Structural Biology - Protein Folding

Question 1

Why is it important to understand protein folding?

Answer 1

Predict the 3D structure from the primary sequence
Understand misfolding related to human diseases
Design proteins with a new function

Question 2

What are intrinsically disordered proteins?

Answer 2

Don’t have a defined 3D / globular structure
Because sequences don’t allow for formation of hydrophobic core
Are very flexible
30% of proteins are intrinsically disordered proteins
A small number can aggregate and cause disease
Neurodegenerative diseases (Alzheimer’s - alpha beta, Parkinson’s - alpha synuclein) and Cancer (P53)

Question 3

Briefly explain the process of protein folding

Answer 3

After ribosomal synthesis, most proteins (but not all) fold into a 3D structure
Spontaneous process
Transition from denatured to native state
Each protein structure has its own free energy
Native (folded) state has minimal free energy due to increased native interactions and increased residue contacts

Question 4

Explain the anfinsen experiment

Answer 4

Proved that the native state is the conformation with the minimal free energy
Denature ribonuclease A (has four disulphide bonds) with 8M urea and beta mercaptoethanol (BME) to unfold the protein to a random coil state with no enzymatic activity
Remove BME witch leads to oxidation of sulfhydryl/thiol groups (reforms disulphide bonds)
Remove urea witch leads to scrambled protein with no activity
Add trace amounts of BME which converts scrambled protein to native state
BME is not a catalyst only breaks disulphide bonds which leads to different conformations of the protein
Conclusion: this process is driven by the conformational free energy that is gained by going into the native structure (active conformation)
Formation of N structure is encoded in the sequence of the protein
Thermodynamics is important in protein folding - every conformation has a different energy but lowest free energy is favoured

Question 5

Explain the chemical stability of protein folding

Answer 5

Chemical stability is the ability of maintaining the chemical structure of native states/polypeptide chain under various conditions (ex. temperature, pH, denaturing agents)
Want intact covalent bonds, oxidation states, metal coordination
Deamination of Asn and Gln residues (to Asp and Glu, respectively)
Hydrolysis of the peptide bond of Asp residues at low pH
Oxidation of Met at high temperature to methionine sulfoxide (Sign of aging in proteins)
Elimination of disulfide bonds
Thiol-catalyzed disulfide interchange at neutral pH (oxidation of disulphides, very reactive)
Oxidation of cysteine residues to thyil radicals, Cys-S·

Question 6

Explain the conformational/thermodynamic stability of protein folding

Answer 6

Proteins fold into the Native structure because it is more stable than the denatured state
Conformational stability is the ability to adopt a well defined conformation instead of a random coil
It is given by the difference in free energy between D and N states DeltaG(D-N)
Non-covalent interactions
Psi and phi in backbone of protein rotate which allow polypeptide to assume various conformations
Tells us about flexibility of secondary structure (alpha helices/beta strands) and how these adapt (compress/elongate) to allow formation of 3D structure
Some conformations are disallowed
Glycine has the greatest accessibility

Question 7

Explain proteins folding in terms of the free energy change

Answer 7

Free energy change upon folding, depends on enthalpy and entropy
Unfavourable entropy change by folding a flexible polypeptide
Favourable enthalpy change from intra molecular side chain interactions (Hydrophobic interactions, Hydrogen bonds, Disulphide bonds)
Favourable entropy change from burying hydrophobic groups in the molecule (Release of water molecules from hydrophobic side chains - strongest force that drives protein folding)
In total net negative delta G

Question 8

Explain the different parameters which influence protein folding

Answer 8

Delta G varies with: pH, Temperature, Pressure, ionic strength, molecular crowding, hydrophobic effect, H-bonds, van Der Waals, disulphide bonds
Example proteins have optimal activity at a specific temperature
Crowding: thick, dense solution like polysaccharides (dextran) gives a more stable protein

Question 9

How does protein stability affect their function?

Answer 9

Average stability (free energy) of a small protein is only 5-15kcal/mol
To fold a protein, need to break interactions in denatured state and from interactions in the native state
Balance between them is very small
So, proteins are not very stable which is important because it allows them to be dynamic to be able to perform a function and allows cells to recycle proteins

Question 10

Explain how covalent interactions (disulphide bonds) determine protein folding

Answer 10

Oxidation forms disulphide bond, reduction breaks it
Process is reversible
Oxidation process can be intramolecular (in same protein) or inter-molecular (with different proteins ex. antibody light and heavy chains)
Cellular enzymes (protein disulphide isomerases) help form disulphide bonds

Question 11

Explain how compaction determines protein folding

Answer 11

Proteins are compact
Compactness = amount of surface area of an object relative to a perfect sphere of comparable volume (perfect sphere = 1 but other object will have <1)
Alpha helices and beta sheets are very compact
Folding is directed by internal residues
Hydrophobic effect drives folding (hydrophobic amino acids cluster together in core of protein to minimise exposure to water)
Interactions between amino acids drive folding (ionic, vdw, disulphide)
Need a good balance between compactness and flexibility to allow function

Question 12

Explain the hierarchical pattern of protein folding

Answer 12

Folding follows a hierarchical pattern (step-wise)
Subdomains form spontaneously
Domains are stable, independent folding units
Large proteins can contain several domains
Tertiary structure forms when these pack together

Question 13

Explain the adaptability of proteins when folding

Answer 13

Proteins are adaptable
ex. rigid hydrophobic core/packing of non-polar side chains
Large mutations don’t affect backbone of protein
Example: Phenylalanine (aromatic) to alanine mutation in T4 lysozyme showed that a benzene was occupying position of side chain and structure was preserved after mutation

