Protein Folding

Question 1

Folding landscape

Answer 1

1) Representation of different conformational states
-All have an associated free energy
2) In folding process -> decrease in nº of conformations, free energy, and nº of contacts (native or non-native)

Question 2

Full native conformation

Answer 2

-Only native contacts
-Protein reaches minimal free energy conformation

Question 3

Intrinsically disordered proteins (IDPs)

Answer 3

1) Don’t reach folded conformation
2) no hydrophobic core as w/globular proteins
3) More abundant than thought; up to ~30% of eukaryotic proteins
4) Many related to diseases
5) Have v. important functions
6) Complement functional repertoire of ordered proteins
7) Remarkable binding properties
8) Examples:
-Cancer: p53
-Alzheimer’s: ß -amyloid protein
-Parkinson’s: α-synuclein
-Taupathies: Tau
-Prion diseases: prion protein

Question 4

Protein folding problem

Answer 4

1) Why does it take that particular structure? Why does it fold at all? Why does it fold so fast?
2) help predict process that proteins go through to get native conf
3) Understanding this will:
-Combat misfolding related to human diseases
-Produce new proteins with novel functions
-Predict 3D structure from primary sequence

Question 5

Afinsen experiment

Answer 5

1) Native proteins contain conformation w/ lowest minimal energy
2) All info to reach native structure is in 1º seq

Method:
1) Ribonuclease (14 kDa) w/well-defined sequence pattern of disulphides
2) Denature in 8M urea + ß-mercaptoethanol (BME) to unfold protein
3) Protein had no catalytic activity = completely unfolded
4) Remove BME led to formation of mostly non-native disulphides
5) Remove urea gives completely unfolded protein
6) Adding trace amounts BME = protein recovers catalytic activity
7) Conclusion: BME breaks diS bonds = allows protein to explore other conformations w/smaller energy to achieve native structure

Question 6

Protein stability

Answer 6

1) Native state is more stable than denatured state
2) Protein stability = the conformational stability of the N is given by the difference in free energy between D and N: ∆G_D-N

Question 7

Chemical stability

Answer 7

1) Ability of maintaining chemical structure of the native states (e.g. intact covalent bonds, oxidation states, metal coordination, etc)

Processes include:
1) Deamination of Asn/ Gln to Asp/Glu
2) Hydrolysis of peptide bond of Asp at low pH
3) ox. of Met at high temperature to met sulfoxide
4) Elimination of diS bonds
5) Thiol-catalyzed diS interchange at neutral pH
6) ox of Cys residues to thyol radicals, Cys-S* -happens when thiol in Cys loses protons but keeps ē -> v. reactive species that can form non-specific bonds (dangerous)

Question 8

Conformational stability

Answer 8

1) Ability to adopt well-defined conformation rather than random coil (D)
-Associated with 2º/ 3º structure
2) Local structure adopted by backbone atoms described in phi and psi angles
-Phi and psi rotate allowing various conformations
3) Refers only to formation of non-covalent bonds

Question 9

Determinants of protein folding (1)

Answer 9

1) ∆G= ∆H-T∆S
2) 2 parameters: change in entropy vs enthalpy
3) Change in entropy = changes in conformations = reduction in entropy when folding protein to N = unfavourable
4) Enthalpy contribution from intra-molecular side-chain interactions (e.g. hydrophobic, diS, etc) = favourable to stability of protein
5) Major favourable energetic contribution to protein folding/stability of N comes from expulsion of water from hydrophobic core
6) All interactions have to be broken to form new interactions in N

Question 10

Parameters influencing protein folding

Answer 10

1) Stability of protein depends on pH, temp, pressure, ionic strength, crowding, amount of salt, etc
2) max activity of protein correlated to protein stability
3) Crowding = putting protein in solution w/co-solvents that mimic cellular environment
-Apoflavodoxin in normal buffer has thermal stability of 48ºC; but in 400mg/ml of Ficoll, temperature increases -> diff for each protein
-Protein inside cell is more stable just because it’s surrounded by proteins at high concentrations that act as solvent
4) Non-covalent interactions
-Average stability of monomeric small protein ~5-15 kcal/mol
-For a protein to fold, need thousands of these interactions
5) If too stable = v. rigid = cannot perform function i.e. to perform catalytic function = need conformational changes to interact w/other proteins to form complexes

Question 11

Determinants of protein folding (2)

