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Biology 2B03 > Module 2 - Protein structure and function > Flashcards

Module 2 - Protein structure and function Flashcards

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1
Q

function of chaperones

A

to prevent inappropriate interactions between amino acid resides and increase the efficiency of protein folding

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2
Q

two types of chaperones

A

molecular chaperone, chaperonins

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3
Q

molecular chaperones

A
  • bind to hydrophobic R groups and prevent the developing polypeptide from associating with other proteins, from folding prematurely, and from aggregating with other hydrophobic residues
  • they function within a protein or amongst proteins
  • heat-shock proteins (HSPs)
  • ex: Hsp70 in cytosol and mitochondria, BiP in endoplasmic reticulum, DnaK in bacteria
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4
Q

heat-shock proteins (HSP)

A

produced in response to exposure of stressful conditions

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5
Q

Hsp70

A
  • contains 2 domains: nucleotide-binding domain, substrate-binding domain
  • hydrophobic patch on substrate-binding domain allow it to wrap around hydrophobic parts on unfolded proteins
  • ATP hydrolysis changes conformation of Hsp70 chaperone, hence changing the shape of the target protein
  • this change in shape of the target protein allows it to fold properly
  • ATP hydrolysis is stimulated by co-chaperone, DnaJ/Hsp40
  • ADP is released from Hsp70 by the nucleotide exchange factor, GrpE/BAG1
  • new ATP arrives to fill nucleotide-binding domain
  • folded protein is released and Hsp70 is ready to repeat process as needed
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6
Q

chaperonins

A
  • large cylindrical macromolecule assembly that forms an isolation chamber for newly synthesized polypeptides that allow them to fold without interference from other macromolecules
  • ex: TCiP in cytosol, GroEL in bacteria or chloroplast, Hsp60 found in mitochondria
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7
Q

structure of chaperonins

A
  • made up of 2 GroEL subunits and 1 GroES subunit

- the two subunits that make up GroEL form the two independent folding chambers

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8
Q

GroEL

A
  • large subunit
  • multiple proteins form walls that are attached to one another at the bases
  • walls are made up of 7 Hsp60 subunits
  • Hsp60 has 3 domains: apical domain, intermediate domain, equatorial domain
  • 7 ATP molecules are needed for one GroEL chamber at any time
  • the two chambers are alternately used
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9
Q

GroES

A
  • small subunit

- caps

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10
Q

folding process within the chaperonin

A
  • the top chamber binds to an ATP and a new substrate peptide
  • the new GroES cap binds to the top of the GroEL chamber, allowing for the isolation of the substrate peptide
  • the chamber remains closed during the folding process
  • conformational change enlarges the chamber, giving room for the peptide to fold
  • these conformational changes are observes in GroEL upon association with GroES
  • ATP hydrolysis allows GroES cap to come out and for the protein to diffuse out
  • if protein was not able to fold completely, the process repeats itself
  • the 2 GroEL chambers are alternately used. this means that the bottom one will now be used
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11
Q

what proteins must cells degrade?

A

misfolded proteins, denatured proteins, proteins at too high concentrations, proteins taken up into the cell, regulated proteins

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12
Q

process of protein degradation

A

1) tagging of the protein by attachment of ubiquitin molecules
2) degradation of tagged protein into short peptides (7-8 residues) by the proteasome

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13
Q

ubiquitin

A

small protein, 76 residues

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14
Q

ubiquitinylation

A
  • the addition of ubiquitin to a protein for targeting that protein for degradation by proteasome
  • 3 enzyme system: E1, E2, E3
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15
Q

E1

A
  • ubiquitin activating enzyme

- recognizes free ubiquitin in cytosol and receives it

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16
Q

E2

A
  • ubiquitin conjugating enzyme

- facilitates attachment of ubiquitin to target protein

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17
Q

E3

A
  • ubiquitin ligase

- recognize specific target for degradation and attach ubiquitin to it

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18
Q

ubiquitin ligase

A

large family of proteins, each member recognizing a different signal

19
Q

steps in ubiquintinylation

A

1) ubiquitin activated by linkage to E1. requires energy of ATP hydrolysis
2) activated ubiquitin is transferred to Cys on E2
3) E3 recognizes substrate and transfers ubiquitin to lysine side chain of target substrate
4) poly-ubiquitinylation. E3 provides specificity of degradation

20
Q

ligand

A

molecule that is bound by a protein

21
Q

what must ligand-binding demonstrate?

A
  • high affinity and specificity
  • both of these factors are dependent on the molecular complementarity between the ligand and surface of the ligand binding site
22
Q

affinity

A

refers to the strength of binding between protein and ligand

23
Q

specificity

A

ability of protein to preferentially bind to one or small number of molecules

24
Q

molecular complementarity

A
  • this is dependent on non-covalent interactions between facing surfaces
  • shapes of surfaces must match for non-covalent interactions to occur
  • for shapes that do not fit, thermal motion will break them apart
  • for shapes that do fit, accumulated non-covalent interactions allow the surfaces to stay together despite the thermal motion
25
Q

binding affinity

A
  • measured by association constant for the binding equilibrium (Keq)
  • L + P <> LP
  • high Keq = high affinity = reaction tends toward right = low Kd
  • high Kd = low affinity = reaction tends toward left = low Keq
26
Q

catalysis

A
  • enzymes bind their ligands (i.e. substrates) and promote a chemical reaction between them
  • speeds up reaction by bringing reactants closer together
27
Q

2 functional regions of enzyme active site

A

1) binding site/pocket - determines specificity

2) catalytic site - promotes reaction

28
Q

Vmax

A
  • maximal velocity of a reaction at saturating substrate concentration
  • this will be achieved when all substrate-binding pockets of all enzymes are filled
  • substrates that have different affinities will require different amounts of substrate to reach the same Vmax
29
Q

Km

A
  • Michaelis constant
  • concentration of substrate at which reaction velocity is half-maximal
  • measure of the affinity of an enzyme for the substrate
30
Q

what happens at different enzyme concentrations?