Question 14

Explain the sequence versatility of proteins (conservation of sequence and folding)

Answer 14

20% sequence identity (80% difference) between 2 proteins = can have the same fold/structure
But can also have the opposite: 88% identity and a different structure
Only some residues are important in determining the unique structure

Question 15

List different techniques to measure protein stability

Answer 15

Absorbance ex. trp, tyr, chromophoric probes (but only gives small signal)
Fluorescence ex. trp, fluorophoric probes, aromatic residues (big signal)
CD
NMR
Differential scanning calorimetry (DSC) to determine delta H of unfolding
Catalytic activity

Question 16

Explain the protein denaturation curve

Answer 16

Denaturation: loss of native structure integrity and loss of activity
Proteins can be denatured by: Heat or cold, pH extremes, organic solvents, chaotropic agents (urea, guanidinium hydrochloride)
Denaturation curve is sigmoidal
Tm = melting point, when protein is 50% denatured and 50% folded
D50% = denaturing concentration at which 50% is denatured and 50% is folded
Denaturation leads to a decrease in fluorescence intensity and increase in wavelength maxima
Ex. Scan for different concentrations of urea and take each point at which difference is the largest to form a sigmoidal curve

Question 17

Explain how circular dichroism (CD) works

Answer 17

CD measures the differential absorption of left and right circularly polarised light by optically active media
De = eL – eR (e=molar absorbance)
Chiral molecules (handedness) are either L or R and are optically active
Circularly polarised light can be clockwise or anticlockwise
Polarised light interacts with chiral molecules, there is differential absorption
Chiral molecules absorb circularly polarised light in only one direction
Light is absorbed at diff wavelengths by the protein
Molar ellipticity (difference in absorption between left handed and right handed circularly polarised light)
If light is absorbed equally in both directions (eL=eR) get linearly (plane) polarised light, molar ellipticity=0
If light is absorbed more in one direction than the other (eL<eR), get elliptically polarised light
CD spectrum gives secondary structure information
Different secondary structure (alpha helix, beta sheet, random coils) have different CD signatures
CD spectrum shows molar absorption difference (of left and right handedness) vs wavelength

Question 18

Explain the background of circularly polarised light

Answer 18

Light (electromagnetic radiation) is an oscillating wave of E-fields (electric) and B-fields (magnetic)
Focusing only on E-field
Oscillations can be Planar (horizontal and vertical components in phase, only in one plane) or Circular (horizonal and vertical out of phase, in 2 planes) or Elliptic (a mix)
Circular polarized light is 2 waves in different planes and one has a 90 degree phase shift
The phase shift can be -90 degrees or +90 degrees
Circular polarised light is chiral (handedness)
Left circularly polarized light = counter clockwise
Right = clockwise

Question 19

Pros and cons of CD spectra

Answer 19

Pros:
CD spectrum recorded in the far-UV (190-240nm)
3D fold of secondary structure gives a particular CD signature
Allows monitoring of folding and unfolding
It can be used to monitor protein folding but also biological activity
Cons:
Lack of structural resolution (Only shows secondary structure, not at amino acid level)
Often over interpreted so need to combine with other techniques

Question 20

Explain what the levinthal paradox is

Answer 20

Assume 3 conformations per amino acid in the denatured state (psi and phi angle allowed conformations)
For 100 amino acid protein there are 3^100 potential conformations
If the chain can sample 1x10^12 (trillion) conformations/sec it will take 2x10^28 years to reach the native state
There are too many different possible conformations for a protein to fold by a random search of conformations
A protein folds by following defined pathways
Most single domain proteins fold in milliseconds to seconds

Question 21

Explain what makes a protein fold so fast and how intermediates can be trapped

Answer 21

A series of conformations are encoded in the protein sequence
Search for minimum energy conformation is not random
Direction towards native structure is a funnelled energy landscape
Free energy decreases down the diagram
At bottom (lowest free energy) is the native state
Energy landscape has local minima
Partially folded proteins (intermediates) can be trapped
This will slow down/inhibit folding
If hydrophobic groups are exposed, protein will aggregate or stick to other proteins

Question 22

Explain how FRET works and when it is used

Answer 22

FRET (fluorescence resonance energy transfer) measures two state folding (no intermediate species D<–>N, single domain proteins)
Two fluorophores are labelled on molecules of interest
One fluorophore is the donor, one is the acceptor
Donor absorbs light at a specific wavelength and emits it at a longer wavelength in the visible spectrum
Acceptor has absorption spectrum that overlaps with emission spectrum of donor
Excite donor with specific absorption wavelength
Donor can transfer energy to acceptor when they are in close proximity (1-10nm)
Acceptor fluorophore is exited and emits light at its characteristic wavelength
EXAMPLE
Red fluorescent protein absorbs yellow light and emits red light (that has lower energy) - Acceptor
Green fluorescent protein absorbs blue and emits green - Donor
When close together, green donor will pass energy to acceptor chromophore and will emit in red

Question 23

How is FRET used to determine protein folding?

Answer 23

FRET used for two state folding
In D state: separation between dyes is larger and transfer efficiency is small
In N state: separation is smaller and transfer efficiency is large
At equilibrium, still movement between D and N states (protein is folding and unfolding)
At D50 protein will be 50% denatured and 50% native
Denaturation is reversible, corresponds to jumps in FRET
No intermediate steps in FRET
Process is highly comparative

Question 24

How is FRET measured with a device and what is the result of it in graphical form?