Answer 11

1) Covalent interactions
-diS bond formation = reversible process
2) Compaction
-a-helices and b-sheets individually are v. compact
3) Folding is directed by internal residues related to hydrophobic core
-Most surface mutations do not affect folding; may affect solubility, stability
-Balance between compactness and flexibility; surface residues may have greater flexibility but have hydrophobic core that is rigid
4) Hierarchy
-Domain = sequence that can be folded on its own; soluble in solution
-Subdomains = form independently from domains; form 3º or 4º structure
5) Adaptability
-Hydrophobic cores are adaptable to changes
-Mutations can be accommodated with local shifts without affecting fold of backbone
6) Sequence versatility
-Conservation of sequence and folding
-20% amino acid sequence identity = same overall fold = but have huge variability
-Certain residues are key to maintain conformation of shape

Question 12

Techniques to measure protein stability

Answer 12

1) Methods distinguish betw. D and N state
2) Absorbance (v.small diff betw. D&N)
3) Fluorescence
-Most common chromophore used
- greatest change in signal between N and D
-greatest S:N
-Rely on aromatic side chains (Trp, Tyr, Phe)
4) Circular Dichroism (CD)
5) NMR
6) Differential scanning calorimetry (DSC)
7) Catalytic activity (does not give thermodynamic parameter)

Question 13

Protein denaturation

Answer 13

1) unfold proteins w/heat or cold
-just before 0º, small fraction will unfold
- more common heat denaturation = stronger signal
2) pH extremes
3) Organic solvents
4) Chaotropic agents
-Most common after heating
-E.g. urea, guanidium hydrochloride
-Sigmoidal curve related to cooperativity
-Refer to individual Tms to check protein stability
-D50 parameter = concentration at which 50% of protein is denatured

Question 14

Circular dichroism (CD)

Answer 14

1) Measures the molar absorption difference (∆ε) between left and right circularly polarized light
2) Circular polarized light = shift phase by quarter cycle (rotating circularly; forms helical shape)
-For opp direction, shift by 45º
3) In CD have sample and 2 circularly polarized lights; sample only absorbs one of them in only one direction; measure how one was absorbed preferentially to other
4) Spectropolarimeter
-At different wavelengths get specific pattern
-a-helix min = 208 and 222nm
-b-sheet peak = 208nm
-Most cannot read below 200nm
5) To measure stability of a protein, take frequencies, measure CD only at particular wavelength (at region w/greatest signal between random coil and folded protein)
6) CD monitors protein folding and biological activity
-Can tell you proportion of a-helix or b-sheet in protein
7) Disadvantage: spectra does not give you AA level information

Question 15

Planar vs circular polarized light

Answer 15

1) Planar = horizontal and vertical components in phase
2) Circular = horizontal and vertical components out of phase
-Direction of rotation depends on relative phase
-Chiral molecules (e.g. L-amino acids) are optically active = have different refractive indices and different extinction coefficients for L and R circularly polarized light
-Optical activity also affected by environment i.e. location in secondary structure elements
3) Mix = elliptical
4) Combining 2 circularly polarised lights in different directions = linearly polarized light in particular direction (just a single plane)
5) Only circularly polarized light in one direction = protein absorbs = circularly polarized light with smaller amplitude/signal
-In 2 directions = add contributions of two circles (big circle for light that was not absorbed + small circle for light that was absorbed) = ellipse

Question 16

Ellipticity

Answer 16

1) Difference in absorbance does not tell you which of the two lights was absorbed preferentially
2) magnitude of θ tells you the difference in absorption between the two lights

Question 17

The Levinthal Paradox

Answer 17

1) Folding of protein is not result of random search; must always follow defined pathways
2) Impossible in timescale to randomly sample every conformation until lowest E one is found

Question 18

What makes a protein fold so fast?

