Name this Structure

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  • protease inhibitor approved for the treatment of relapsed mutliple myeloma 


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


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 


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


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


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. 



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)


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