Answer 24

Stopped flow device:
Syringe with denatured and non-denatured protein
Are mixed together to initiate refolding or unfolding
Quartz cell and pass through fluorescence detector
Measure difference in signal between N and D state
Get single exponential relaxation curve for 2 state process (look at notes)
Can measure folding and unfolding rate to get delta G of protein
DeltaG=-RT(kfolding/kunfolding)

Question 25

How do absorbance/fluorescence vs time curves for D to N states look like for 2 state and 3 state folding?

Answer 25

For 2-state (D-N): Single exponential curve Subtract experimental data from theoretical exponential curve to get a straight line If points are close to the X-axis fit is good For 3 or 4 state proteins: add up multiple exponentials Formation of intermediates occurs at different speeds Fit data to double exponential curve (D-I-N) or fit curve to triple exponential curve (D-I-I-N) to see which one gives a straight line Formation of intermediate occurs very quickly (4ms) Formation of N state takes longer (400ms) Clear separation of formation of intermediate and N state

Question 26

Explain the principle of the F value analysis, what it is used for and assumptions

Answer 26

Experimental method to study the structure of the TS at residue level Specific probes are used to distinguish between D and N state (CD, fluorescence, NMR) Series of conservative deletion mutations that deletes sections of protein one by one Removal of methyl groups T to S, I to V (1methyl) V to A (2 methyl) I/L to A, F to L, X to A (3 methyl, more aggressive) Any mutation should end up with alanine and not glycine as it has no side chain which affects conformation of the backbone Compare differences in thermodynamics and folding rates between Wild type and mutant Measure F values at different time points to make kinetic profiles Assumptions: Assume that mutations don't affect the structure of the N state and don't create new interactions during folding

Question 26

Explain transition state theory and folding rate

Answer 26

Occurs for all chemical reactions Used to analyse two state folding Free energy (G) vs reaction coordinate N state has lower free energy than D state Energy barrier from D to TS Structure of TS tells us what interactions need to form to make the N state TS has high energy: very short lived and unstable so can't be characterised structurally Folding rate is proportional to the exponential of the negative activation free energy (energy difference between TS and D state) k =exp(-DeltaG(TS–D)) Use mutational analysis to determine two-state folding kinetics (and structure of TS)

Question 27

How is F value analysis used for a 2-state folding process and how is the F value interpreted?

Answer 27

Mutations change the thermodynamic stability of N state DeltaDeltaG(D-N) = DG(D-N(mut)) - DG(D-N(wt)) Folding rate (k) is related to activation free energy (energy diff between TS and D) k = exp(-DeltaG(TS–D)) The faster the folding rate, the less stable Some mutations can change the folding rates (k) Measure folding rate of mutant and wild type and get difference in delta G DDG(TS–D) = -RT ln (kwt /kmut) The F value is the fraction of protein that achieved the native conformation at a specific folding intermediate during the folding process Ratio between delta delta G TS-D and N-D When F=1, mutated residue is native, that region is the folding nucleus of the protein (affects kinetic and thermodynamic stability), structured in TS When F=0, mutated residue is non-native, unstructured in TS, is only formed in N state but not in TS (didn't affect folding rate)

Question 28

Explain the 2 competing folding pathways of D1pPDZ and how the F value is used for structural refinement of TSE?

Answer 28

PDZ domains are small domains Adopt a six stranded beta sandwich fold with two alpha helices (bbbabbab) D1pPDZ =PDZ domain of the D1 C terminal processing protease (D1p) Has kinetic competition between 2 folding pathways: to native and to misfolding to off pathway metastable intermediate Analysed by circularly permutated protein: change location of N and C termini When mutants were produced, only got stable I state N state is unstable so no advantage in forming it Misfolded intermediate has alternative packing of N terminal of beta hairpin TSE = transitional state ensemble, a protein that forms a stable non native intermediate (intermediate that doesn't lead to formation of N state)

Question 29

What Is the average protein concentration in a human?

Answer 29

Average MW of proteins in human cell = 50kDa Average protein concentration = 20-30% (very high) Average protein concentration (molar) = 4-6mM (varies with cell type) But many will not reach more than 0-5mM

Question 30

What different factors affect protein folding in vivo?

Answer 30

Protein is surrounded my many other proteins, lipids, polysaccharides in vivo Crowding Protein aggregation Cellular components Compartments

Question 31

What are chaperones and what are the different types?

Answer 31

Proteins that (mostly) use ATP to bind unfolded or misfolded proteins and set them on the folding pathway again Bacterial trigger factor: no ATP needed, ribosome associated, acts early HSP70: requires ATP, eukaryotes, bacterial homologue/name in E. coli is DnaK, reverses denaturation/aggregation, works with HSP40 (DnaJ in E. coli) Chaperonins (HSP60): have bacterial and eukaryotic homologues, large, multi-subunit, cage-like (Type I for bacteria, mitochondria, chloroplasts, Type II for archaea/eukaryotes) HSP90 (eukaryotic): facilitates late stage folding of signalling proteins, unique regulatory role and induces active conformation Nucleoplasmins: decameric, acidic, nuclear (mostly in nucleus), assembles nucleosomes Protein disulphide isomerase (PDI) Peptidyl-prolyl isomerase (PPI) HSP70 and HSP60 are needed by E.coli to survive above 30 degrees

Question 32

What is the structure of the ribosome and what does S mean?