Answer 18

1) Timescale: ms-s
2) Not random; direction toward native structure is funneled
3) Energy landscape has local minima
4) Too many different minima= non-productive folding = decelerate folding = can prevent protein from reaching native state
5) Minima produce intermediates partially structured w/no optimal stability and exposed hydrophobic regions = interaction w/other components in cell = aggregation = toxic
6) Smooth folding pathways avoid minima (make as small as possible) =efficient folding

Question 19

2-state folding: single molecules

Answer 19

1) Folding directly from D to N state w/o stable intermediates
2) Discovered by forster resonance energy transfer
i) Green donor and red acceptor dyes attached to N and C of two-state protein
ii) N = high transfer efficiency = termini are close
iii) D = larger separation between dyes = transfer efficiency is small
iv) Folding/unfolding events correspond to jumps in FRET efficiency
v) Distance dependent = less than 60nm apart

Question 20

2-state folding: protein ensemble

Answer 20

1) Stop flow device
2) syringe 1: protein + denaturant
3) Syringe 2: buffer + no protein & denaturant
4) Piston pushes both solutions; mix; travel through capillary tube; detector; change in fluorescence over time
5) Cannot use fluorometer; folding is too quick
6) Region w/ no data = time taken by device to mix samples; fit data to single exponential; predict where it goes

Question 21

2-state vs 3-state folding intermediates

Answer 21

1) Residual plot = subtract experimental data from ideal curve
-Tells us how good is the fit to our data
2) Single exponential behaviour = protein follows 2-state mechanism of protein folding (no stable intermediates)
3) Curve is combination of 1+ exponentials (some faster than others) = signalling presence of folding intermediates only observable by kinetic experiments
4) Intermediates need to fold faster to give N = faster exponential
5) N = slower exponential
6) Still no good fit = add another exponential = protein has 2-folding intermediates
7) No intermediates in eq. conditions; they are formed and absorbed by N too quickly

Question 22

Transition state theory

Answer 22

1) TS v. short lived = cannot be observed directly = v. difficult to characterise structurally (no NMR, crystallisation, etc)
2) TS is betw. D and N; has much higher free energy
3) TS has critical contacts that decide if protein can go from N to D (vice-versa)
4) TS theory: direct correlation between folding rates and energy difference between D and TS
5) More stable TS = faster folding rate
6) K ∝ exp(-∆G_(TS-D))
7) 2-state folding kinetics is explored via mutational analysis

Question 23

Mutational analysis

Answer 23

1) Avoid mutations that include new groups in AA side chain
2) Use conservative deletion mutants = remove methyl group, charge, or ring (do not add extra groups)
3) Assumptions:
i) Mutants decrease stability but do not affect structure of native state
ii) Mutants do not create new interactions
4) Default mutation to Ala = remove most of side chain but left w/b-carbon; if mutate b-carbon, then affect protein conformation
5) Compare differences in thermodynamic stability and folding rates between wild type and mutants

Question 24

Protein folding in vivo

Answer 24

1) Experience molecular crowding
2) Av. MW of proteins in human cell: 50kDa
2) AV. [protein] per cell: 20-30%

Question 25

Chaperones

Answer 25

1) Quality control mechanisms to assist protein folding in vivo
2) Proteins that use ATP to disrupt mis-folded proteins and set them on folding pathway again

Question 26

Chaperone types

Answer 26

1) Bacterial trigger factor (ATP not needed)
-Expression levels increase when expose bacteria to cold shock
-Ribosome associated: acts early
2) HSP70 (prokaryotic + eukaryotic)
-Family of proteins
-Reverses denaturation/aggregation
-Works with HSP40
3) Chaperonins (HP60)
-Large, multi-subunit, cage-like
-Type I: bacteria, mitochondria, chloroplasts
-Type II: archae/eukaryotes
4) HSP90 (eukaryotic)
-Family of proteins
-Facilitates late-stage folding of signalling proteins
-Unique regulatory role = induces active conformation
5) Nucleoplasmins
-Help assemble nucleosomes
6) Protein disulphide isomerase (PDI) –family
7) Peptidyl-Prolyl Isomerase (PPI) –family

Question 27

E. coli ribosome

Answer 27

1) 25nm diameter; 2.52MDa mass ~20% of cell mass
2) Two unequal subunits that dissociate at +1mM Mg2+
-30S subunit; 0.93MDa; 21 proteins; 16S RNA
-50S subunit; 1.59MDa; 31 proteins; 2x rRNAs (5S and 23S)
3) ~60% RNA
4) rRNA has catalytic activity to fuse peptides w/help of other proteins

Question 28

Protein synthesis

Answer 28

1) Initiation = binding of mRNA and initiator aminoacyl-tRNA to small subunit, followed by binding of large subunit
2) Elongation = synthesis of all peptide bonds w/help of tRNAs bound to acceptor (A) and peptidyl (P) sites
3) Termination = tRna recognises ‘stop’ codon