A
  • decrease in enzyme concentration will decrease reaction rate
  • changes Vmax
  • no change in Km
31
Q

what is PKA?

A
  • protein kinase A
  • adds phosphate group to target protein
  • has 2 substrates: target protein, nucleotide ATP
  • 2 domains: small domain (i.e. glycine lid) and large domain
  • these 2 domains together form a nucleotide-binding pocket for ATP and a substrate-binding pocket for the target peptide
  • 2 domains also known as kinase core
  • the target peptide is recognized by glutamic acid residues found in the large domain
32
Q

how does PKA work?

A
  • ATP and target peptide bind to the PKA when it is at open-conformation
  • once binding occurs, the domains move together so that the glycine lid can trap the substrate
  • this brings substrates close together, allowing for phosphorylation: ATP transfers phosphate to target peptide
  • now ADP and phosphorylated peptide have different shapes
  • these different shapes have a low affinity for binding sites of PKA, and thus the PKA switches to open conformation and ADP and phosphorylated peptide are released
33
Q

general mechanisms for regulating protein conformation

A

allosteric regulation, covalent modification, proteolytic cleavage, signal-induced regulation of protein levels, compartmentalization, enzyme complexes

34
Q

allosteric modulators

A
  • type of mechanism for regulation of protein conformation
  • small molecules that bind to sites other than the active site of a protein to modify function
  • also known as effector molecules
  • can have a positive or negative effect on protein activity
35
Q

cAMP

A
  • allosteric activation in PKA
  • PKA switches between 2 conformations: active monomer, inactive monomer
  • inactive PKA contains 2 regulatory subunits: regulatory subunits (R), catalytic subunits (C)
  • pseudo-substrate binds to C in tetramer PKA, making it inactive
  • allosteric activator, cAMP, binds to R, resulting in a conformational change in R
  • this shape change of R causes a shape change in the pseudo-substrate, so that it no longer binds to C. this makes the PKA active again
  • at low cAMP concentrations, PKA is inactive. at high cAMP concentrations, PKA is active
36
Q

CTP

A
  • allosteric inactivation in aspartate transcarbomylase
  • aspartate transcarbomylase is regulated by the allosteric inhibitor, cytosine triphosphate (CTP)
  • aspartate transcarbomylase made up of 6 regulatory subunits and 6 catalytic subunits
  • regulatory subunit contains modulating binding site for CTP
  • when CTP binds to it, conformational changes occur, converting it into the inactive conformation
  • active R state and inactive T state depends on the CTP concentration
37
Q

negative feedback in allosteric regulation

A

an enzyme that catalyzes an early step in a multistep pathway is inhibited by the final product in the pathway

38
Q

allosteric inhibitors/activators of PKA

A
  • CTP: allosteric inhibitor, increases Km, reduced affinity

- ATP: allosteric activator, decreases Km, increased affinity

39
Q

co-operative allostery

A
  • special kind of allosteric modulation
  • binding of one ligand molecule affects binding of subsequent ligand molecules
  • forms an S-shaped sigmoidal curve
  • ex: haemoglobin
  • as substrate concentration increases, Km decreases
40
Q

increasing reaction velocity with monomeric enzymes vs. allosteric enzymes

A

allosteric enzymes require a much smaller change in ligand concentration to achieve the same increased reaction rate than a monomeric enzymes would

41
Q

co-operative allostery: haemoglobin

A
  • oxygen is the substrate and the allosteric activator
  • we need high affinity of oxygen in the lungs and a low affinity for oxygen in the tissues
  • haemoglobin has 2 states: T state (inactive site) and R state (active state)
  • T state has low affinity for oxygen while R state has high affinity for it
  • when oxygen binds to 1 subunit, it changes the conformation of the other 3 subunits to R state, allowing for oxygen to bind to it
  • allosteric inhibitor: 2,3-BPG
  • 2,3-BPG decreases the haemoglobin’s affinity for oxygen when it is bound to the effector site
  • 2,3-BPG is found in high concentrations in the tissues
42
Q

covalent modification

A
  • type of mechanism for regulating protein conformation
  • phosphorylation: an “on-off” switch for enzymes via the addition or removal of chemical groups
  • amino acids targeted: serine, threonine, tyrosine
43
Q

proteolytic cleavage

A
  • allows cell to make a lot of protein in an inactive conformation and then rapidly cleave polypeptide at different points to activate it
  • not reversible
44
Q

protein complexes

A
  • enzymes are separate: reaction is dependent upon diffusion
  • enzymes are associated: reduces diffusion
  • when the enzymes are associated, it reduces diffusion and hence increases efficiency
  • different ways to accomplish the association of enzymes: multimeric complex, assembly on a scaffold

Biology 2B03 (5 decks)

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