Answer 32

E. coli ribosome is 25 nm diameter, 2.52 MDa (2,520kDa) in mass Two unequal subunits that dissociate at < 1mM Mg2+ S is a unit to describe the speed at which a molecule migrates under significant force Bigger molecules travel faster under significant force 50S = travels 50 micro metres per second under a force of 1 million G 30S subunit is 0.93 MDa with 21 proteins and a 16S rRNA 50S subunit is 1.59 MDa with 31 proteins and two rRNAs: 5S and 23S Ribosomes are 60% RNA 20,000 ribosomes in a cell, 20% of cell's mass

Question 33

What are the three phases of protein synthesis?

Answer 33

Initiation: binding of mRNA and initiator aminoacyl-tRNA to small subunit, initiation factors bind, then large subunit binds Elongation: synthesis of all peptide bonds, tRNA binds acceptor (A) and peptidyl sites (P) Termination: termination tRNA recognizes stop codon

Question 34

What is the trigger factor and why is it important in protein synthesis?

Answer 34

Newly synthesized proteins leave the ribosome through a narrow tunnel in the large subunit (peptidyl transferase centre, PTC) When synthesis is still ongoing, nascent protein chains may form misfolded intermediates These are dangerous to the cell: exposes hydrophobic regions leading to aggregation OR can be substrates for proteolysis Trigger factor is an ATP dependent chaperone (in bacteria) + displays PPIase activity It binds to ribosomal protein L23 and receives the synthesized protein Allows protein to fold by ex. recognizing hydrophobic regions If protein is very long more than one Trigger Factor binds to the protein

Question 35

What is the structure of the trigger factor and individual domains?

Answer 35

N-terminal domain: Has some chaperone activity, L23 ribosome binding Linker: links N and P domain P domain: Peptidyl-prolyl activity, Auxiliary (some) chaperone activity C-terminal domain: Main chaperone activity Protein is constitutively expressed, but increased expression at low temperatures (when cell is cold shocked) - it is NOT a heat shock protein Domains are flexible (rotation) and each domain is flexible on the inside (local flexibility) Allows protein to bind substrates with other conformations

Question 36

What are heat shock proteins?

Answer 36

Heat shock proteins are one of the biggest families of chaperones Are expressed at low levels normally in the cell Are overexpressed when the cells are under high temperatures Prevent aggregation and misfolding Bind to nascent polypeptides to prevent premature folding Facilitate membrane translocation/import by preventing folding prior to membrane translocation Facilitate assembly/disassembly of multiprotein complexes (ex. nucleosome)

Question 37

Explain how trigger factor, DNaK and GroEL work together in a chaperone network

Answer 37

In bacteria Protein is synthesized and bound by trigger factor Then chaperone DnaK (Hsp70) in bacteria binds protein from trigger factor and allows it to fold If DnaK fails it is bound by chaperone GroEL

Question 38

Explain the cycle of how HSP70 and J-domain proteins interact

Answer 38

HSP70 is an ATP binding protein N terminal nucleotide binding domain (NBD) that binds ATP C-terminal substrate binding domain (SBD) beta and alpha Alpha and beta are not allowed to interact when protein is in the ATP form low affinity for substrates in ATP form (like open spring) Hsp40 (J-domain proteins) More than 40 J-proteins in the human genome Modulates ATPase activity of Hsp70 Turns ATP to ADP in Hsp70 Hsp70 in ADP form traps binds the ligand/substrate with high affinity (like spring closing) Hsp70 only binds a small region of the protein Hsp70 is the most abundant chaperone and contains many orthologues Allows protein to fold Nucleotide exchange factor ex. GrpE Exchanges ADP for ATP Release of the substrate in the folded form Cycle restarts

Question 39

What is the structure of GroEL/GroES?

Answer 39

Multiple heptameric rings Each monomer in heptameric ring is 60kDa Except top ring, has monomers of 10kDa 2 monomeric rings form an internal chamber that binds the ligand This isolates protein from crowded medium of the cell and allows the protein to refold GroEL: active state (middle ring). Cis monomer so it is near GroES. Elongated to form a bigger chamber GroEL: resting state (bottom ring). Trans monomer - it is far from GroES GroES: top ring/cap, smaller look at image in notes

Question 40

What is the mitochondrial form of GroEL/GroES?

Answer 40

GroEL/GroES: prokaryotic HSP60/HSP10: mitochondria/chloroplast

Question 41

Explain the mechanism of the active and inactive states of GroES/GroEL

Answer 41

Inactive state (trans) without GroES: Small chamber Exposed hydrophobic patches: allows unfolded protein to bind to at least 2 hydrophobic patches Active state (cis) with GroES: Hydrophobic patches are hidden in GroEL --> large, hydrophilic cavity Allows protein to fold on its own Can fold proteins up to 60kDa

Question 42

What is the GroEL/GroES cycle?

Answer 42

Anti cooperative binding of ATP (fast) Only one of the two heptamers of GroEL will bind ATP at a time Mis/unfolded polypeptide binds (slow) GroES binds (very slow) and pushes unfolded protein into the chamber Then other heptametic ring binds ATP in trans ATP --> ADP to relase 7Pi Improperly folded protein binds to the other chamber Then folded protein + GroES + 7ADP are released Turn 180 degrees and cycle starts again GroES can only bind one GroEL Look at diagram in notes

Question 43

How does GroEL/GroES work on E. coli proteins?