Question 29

Trigger factors

Answer 29

1) Newly synthesised proteins leave ribosome through tunnel in large subunit in unfolded state = sensitive to aggregation and degradation
2) New chain = exposed to proteases = recognise hydrophobic/unstructured regions
-To avoid this: trigger factor
3) Trigger factor = ATP-independent chaperone
4) Has domain that binds directly to protein L23 = in exit channel of ribosome
5) Binds hydrophobic regions; helps protein to fold
6) More than one chain of TF can bind same polypeptide
7) When protein is folding and has no exposed hydrophobic regions; released from TF

Structure:
1) N-terminal domain: L23 binding + some chaperone activity
2) C-terminal domain: main chaperone activity
3) Linker polypeptide: peptidyl-prolyl activity (allows trans/cis isomers to reach right conformation) + auxiliary chaperone activity
4) Has intradomain and interdomain flexibility to accept polypeptides in different conformations
5) Only in prokaryotes

Question 30

Heat shock proteins

Answer 30

1) Main family of chaperones
2) Overexpressed upon heat and stress = favour protein renaturation/degradation
3) Exist at basal concentrations in cell
4) Cell exposed to heat = induce unfolding and aggregation = heat shock proteins prevent this
5) Bind to new polypeptides to avoid premature folding
6) Facilitate assembly/disassembly of multiprotein complexes (e.g. nucleosome)

Question 31

E. coli chaperone network

Answer 31

1) Polypeptide -> trigger factor -> if not able to fold protein correctly -> chaperone DnaK (homologue of eukaryotic HSP70) -> still no success -> chaperone GroEL
-2) Eukaryotes don’t have trigger factor so directly to HSP70; other proteins connect HSP70 directly to exit channel of ribosome

Question 32

HSP70

Answer 32

1) N-terminal nucleotide binding domain = binds ATP
2) 2x C-terminal domains (α,β are far apart) = bind substrate
3) HSP-like proteins (or DnaJ in E. coli) accelerate rate of ATP to ADP catalysis = changes conformation of HSP70 to bound form = traps polypeptide in ADP form
4) Nucleotide exchange factors (NEFs) = exchange ADP to ATP = opens structure again = substrate allowed to fold = released

Question 33

GroEL/GroES (Chaperonin Type II)

Answer 33

1) 184Å tall; 3 domains; big chamber
2) 2x Heptameric ring (GroEL) -> 7 subunits
-Each is 60kDa (HSP60)
-71Å (bottom) + 80Å (middle)
3) 1x Heptameric ring on top (GroES)
-10kDa (HSP10)
-33Å (top)
4) Trans/closed = inactive conformation
-Small hydrophilic cavity
-Rim exposes hydrophobic patches where unfolded proteins bind = signals GroES to bind
5) Cis/open = active conformation
-GroES replaces hydrophobic patches = pushes protein inside cavity
6) Alternating use of each ring; always something inside of either ring
7) Helps fold ~10% of all cytosolic proteins
-85 are dependent on activity of GroEL/ES to fold
-Many are α,β proteins = have long range interactions
-GGIVLTGSA motif similar to motif recognised in unfolded protein PxHHHxPxP

Question 34

GroEL/GroES cycle

Answer 34

1) Anti-cooperative ATP binding
-When one ring binds ATP, other is not allowed to bind ATP
-Allows interaction w/misfolded or partially-folded proteins
-ATP binding = quick
-Polypeptide binding = slower
2. Signal for GroES to bind
-Slower
-Pushes unfolded protein into chamber
3. Once structure forms, ATP allowed to enter in trans ring
-Promotes formation of ADP in cis
4. Protein trying to refold at the same time
-Release 7xPi
5. Stays in conformation until another protein binds

Question 35

HSP90

Answer 35

1) V. abundant, flexible, highly dynamic
2) Mainly acts at late stages of substrate folding (e.g. kinases, signalling proteins)
3) Mechanism:
i) Dimerize via C-terminal domain
ii) N-terminal domain binds transiently as it binds ATP + substrates
4) Function affected by post-translational modifications (e.g. phosphorylation, acetylation)
5) Details of substrate binding = not clear!
6) Needs different co-chaperones
7) Substrates bind at different steps in cycle when HSP90 is in diff. conformations

Question 36

Protein disulphide isomerase (PDI)

