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Biology 2B03 > Week 3 + Module 2 > Flashcards

Week 3 + Module 2 Flashcards

(54 cards)

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1
Q

Chaperones

A
  • incompletely folded proteins are helped to fold by chaperone proteins
  • prevent inappropriate interactions between amino acid residues and increase the efficiency of protein folding
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2
Q

Protein folding

any rules

A

we believe there are rules

wrong

CHAOS

  • internal and external stressors promote unfolding and misfolding
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3
Q

Anfinsen experiment

A

protein folding is…

  • spontaneous
  • reversible
  • unique
  • break h-bonds and disulphide using urea and mercapto-ethanol
  • dialysis to remove them
  • refolding
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4
Q

Anfinsen in-vitro vs. in-vivo

A

Probability of correct folding after removal of denaturant increases with

  • low temps
  • low protein concentrations

(= less molecular interactions/due to thermodynamic movement)

In cell (in-vivo)
- not usually the case that those conditions are met

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5
Q

Free energy funnel

A
  • free energy surface proteins explore as they move to native state by forming intramolecular contacts
  • accelerated by chaperones

Native State → Partially Folded
- requires more activation energy
- more stable as native

Partially Folded → aggregates
- requires less energy
- less stable

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6
Q

Anfinsen’s dogma / thermodynamic hypothesis

A
  1. due to thermodynamic molecular forces, polypeptides automatically assume unique, stable conformations (environment)
  2. every sequence of amino acids auto-assemble into a unique, defining conformation (nature)

there are exceptions

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7
Q

Chaperonins

A

Large cylindrical macromolecular assemblies that form an isolation chamber for newly synthesized polypeptides that allows them to form without interference from other macromolecules

  • multimeric
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8
Q

Examples of chaperonins

A

TCiP - eukaryotic cytosol
GroEL - bacteria or chloroplast
Hsp60 - mitochondria

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9
Q

GroEL / GroES chaperonin

A
  • 2 independent folding chambers
  • only one chamber used at a time
  • allows folding inside, doesn’t DO the folding
  • separate cap used for top/bottom chambers
  • ATP to ADP hydrolysis used for energy
  • switches chamber used each time
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10
Q

Cap-binding changes

A

GroES cap
- cause GeoEL to shift to larger “relaxed” conformation to allow room for folding

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11
Q

GroEL subunits

A

3 domains
- apical
- intermediade/hinged
- equitorial

made of 7 Hsp60 subunits

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12
Q

Molecular chaperones

A
  • monomeric
  • bind to hydrophobic
    • bind to a short segment of a protein substrate and stabilize unfolded proteins, preventing aggregation and degradation

don’t assist or guide, more like block improper interactions between potentially complementary surfaces

ex. HSP70

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13
Q

Heat shock proteins - molecular chaperones

(3)

A

HSP70 - in cytosol and mitochondria

BiP - in ER

DnaK - in bacteria

bind to hydrophobic R groups and prevent the nascent polypeptide from associating with other proteins or from folding prematurely, and from aggregating with other hydrophobic molecules

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14
Q

How do Hsp70 family of heat-shock proteins work

A

HSP70
- 2 domains
1. nucleotide binding
2. substrate binding

A. ATP binds to nucleo domain
B. Hydrophobic residues bind to hydrophobic particles (sub domain)
C. DnaJ/HSP40 co-chaperone stimulates ATP to ADP hydrolysis
D. Changes chaperone conformation and lets protein fold
E. ADP released and new ATP comes, releasing folded protein

  • helps gain thermotolerance at an accelerated rate
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15
Q

Bip/GRP78 during ER stress - molecular chaperone

A

n/i

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16
Q

AATD - a1 antitrypsin deficiency

hint: liver

A
  • a1 antitrypsin comes from liver and usually coats lungs to protect from neutrophil elastase (inhibitor)

neutrophil elastase
- produced by WBCs to break down harmful bacteria

  • misfolding keeps it stuck in the liver
  • high likelihood of emphysema, wheezing, shortness of breath, asthma
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17
Q