Answer 43

85 cytosolic proteins are are strictly dependent on GroEL/ES to fold These proteins usually have more complex tolpologies Many are alpa/beta proteins some are a/b barrels that are stabilised by long-range interactions

Question 44

What is Hsp90?

Answer 44

Hsp90 is an abundant, very flexible, highly dynamic protein Binding of substrates is very transient and requires many factors Acts mainly at late stages of substrate folding Stably dimerizes via its C term domain Dimerises transiently via its N term ATPase domain - when ATP is bound Different co-chaperones help binding to different ligands Hydrolysis of ATP to release folded ligand PTMs (e. g. phosphorylation, acetylation) affect its functional state Details of substrate binding (and mechanism) still under study

Question 45

What is protein disulphide isomerase (PDI) and how does it function?

Answer 45

PDI catalyses formation and exchange of disulfide bonds Shuffles disulfide bonds and allows protein to explore different conformations before it forms the native disulfide bonds (similar to anfinsen experiment) Binds exposed hydrophobic regions Allows protein to find most thermodynamically stable pairs Has broad substrate specificity - diverse disulphide containing proteins Reduced PDI (thiol) generated by oxidised glutathione GSH breaks misfolded disulfide and leads to formation of oxidsed PDI (which is regenerated by reduced glutathione GSSG) Breaking of disulfie allows protein to form a different disulfide Cysteines in conserved motif (CxxxC) Broad hydrophobic cleft: peptide binding and chaperone activity

Question 46

What is peptide propyl isomers (PPI)?

Answer 46

In proline, cis trans isomerization is rate limiting in the folding of proteins (need to have correct conformation) PPI binds to X-proline bond and accelerates cis-trans isomerisation by 300-fold Twists the peptide bond so that backbone C, O, N atoms are no longer planar Use HSQC spectrum to determine if protein is correctly folded Amino acids that are next to proline can have more than one conformation

Question 47

Give examples of proteins that are degraded in the cell and their half-life

Answer 47

In the cell, proteins are degraded at different rates Rate depends on the stability and function of protein Ornithine decarboxylase: half life of 11 minutes Produces antioxidants: polyamines, important for DNA stability, cell division Produces compounds that increase cell growth and reduce apoptosis Can be dangerous to the cell so protein is very tightly controlled P53 had half life of 5-20minutes Can cause cell cycle arrest/apoptosis Albumin half life of 3 weeks Osmotic pressure in serum cells Carrier of metabolites Haemoglobin lasts as long as red blood cell (a month) Structural proteins Inert, non catalytic In structures that protect them from degradation Ex. gamma crystallin in eye lens (70 years), collagen (110 years) Degradation by CHAPERONES

Question 48

What is protein turnover and what are the different systems that are responsible for it?

Answer 48

Protein turnover is the balance between synthesis and degradation Ubiquitin proteasome system (UPS) degrades cytoplasmic and nuclear proteins Lysosome endosome system degrades ER proteins, extracellular, cell surface Aggresome Catabolism: synthesis < degradation Anabolism: synthesis > degradation

Question 49

What is the structure of ubiquitin and how does it attach to substrates?

Answer 49

Ubiquitination: Most important mechanism to regulate protein degradation Ubiquitin: small protein of 76 amino acids, used to tag proteins for degradation in proteasome pathway Degrades cytoplasmic and nuclear proteins Ubiquitin attaches to substrate via C-terminal glycine 76 Proteins are linked to ubiquitin via isopeptide bond between carboxyl of C-terminal glycine 76 and amine side chain of lysine in substrate

Question 50

What are the different functions of the ubiquitin lysines? How are polyubiquitin chains formed?

Answer 50

Ubiquitin has 7 lysines that are responsible for different cellular functions G76 binds substrate K48 polymerises Ub Lysine 48 Ub1 binds to C-terminal G76 of Ub 2 to form polymer Chain of 4 Ub = signal for protein to be degraded Polyubiquitin chains: Can form linear or branched patterns

Question 51

What is the mechanism of attaching a ubiquitin to a target molecule?

Answer 51

Ubiquitination at the C-terminal of Gly76 E1 (ubiquitin activating enzyme) uses ATP to adenylate Ubiquitin, release of Ppi E1 forms thioester bond with ubiquitin, release of AMP Ubiqutin transferred form E1 to E2 (ubiquitin conjugating enzyme) Complex is recognized by E3 (ubiquitin protein ligase) E3 complex binds the target E3 ligases have specificity for certain motifs that allows it to bind the target A protein tagged with a single Ub molecule becomes a substrate for other ligases to attach additional Ub molecules

Question 52

Give examples of diseases that use E3 ligases

Answer 52

More than 600 E3 ligases in the human genome Parkin: E3 ligase, involved in destruction of alpha-synuclein (aS) Knockout of parkin = accumulation of aS leads to Parkinsons disease HPV: produces E3 protein that targets p53 in the host 90% of cervical cancers are related to this E3 ligases are important drug targets

Question 53

Explain the ubiquitin-proteasome system (UPS)

Answer 53

DNA --> RNA --> Ribosome --> Folding --> Native protein --> Denaturation/damage --> Misfolded --> Hsp40/Hsp70 assist folding If chaperones are not successful in refolding the protein: Chip (E3 ligase) binds to chaperone-protein complex and ubiquitinates it Ubiquitination is recognized by region of proteasome Cleaves ubiquitin Misfolded protein into chamber where it is degraded

Question 54

What is the function of the proteasome?