Answer 36

1) Catalyst that promotes formation of new diS bonds from misfolded diS bonds
2) Common motif: Cys-Gly-His-Cys
3) Creates thiolate intermeditate (S- good nucleophile; no H+)
4) Attacks S to form diS; other S becomes thiolate; takes proton from thiol of second Cys; PDI forms diS by breaking the misfolded one
5) Process is reversible; have reduced and oxidised glutathione
6) Glutathione helps protein break non-native diS or form native diS in absence of ATP (red. PDI generated by ox. Glutathione)

Question 37

Peptidyl Prolyl Isomerase (PPI)

Answer 37

1) In absence of ATP, allow S to switch betw. cis-trans conformations
2) cis-trans prolyl isomerisation is RDS in in vivo protein folding; PPi accelerates this
3) For most AAs, trans conf of peptide bond = most energetically fav
-Not the case for prolyl
-Sometimes cis conf preferred = constrained by rest of structure
4) Important when purifying protein; if in absence of chaperones, some will not reach correct prolyl conformation thus not completely active
5) protein specific; changes can be localised to small region of protein
6) Some cases, protein unable to fold if incorrect isomer of prolyl

Question 38

Protein turnover

Answer 38

1) Balance betw. protein synthesis and degradation
-Respond to different cellular needs/signals at different times
2) Protein degradation depends on stability and activity
3) examples:
i) ornithine decarboxylase HL 11min
-Produces antioxidants (polyamines) important for DNA stability and cell division
-Products of metabolic pathway starting w/ODC increase cell growth/reduce apoptosis
ii) p53 (expressed at v.low levels) HL 5-20min
-Sometimes undetectable; requires activation signal like DNA damage
-Stabilization induces overproduction of p53
-If p53 not produced = induces cell-cycle arrest = maybe apoptosis
iii) Albumin HL 3 weeks
-Carrier of metabolites
iv) γ-crystallin (eye lens protein) HL +70 years
v) Collagen HL +110 years

Question 39

Mechanisms for protein degradation

Answer 39

1) Ubiquitin-proteosome pathway degrades proteins from cytoplasm and nucleus
2) Endosome-lysozyme pathway degrades extracellular, cell-surface and ER proteins

Question 40

E3 ligases

Answer 40

1) Variety of E3 ligases (+600); diff ligand specificities for diff proteins
2) Parkin E3 involved in destruction of a-synuclein; mutation of parkin = aggregation of a-synuclein = Parkinson’s
3) HPV encodes E3 targeting p53 in host; 90% of cervical cancers associated w/this activity

Question 41

Ubiquitin-proteosome pathway

Answer 41

1) main method
2) Ubiquitin = small protein (76aa); v.soluble/ abundant/ stable; used to tag proteins for degradation
3) Ub cov. attached to S for degradation; cellular fate depends on Lys used to tag S
4) C-terminal of Ub Gly76 reacts with S Lys-side-chain = isopeptide bond
-Isopeptide bond = amide bond between 2 amino acids, at least one is a side chain, other can be backbone group
5) Ub is oligomerized using another Lys; if using Lys48 (4+ Ub molecules) = sign for degradation
6) Can have different patterns of ubiquitination: linear or branched (if +1 Lys used)
7) E1 (Ub activating enzyme) adenylates Ub using ATP; then cov. bind Ub (forms thioester bond); passes Ub to E2
8) E2 (Ub-conjugating enzyme) sits on top of E3 (Ub-protein ligase)
9) E3 binds target for ubiquitination = induces formation of stable amide bond between Ub and target
10) Protein synthesised by ribosome
-Either N then D if unstable or mutation introduced
-Or directly misfolded
11) Protein follows many rounds of chaperones; if they are unable to fold protein correctly, one of E3 ligases binds to chaperone leading to ubiquitination of that protein = signal to send protein to proteasome for degradation