Treatment of AAT deficiency

+ what is the mutation

A

SERPINA1 encodes AAT
- 75+ mutations causing this
- Glutamate -> lysine @ position 342

  • give person IV AAT or lung/liver transplant eventually
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18
Q

Bip/Grp78 and prostate cancer

A

Antibodies against NH2
- proliferative effects on tumor, anti-apoptotic
= make tumor worse

COOH terminal domain ligation
- operates through p53
- triggers apoptosis

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19
Q

Bacterial vs. mammal chaperonins

GroEL

A

Bacterial:
- Has detachable lid
- GroEl chamber = 7 units each (14 in total)

Mammalian/Eukaryotic:
- Has built-in spiraling-closure lid
- GroEL chambers spin towards the inside to close the chamber and (v.v)
- There are 8 subunits/chamber = 16 in total
- Can have 8 to 9 homomeric or heteromeric subunits in each ring
- ATP hydrolysis triggers lid closing

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20
Q

What up-regulates HSPs

A
  • elevated heat
  • cold shock
  • anoxia
  • chemical exposure
  • desiccation
  • ageing
  • degenerative disorders
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21
Q

Proteotoxic stresses

A
  • stresses that increase the fraction of proteins that are in an unfolded state
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22
Q

Protein degradation - what’s degraded

Study These Flashcards
A

cells degrade:
- misfolded proteins
- denatured proteins
- proteins at too high a concentration
- proteins taken up into the cell
- regulated proteins

so they don’t aggregate and form harmful complexes

23
Q

Protein degradation - process

Study These Flashcards
A
  1. tagging by ubiquitin
  2. degradation of the tagged protein into short peptides (7-8 residues) by the proteasome
24
Q