Answer 54

Large proteolytic complexes in eukaryotes, archaea, bacteria In eukaryotes: are found in nucleus and cytoplasm Degradation process gives peptides 7-8 amino acids long

Question 55

What is the structure of the proteasome?

Answer 55

Cylindrical complex Core particle (blue): 20S, 4 stacked heptameric rings (abba) forming pore/chamber Regulatory particle (red): 19S, regulates entry to destruction chamber, recognizes ubiquitin, sometimes uses ATP Each ring has 7 domains/proteins

Question 56

What is the proteasome mechanism in eukaryotes?

Answer 56

7 beta subunits/isoforms beta 1, beta 2, beta 5 contain N-terminal catalytic threonines, 6 active sites Before CP autocatalytic maturation of precursor, these are protected by pro-peptides Threonine in active site are in different conformations/environment so have different substrate specificities 3 proteolytic activities: chymotrypsin like, trypsin like, peptidyl glutamyl peptide hydrolysing like Regulatory particle (RP) caps 20S CP: recognizes and unfolds ubiquitinated proteins before degradation RP removes Ub from substrates to recycle Some RP's use ATP hydrolysis to provide energy for protein unfolding

Question 57

What is the proteasome mechanism in bacteria?

Answer 57

All 14 beta subunits (same isoform) have proteolytic activity Bacteria don't have ubiquitin so simpler structure Not all bacteria have proteosomes Mycobacteria have ubiquitin like protein (Pup): binds so proteasome substrates, is essential for degradation Inner two rings of seven beta subunits Seven protease (x2) active sites Protein enters central pore for degradation Outer two rings contain seven alpha subunits A gate through which proteins enter the barrel Are controlled by binding to cap structures or regulatory particles - recognize protein substrates and initiate degradation process

Question 58

What external factors can affect protein quality?

Answer 58

Oxidants in the air, ozone, nitric oxide, carbon monoxide Radiation like UV Ionizing radiation

Question 59

How do reactive species affect protein quality/induce misfolding?

Answer 59

Reactive species can modify amino acids within proteins, damage prosthetic groups or oxidise key transition metal centres Introduces extra charges/changes charges = increases local hydrophilicity Ex. in hydrophobic core or surface Two of the same charges next to each other = unfolding Can modify other amino acids leading to partial unfolding and exposure of hydrophobic regions leading to degradation E3 ligases recognise degrons (degradation motifs)

Question 60

What internal factor can affect protein quality?

Answer 60

Oxygen is the final acceptor in the ETC (respiration) to form water Oxygen reacts with leaked electrons from respiration to form superoxide radicals (O2-) Superoxide radicals react with/reduce Iron or Copper ions in the cell (Fe3+ --> Fe2+) Superoxide radicals interacts with protons to form hydrogen peroxide, H2O2 H2O2 reacts with Fe2+ and Cu+ to form highly reactive hydroxyl radicals (OH)

Question 61

Explain the two step response of cells to oxidative stress/protein damage

Answer 61

Cells are often exposed to low level of oxidative stress/protein damage A large stress leads to accumulation of damaged proteins (bad for the cell) Cells have a two-step transient response 0.5 - 5h after oxidative stress exposure: capacity to degrade oxidised proteins increases (protein synthesis independent) and reduction in ATP dependent proteolytic capacity (broad response) 5h - 48h after: protein synthesis dependent increase in proteolytic capacity and ATP stimulated proteolytic capacity (more specific response)

Question 62

How does dissociation of the proteasome allow response to protein damage?

Answer 62

Dissociation/disassembly of 26S proteasome into free 19S regulators and 20S core proteasomes 20S proteosome without RP allows for indiscriminate protease activity in first 5 hours 3-5h after: 26S proteasome re-forms from free 19S regulator and 20S core proteasome Chaperones are involved in process: Hsp70 stabilises 19S regulator after dissociation from 20S and reassembles functional 26S In yeast: Ecm29 binds 19S regulator after dissociation, required to degrade proteins

Question 63

What is the related autophagy pathway?

Answer 63

Different pathway to degraded proteins Basal/micro autophagy: non-selective, bulk clearance of misfolded proteins Defective organelles or proteins are recognized by lysosome and degraded Ex. Starvation induced Vs aggresome-autophagy pathway is specialised

Question 64

What is the aggresome-autophagy pathway?

Answer 64

Specialized, selective clearance of misfolded or aggregated proteins Small amount of misfolded proteins --> refolded by chaperones OR tagged with Lys48 linked polyubiquitin chains --> degradation by proteosome If chaperone and proteosome systems are overwhelmed --> aggregated protein--> cytotoxicity If amount of aggregated protein is too high --> PD linked E3 ligase parkin + E2 enzyme -->protein ubiquitinated using K63 (instead of K48) --> recognized by HDAC6 --> binds dynein motor complex --> transport towards MTOC to form aggresome Binding to p62 --> aggregate is surrounded by autophagic membranes to form autophagosome --> fuse with lysosome for degradation

Question 65

What is chaperone mediated autophagy in lysosomes?

Answer 65

Lysosomes have low pH (pH 5.5) and contain different proteolytic enzymes (serine proteases, aspartic proteases) Substrate containing specific motif (KFERQ) is recognized by a homologue of Hsp70 (Hcp70) Substrate is recognized by receptor on surface of lysosome Another chaperone (lysosomal Hsp70) pulls the substrate into the lysosome where it is degraded Only happens to proteins that contain specific motif

Question 66

How do misfolded proteins lead to diseases via the formation of off-pathway intermediates?