Question 42

Proteasome

Answer 42

1) Large proteolytic complex in all eukaryotes (nucleus & cytoplasm), archaea and some bacteria
2) Degrades peptides to ~7-8aa
3) Cylindrical complex
-Core particle (20S) = 4 stacked rings (αββα) forming central pore
-Regulatory particle (11S) = regulates entry to degradation chamber
-Each ring composed of 7 individual proteins
4) Eukaryotes have 7 different ß subunits
-Only ß1, ß2, and ß7 are catalytic; have N-terminal threonines in 6 active sites ;Thr in different environments/conformations gives different specificities
-Produced as precursors that are cleaved autocatalytically
5) Bacteria all 14ß subunits (same isoform) exhibit proteolytic activity
6) Regulatory particle (RP) recognizes unfolded proteins -> removes Ub -> some have ATPase activity to push protein to unfold into degradation chamber
7) In eukaryotes, RP caps core particle
8) Eukaryotes: 3 proteolytic activities: chymotrypsin-like, trypsin-like, and peptidyl-glutamyl peptide-hydrolysing (PGPH)-like (for broad substrate specificity)
9) Bacteria: do not have Ub and not all have proteasomes
-Other systems for less specific protein degradation
10) Mycobacteria express prokaryotic Ub-like protein (Pup); also has degradation chamber in inner part of proteasome; also have αβ subunits
-“pupylation” = Pup cov. links to many known proteasome substrates in mycobacteria; essential for degradation
-Inner 2 rings made of 7ß subunits hosting 7 protease active sites
-Target protein must enter central pore before its degraded
-Outer two rings contain 7α subunits = gate through which proteins enter barrel
-α subunit controlled by binding to cap structures or regulatory particles that recognize protein substrates and initiate degradation

Question 43

Factors affecting protein quality

Answer 43

1) external oxidants and reactive species
-Oxidants present in air (pollution), ozone and by various other chemical agents
-Exposure to radiation (e.g. UV)
2) internal oxidants and reactive species produced during metabolism
i) Free ē not forming water= react w/O = superoxide radicals
ii) Superoxide radicals reduce iron/copper ions in the cell; can be used by superoxide dismutase to form hydrogen peroxide = reacts w/Fe2+ and Cu2+ forming highly reactive OH radicals
iii) Both radicals dangerous = affect structure of protein and DNA
iv) Other radicals e.g. N, can also affect structure in diff ways
v) Overall effect = introduced charges induce partial unfolding = can expose hydrophobic aa in protein core = increase local hydrophobicity = exploited by cellular machinery involved in protein degradation
3) E3 ligases = recognition of degrons (degradation motifs e.g. modify side-chains/proteins in different ways)

Question 44

Role of oxygen in biological systems

Answer 44

Final acceptor in electron transport chain (respiration) for formation of water

Question 45

Regulation of proteasome activity under stress

Answer 45

1) large cellular stress (e.g. oxidative)= greater accumulation of damaged proteins = highly detrimental to the cell
2) Cells have 2-step transient response:
i) 0.5h-5h after oxidative stress exposure = capacity to degrade oxidized proteins increases; non-specific way; does not synthesise more proteins; does not use ATP to promote activity
ii) 5-48h after exposure = synthesise proteins and use of ATP to promote proteolytic capacity; more specific because it recovers regulatory particle
3) 2-step response = product of disassembly of 26S proteasome into free 19S regulators and 20S core proteasomes
4) 3-5h after initial stress, 26S proteasome re-forms from free 19S regulator and 20S core
-Several chaperones involved
-(mammals) Hsp70 stabilises regulator after disassociation from core for re-assembly
-(yeast) Ecm29 binds regulator following dissociation; required for increased survival and ability to degrade damaged proteins

Question 46

The aggresome-autophagy pathway

Answer 46

1) Crucial defence system against toxic build-up of misfolded proteins
2) V. extensive oxidative damage forms v.toxic aggregates; try to form single large aggregate and tackle that
3) Basal autophagy = specialized induced autophagy mediates non-selective bulk clearance of misfolded proteins along w/normal cellular proteins and organelles
4) Happens in cytoplasm; similar pathway in nucleus

Pathway:
1. Native protein = misfolded = refolded by chaperones or tagged w/Lys48-linked polyUb chains for degradation by proteasome
2. When chaperone/proteasome systems fail = misfolded proteins form oligomers & aggregates = cause cytotoxicity
3. Under conditions of proteasomal impairment, PD-linked E3 ligase parkin co-operates w/E2 enzyme Ubc13/Uev1a to mediate Lys63-linked polyUb of misfolded proteins
4. Lys63-linked polyUb chains promote binding to HDAC6 = link misfolded proteins to dynein motor complex for retrograde transport towards MTOC (microtubule-organising centre) to form aggresome
5. Also promote binding to p62 = facilitate recruitment of autophagic membrane to aggresome for formation of autophagosome
6. Fusion of autophagosome w/lysosome allows degradation of misfolded and aggregated proteins by lysosomal hydrolases