Ubiquitinylation

Study These Flashcards
A

E1: Ubiquitin activating enzyme
- recognizes and picks up ubiquitin

E2: Ubiquitin conjugating enzyme
- attaches ubiquitin to target protein

E3: Ubiquitin ligase
- each member recognizes a different signal

25
E1, E2, and E3
1. ubi activated by linkage to E1 2. activated ubi is transferred to cys on E2 3. E3 recognizes substrate and transfers ubi to lysine side chain of target substrate 4. poly-ubiquitinylation = degradation
26
Proteasome complex + degradation
- core forms a central hollow cylinder with proteolytic activity - caps on each end form openings for polypeptides to be threaded through - polyubiquitinated protein threaded into proteasome and broken into 2-24 aa peptides
27
Proteasome issue example - spinocerebellar ataxia
- mutation in ataxin-1 gene creates misfolded protein - tagged with ubi but can't be unfolded in proteasome - builds up into lethal aggregates and prevents degradation of other proteins
28
Ligand
A molecule that is bound by a protein (aka a substrate)
29
Ligand binding must demonstrate
1. specificity - ability of a protein to preferentially bind to one or a small number of molecules and not others - bind only specific ligands 2. high affinity - strength of binding between protein and ligand both depend on molecular complementarity
30
Molecular complementarity
- fit like puzzle pieces - allows favourable non-covalent interactions to form aka polars like hydrogen, dipole, LDFs - depends on protein shape and amino sequence
31
CAMP ligand-binding pocket
- cyclic AMP - 6 amino acids with side chains facing pocket - only CAP fits in there
32
Binding affinity
The free energy of interaction between a protein and its ligand can vary greatly Association constant for binding equilibrium (Keq) high Keq = high affinity
33
Binding affinity - reaction
L+P k LP high Keq - favours to the right - complex stays together Keq = [LP]/[L][P] high Kd - favours to the left - complex separates Kd = [L][P]/[LP]
34
Enzyme catalysis
- speeds up rates of reaction - reduces energy of the transition state - low energy favoured = more stable
35
Enzyme active state
Two functional regions 1. Binding site/pocket - determines specificity 2. Catalytic site - promotes reaction
36
Enzyme kinetics - Vmax
Vmax when all enzymes are saturated / in use - takes more substrate concentration for low affinity substrates to reach Vmax - goes down with lower enzyme concentrations
37
Enzyme kinetics - Km
Michaelis constant - measure of affinity of an enzyme for the substrate = concentration of substrate at 1/2 Vmax Km up = affinity down = more substrate needed
38
PKA - what is it
Small domain with glycine lid Hinged connector Large domain - Enzymatic protein - PKA has 2 substrate that binds to it 1. nucleotide (ATP) binding pocket 2. target peptide 1+2 = kinase core - high ATP affinity
39
PKA - process
1. open - substrates bind (ATP pocket + target peptide) 2. closed - glycine-rich lid traps substrates 3. closed - phosphorylation of target peptide using energy from atp hydrolysis 4. open - ADP and phosphorylated peptide released (lower affinity for adp)
40
Protein function regulation mechanisms (5)
1: Allosteric regulation 2: Signal-induced regulation 3: Covalent modification 4: Proteolytic cleavage 5: Enzyme complexes and more
41
Allosteric regulation
- allosteric modulators are small molecules that bind to sites other than the active site of a protein to modify function - change shape of protein - activate or inhibit activity
42
Allosteric regulation - PKA
- allosteric activator cAMP binds to regulatory subunits (R) - produce conformational change - release active catalytic subunits they remove the inhibitor (catalytic subunits)
43
Allosteric regulation - aspartate transcarbomylase
- regulated by the allosteric inhibitor - CTP (Cytosine triphosphate) - when it binds to 6 subunits, causes them to tense and become inactive
44
Allosteric regulation --> negative feedback
- enzyme that catalyzes an early step is inhibited (metabolic path turned off) by the final product - so no energy wasted creating extra product
45
Allosteric regulators - effect on Km
Activator - decreased Km - increased affinity - increased rate Inhibitor - increased Km - decreased affinity
46
Co-operative allostery
- binding of one ligand molecule affects binding of subsequent ligand molecules ex. binding to one subunit of a tetramer changes conformation of all
47
Co-operative allostery - graphs
- sigmoidal curve - cubic function - low affinity at first then reaches Vmax quickly
48
Hemoglobin allostery
Oxygen - substrate - allosteric activator Active state - R-state - high oxygen affinity - in lungs Inactive state - T-state - low oxygen affinity - in tissues - allows for efficient pickup/dropoff
49
Hemoglobin allosteric inhibitor - 2,3-BPG
- in places where oxygen is delivered - makes hemoglobin oxygen affinity lower = releases o2 Fetal hemoglobin -> low 2,3-BPG affinity
50
Covalent modification
- on/off switch for enzymes via addition or removal of chemical groups ex. - acetylation - methylation - phosphorylation
51
Phosphorylation
- targets serine, threonine, tyrosine Kinases add (phosphorylate) Swap OH for phosphate PO4 Phosphatases remove (dephosphorylate)
52
CDK protein - phosphorylation
Phosphorylation is an ACTIVATING event Active - Phosphorylated CDK moves the + AA domain, opening substrate binding pocket and activating CDK - Phosphate group brings negative charges, red-domain forms new ionic bonds to change the shape of the CDK Inactive - CDK cannot bind to substrate due to substrate-binding pocket being blocked by + amino domain
53
Proteolytic cleavage
- irreversible bc broken peptide bonds can't be reformed - 2 cleavages after initial protein folding - creates Ile amino terminal that can fold inwards
54
Protein complexes
Separate enzymes = reaction dependent on diffusion Associated enzymes - when protein complexes are formed - reduces diffusion either... 1. multimeric complex 2. assembly on a scaffold protein

Biology 2B03 (12 decks)

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