Answer 66

Misfolding and aggregation of proteins are associated with diseases Ex. neurodegenerative diseases (alzheimers, parkinsons), non-neuropathic diseases (diabetes type II) Synthesis of a protein by ribosome --> unfolded state --> native state --> can form crystal, fibre or oligomer Unfolded state --> disordered aggregate --> chaperones help to form directly or lysosome/aggresomal degradation Off pathway intermediates: unwanted species that lead to formation of amyloid fibrils Amyloid fibrils are difficult to remove from cells Cause mechanical damage to cells + cell lysis Lead to disease

Question 67

What are amyloid fibrils?

Answer 67

Amyloid fibrils are insoluble protein aggregates that accumulate in extracellular space Can be functional: bacteria and fungi use amyloid fibrils to protect surfaces and interact with other species, important in biofilm formation Or can cause disease: neurodegenerative diseases (Alzheimer's, Parkinson's) Start from different precursor proteins but have same structural features Not all proteins form fibrils in vivo, some only form aggregates which can lead to neurological (accumulate in brain) or non-neurological diseases

Question 68

Give 3 examples of diseases that involve amyloid fibrils

Answer 68

Beta-amyloid: beta amyloid fibrils cause Alzheimer's disease. Beta amyloid is derived from cleavage of larger amyloid precursor protein Alpha-synuclein: Forms fibrils in Parkinson's. Aggregation leads to formation of Lewy bodies Prions: infectious proteins that can cause normal proteins to misfold and aggregate to amyloid fibrils

Question 69

What is Alzheimer's disease and what are the 2 main causes (brief)?

Answer 69

Associated with aging: 1 in 14 people over age of 65 and 1 in 6 over age 80 Leads to mental deterioration and death Cause 1: Amyloid beta peptide Cause 2: Tau protein

Question 70

How does Tau protein lead to Alzheimer's disease?

Answer 70

Tau: tubulin associated unit, important in function of axons, very long Two conformations: paired helical fibril and straight fibril More than 100 tau fibrils in different conformations In Alzheimer's: Tau is hyperphosphorylated and aggregates to form neurofibrillary tangles

Question 71

How does amyloid beta peptide lead to Alzheimer's disease?

Answer 71

Protein degradation and chaperone mechanisms become less efficient over time --> more protein aggregation Beta-amyloid is produced by cleavage/proteolysis of larger amyloid precursor protein (770aa) (by beta and gamma secretases) In Alzheimer's: imbalance in production/degradation of beta-amyloid. Abnormal processing of APP leads to production of beta-amyloid-42 Beta-amyloid forms insoluble plaques in brain tissue and in spaces between neurons in brain --> neurofibrillary tangles and dead neurons Are the plaques the cause Injection of Ab Peptide in monkeys induced the disease Doesn't seem to be an infectious disease Treatments: Levanemab and Aducanumab to stop aggregation (Mab's)

Question 72

How is Parkinson's disease caused by alpha synuclein?

Answer 72

Alpha synuclein function: Release and docking of exocytosis, formation of presynaptic vesicles, Regulating release of neurotransmitters between neurons, Abundant in dendrites in neurons (close to synapses) Misfolding of alpha synuclein leads to aggregation which is toxic Forms Lewy bodies (intraneural aggregates) Inhibits progression of vesicles from ER to Golgi Lyses mitochondria Permeabilize membranes Alpha synuclein aggregate can be secreted + endocytosed so disease can be passed to other cells Can also aggregate in other organs (heart tissue, blood cells): called alpha-synucleinopathies Alpha synuclein can bind membranes Exists in 2 conformations: disordered in cytoplasm OR ordered bound to membrane and forms helices at N-terminal

Question 73

What are prion diseases and give some examples of diseases?

Answer 73

Prion diseases cause transmissible spongiform encelopathies (TSEs) Neurodegenerative disease in humans + animals caused by prion proteins Don't contain genetic material (DNA/RNA) Contain misfolded version of prion protein (PrP) Examples: Scrapie (sheep), Bovine spongiform encephalopathy (BSE), Creutzfeldt Jakob disease

Question 74

How can prion diseases be transmitted?

Answer 74

Transmission of protein between individuals or species Ex. Eating contaminated meat lead to outbreak of BSE in cattle and CJD in humans

Question 75

What is the structure and function of the normal prion protein (PRPc)?

Answer 75

280 aa Membrane anchored Widespread protein observed only in mammals Prion proteins are found in brain Formation of axons/dendrites, neuronal homeostasis, cell signalling/adhesion/protective role against stress Soluble 40% alpha helix 3% beta sheet Protease sensitive

Question 76

What is the structure and effect of the abnormal prion protein (PRPSc)?

Answer 76

Misfolded version of prion protein Forms aggregates and fibrils in the brain Sc= scrapie, prion disease in sheep Chemically identical to PrPC (same sequence) Stable conformational variant of PrPC Self propagating: can induce conversion of PrPC to PrPSc Insoluble 30% alpha helix 45% beta sheet (common for fibrils) Protease resistant (proteolysis removes 67aa at N-terminal to give PrP)

Question 77

How does accumulation of PrPSc lead to tissue damage and what effects does this have?