Question 47

Chaperone-mediated autophagy

Answer 47

1) Lysosomes contain proteolytic enzymes (e..g serine proteases)
2) ~1-15% of cell volume (most abundant in liver & kidney)
3) pH maintained at ~5.5 by proton-pumping ATPase
4) Hsc73 (constitutively-expressed Hsp70 chaperone) + other chaperones binds substrate containing KFERQ motif
5) Complex recognised by LAMP2A (receptor on surface of lysosome) = triggers unfolding and internalisation of protein in lysosome = degraded by proteases

Question 48

Misfolding diseases

Answer 48

1) Misfolding + aggregation of protein molecules related to ~20 diseases from neurodegenerative (Alzheimer’s, Parkinson’s) to non-neuropathic (Diabetes type II).
2) Chaperones try to bring misfolded/aggregated proteins back to native conformation
3) If not possible e.g. species forms non-native contacts = species is precursor for amyloid fibrils = v. stable and difficult to degrade = can become v. big causing mechanical damage and cell lysis
4) Amyloid fibrils start from different precursor proteins but share same structural features
5) Not all proteins form fibrils; some just aggregate
-Not visible because even at [low] toxic to cells so kills them

Question 49

Alzheimer’s disease

Answer 49

1) 1/14 +65; 1/6 +80
2) Function of proteins: chaperones, lysosomes etc slows down = less efficient mechanisms = formation of aggregates
3) Aggregate (1): amyloid-ß-peptide = amyloid plaques forming neurofibrillary tangles surrounded by dying neurons = mental deterioration = death
4) Comes from proteolysis of larger amyloid-ß precursor protein
-Size of peptide depends on protease that digested it
-Most common is 40 amino acid long
5) EM and NMR recently allowed visualization of fibril structure
6) Some studies show some morphological differences in fibril structure due to different combinations of peptides
7) Aggregate (2): Tau (Tubulin associated unit)
-Protein binds to microtubules (important in axons of neurons: template for transport of vesicles and backbone to axons)
-Tau can accumulate because axons last for several decades
-Different shapes of fibrils: straight fibre and paired helical fibre (more b-strand)

Question 50

Parkinson’s disease

Answer 50

-Affects 1/37 people
-α-synuclein
-V. abundant in dendrites (edges of neurons near synapse)
-Strong tendency to bind to membranes
-Switches between 2 conformations: membrane-bound or intrinsically disordered in cytoplasm
-Aggregates called Lewy bodies = v. abundant
-Function of normal condition a-synuclein
-Trafficking of synaptic vesicles = important for communication between neurons
-Help mitochondria to avoid oxidative damage
-Function of toxic a-synuclein
-Lyse mitochondria
-Permeabilize membranes allowing calcium influx
-Affects autophagy and degradation of proteins
-Can cause other diseases: a-synucleinopathies
-e.g. Lewy body dementia = different formations: neurites, glial cytoplasmic inclusions, etc

Question 51

Prion Diseases

Answer 51

1) sheep/goats = cows = people ate meat from infected cows
2) First case of protein acting as infectious agent = Stanley Prusiner
3) Prion Protein (PrP); 280 aa; membrane anchored; no known function
4) 2 conformational states
-PrPC : normal, stable, soluble, 40% a-helix, 3% b-sheet, protease sensitive
-PrPSc : scrapie, self-propagating, insoluble, 30% a-helix, 45% b-sheet, protease resistant
-Chemically identical
5) Scrapie in goats never infected humans; sequence specificity allows transformation from normal variant to pathogenic variant
6) Structure of PrP first NMR = then crystallised w/ therapeutic antibody; different secondary structures

Question 52

Characterisation of main species

Answer 52

1) Amyloid fibrils
-Look v. similar = some cause disease others are functional
-Stable and highly resistant to detergants/denaturants
-Not v. toxic but induce cell lysis when in abundance
-First structure 2005
-Common feature: cross ß-spine
2) Amyloidogenic precursors
-Monomers
-Transient and elusive
-Non-toxic
-Important to understand origin of fibrils as therapeutic targets
3) Pre-fibrillar oligomers
-Most toxic
-Important to elucidate key steps in fibril formation
-Bind/disrupt membranes = affect mitochondria, plasma membrane, etc = trigger apoptotic mechanisms
-Action subject to penetration of cellular membrane