Answer 77

Accumulation of PrPSc in brain leads to formation of holes and gives sponge-like appearance Symptoms are cognitive decline, motor dysfunction Leads to diseases: Familial CJD, GSS syndrome, Fatal Familial Insomnia Mutation can increase likelihood of spontaneous conversion, which can convert other proteins

Question 78

Explain the strain specificity and infections in terms of PrPSc proteins

Answer 78

Different strains of PrPSc have different pathologies and structures Infection between species that are close to each other: Mouse PrP can infect hamster (or cows to humans) Scrapie (from sheep) can't infect humans

Question 79

What is Creutzfeld-Jakob Disease?

Answer 79

CJD is most common prion disease in humans Variant of CJD (vCJD) from infected meat or blood transfusions CJD disease is VERY uncommon (2 people per million) Only 2-5% get vCJD from infections 80% CJD form sporadically

Question 80

Explain the difference between amyloid fibrils, amyloidogenic precursors (monomers) and pre-fibrillar oligomers

Answer 80

Amyloid fibrils: stable, resistant to detergents/denaturants, cause cellular and neuronal death Amyloidogenic precursors: transient, non-toxic, unstable Pre-fibrillar oligomers: most toxic species, affect mitochondria, plasma membrane, trigger apoptotic mechanisms, very unstable and heterogenous in terms of structure, difficult to isolate/study

Question 81

How do amyloids form a cross-beta sheet?

Answer 81

Amyloids form a beta-sheet: hydrogen bonds form between adjacent strands Form a cross-beta sheet: beta strands are oriented perpendicular (90 degrees) to fibril axis and form cross-sectional pattern Intersheet distance: 10 angstroms, varies according to sequence Interstrand distance: Contacts between strands in the same sheet, is constant Forms stable + ordered structure = insoluble fibrils look at image in notes

Question 82

Explain the steric zipper model of amyloid fibrils

Answer 82

Multiple beta sheets stack on top of each other Side chains protrude inside the two beta sheets like a self complementing steric zipper between sheets Forms dry spaces like small hydrophobic cores Within each beta strand, every segment is bound to two neighbouring segments through stacks of backbone and H-bonds Stabilizes fibrils

Question 83

Explain the conjectural plot of amyloid fibrils

Answer 83

Monomers have certain free energy Energy is gained in formation of a fibril Fibrils only form at high monomer concentration as energy barrier is very high Delta G of dissolution is very high so fibrils are hard to dissolve

Question 84

What are intrinsically disordered proteins (IDPs) and what diseases are they related to?

Answer 84

Make up 30% of eukaryotic proteins Some of them are related to aggregation diseases Complement function of folded/ordered proteins Connection to neurodegenerative diseases Abeta - alzheimers Alpha-synuclein - parkinsons Tau - tauopathies Prion protein - prion diseases Cancer - p53 Intrinsic disorder can affect different levels of structural organisation Whole proteins can be disordered Or certain protein regions can be disordered Disordered to different degrees Are in more than one conformation at a time Structural heterogeneity

Question 85

Explain the energy landscapes of ordered Vs IDPs

Answer 85

Ordered: well defined folding landscape Funnel shape High energy conformation --> conformation of minimal energy (native/folded state) IDPS: flat landscape composed of multiple minima Fluctuating conformations of comparable free energy Look at image in notes

Question 86

What are the 4 different states of native proteins?

Answer 86

Random coil: no native/non native interactions Pre molten globule: some secondary structure elements but many unstructured elements Molten globule: expanded form of native state (secondary structure is formed but no tertiary contacts) Native/folded Size exclusion chromatography: 4 different states of native proteins will come out of column at different times

Question 87

What are the sequence properties of IDPs?

Answer 87

Folded: have large hydrophobic amino acids (found in hydrophobic cores) IDPs: NO compact structure Polar/charged amino acids = low hydrophobicity and low driving force for protein compaction/hydrophobic core formation = High net charge so electrostatic repulsion Contain residues that break secondary structure (ex. proline) Look at diagram in notes

Question 87

What different structural techniques can or can't be used to analyse IDPs?

Answer 87

Crystallography needs crystals IDP's don't crystallise Proteins are too flexible for EM NMR is used to see behaviour of amino acids in solution

Question 88

What are unfolded Vs misfolded proteins

Answer 88

Unfolded: random coil, premolten globule, molten globule Misfolded: amyloid-like fibrils made out of off-pathway interactions

Question 89

How is the structure of PrP characterised by NMR

Answer 89

PrP: Unstructured N-terminal, folded C-terminal Helical structure is clear Don't see N-terminal residual structure (small regions of beta strands and polyproline) in NMR

Question 89

How is NMR used to characterise IDPs?

Answer 89

IDP's can be recognized in a H1-N15 HSQC spectrum Each peak corresponds to amino acid in a particular chemical environment Limited dispersion: Amino acids in protein are very flexible and are not in a unique chemical environment Sharp peaks: long T2 relaxation NMR can identify residual secondary structure in IDPs Ex. Polyprolines, residual beta strands, residual alpha helices Look at image in notes

Question 90

How is CD used to characterise IDPs?

Answer 90

Circular dichroism: to measure level of structure of IDP Specific spectrum for alpha helix, beta sheet and random coil Plot a double wavelength CD plot Measure at 200nm (minimum for random coil) and 222nm (minimum for alpha helix) Separate pure random coil (green) from residual structure (pre molten globule conformation) (purple) Polyprolines have their own spectrum (similar to RC)

Question 91

Why are IDP interactions difficult to characterise?

Answer 91

Form fuzzy complexes IDPs can bind to more than one place in the protein So very difficult to characterise interaction Complex won't crystallize and no noise in NMR IDP is swarming around surface of folded protein