Question 53

Cross ß-spine

Answer 53

1) Known since 1935 -information from diffraction pattern
2) Fibrils form parallel b-sheets
3) ~10Å intersheet space between them
-Varies dependent on sequence of fibril
4) 4.7Å interstrand space
-Constant -backbone atoms form these interactions
5) Axis of fibril is orthogonal to contacts between strands

Question 54

First atomic resolution structure of amyloids

Answer 54

1) Steric zipper model
2) Double b-sheet; parallel stacked
3) Side chains protrude inside b-sheets to form dry, hydrophobic core
4) Each b-strand bound through backbone stacks and side-chain H-bonds
5) Proposed that intermediate is more stable than native state ie. has lower free energy
-Difficult to denature so must be v. stable

Question 55

Impact of neurodegenerative conditions

Answer 55

1) Overtime people w/these conditions will grow (x2 or x3 by 2050)
2) Huge burden for healthcare system

Question 56

How to define disorder

Answer 56

1) Affects different levels of protein structural organization
2) Proteins can be disordered to a different degree

Question 57

Different functional roles – disordered vs ordered proteins

Answer 57

Disordered proteins:
1) Induced folding: can only acquire particular conformation when bound to other protein
2) Conformational ensemble: experience different conformations at the same time
3) Form fuzzy complexes
4) Structural heterogeneity
5) Binding plasticity: same protein can bind many different ligands

Ordered proteins:
1) Protein engineering: mutate particular AA = particular change in stability
2) 3D structural analysis: 3D structures are fixed
3) Enzymatic catalysis

Question 58

Energy landscape

Answer 58

1) Ordered protein: funnel-shaped
-Single native state w/ minimal free energy
2) IDP: no single minimum
-Fluctuating between different structures so multiple minima
3) Consider different states: native state, random coil, molten globule, pre-molten globule, formation of fibres, etc

Question 59

Structure of proteins

Answer 59

1) IDP’s = not completely denatured domain
2) Both ordered and IDP’s can experience other conformations:
i) Molten globule (helices and strands are more unwound = wider radius of gyration)
ii) Pre-molten globule: secondary structure propensities and some completely disordered regions
iii) Random coil: only some identifiable structures because they are as stable in native as denatured state
3) Different conformations characterized by size exclusion chromatography
-Elute at slightly different times –may overlap a bit
-Depend on hydrodynamic radius

Question 60

Characterizing IDPs

Answer 60

1) Sequence = easiest way to spot IDP
2) Globular proteins prefer large hydrophobic residues = need for hydrophobic cores
3) IDPs avoid large hydrophobic residues = helps avoid aggregation = want charged and other polar residues that can interact w/water = want residues that break 2º structure ie. Gly, Pro
4) Overall exposure to solvent of IDPs will be greater than globular proteins
5) Plot mean hydropathy vs net charge of that protein
-Mean hydropathy = sum of normalized hydrophobicities of all AAs
-IDPS more hydrophobic; bigger net charge than ordered proteins
-Low hydropathy = low driving force for protein compaction
-High net charge = strong electrostatic repulsion

Question 61

Studying structural properties of IDPs

Answer 61

1) no crystallography
2) NMR = can only analyse some things
i) How an unfolded protein looks like
ii) Learn there is dispersion of resonances between 2 frequencies (H and N); dispersion describes chemical shift of each residue = IDPs have limited dispersion
iii) IDPs have sharper peaks = each AA behaving as if part of smaller molecule = tumble more quickly = slower T2 relaxation
iv) Obtain structure of secondary structure (a-helices; extended-ß strands; poly-prolines –more flexible helical structure; comes from segments w/many Prolines; every 3 residues have turn of 360º
v) Obtaining backbone resonances of protein = get characteristic chemical shift for a-helix vs b-sheet
3) Circular dichroism = characterise 2º structure
-can separate proteins in random coil vs pre-molten globule conformation

Question 62

Protein-protein interactions (PPIs)

Answer 62

1) PPI Network
2) Dot = protein; line = interaction
3) Most proteins only have 1/2 interactions; some have multiple  IDPs
4) Fuzzy complexes = multiple interaction proteins; when interacting, remain as ensemble of different conformations; can measure affinity of interaction (v. strong; not able to crystallise)