BC5 old exams

Question 1

How is spontaneous protein folding thought to overcome the Levinthal paradox? An exhaustive search through the vast number of possible conformations (~10¹⁰⁰ conformations for a protein with 100 residues) during protein folding would take ages.

Answer 1

1. Local propensity for secondary structure formation encoded by amino acid sequence, especially alpha-helices and beta-hairpins. These secondary structure elements might also act as a nucleus. (1)
2. Hydrophobic collapse by excluding hydrophobic sidechains from aqueous solvent. This quickly bypasses a very large number of extended conformations. (1)
3. Nucleation at key contacts of the future native structure, for example a beta-helix-beta motif to initiate beta-sheet formation/extension. This would essentially be like a folding trajectory, afterwards folding intermediates become progressively more stable during compaction. (1)

Question 2

Amyloids: (3 points)

1) What are the three biophysical hallmarks of protein amyloids?

2) Explain the mechanism for amyloid formation! What is the rate limiting step, and how can this step be by-passed?

Answer 2

1)

1. Answers:
   
   1. Binding of specific dyes like Congo Red or Thioflavin-T (0.5)
   2. Fibrillar structure in electron microscopy. (0.5)
   3. Cross-beta structure in X-ray fiber diffraction (0.5)

2)

1. Nucleation mechanism: Formation of nucleus, followed by linear extension. Possibly, fragmentation of growing fibers. (0.5)
2. Nucleus formation is rate-limiting. (0.5)
3. Seeding with amyloid fiber fragments (Prion hypothesis). (0.5

Question 3

In which compartments of the eukaryotic cell can “Brownian ratchets” be found? How do to the drive protein translocation? Would this system work to secrete proteins through the bacterial inner membrane? Why or why not? (3 points)

Answer 3

1. ER-Membrane/inner Mitomembrane/(Chloroplast) (1)
2. Hsp70 binds translocating protein and prevents backsliding (Hsp40 recruits Hsp70 to translocating protein). (1)
3. No, no ATP in periplasm.

Question 4

1. Topic: Insertion of membrane proteins (3 points)
   
   1. SNAREs are membrane proteins with a C-terminal transmembrane (TM) domain and a long cytoplasmic N-terminal domain. By which targeting and translocation machinery are these proteins inserted into the ER-membrane? Is SRP involved in the process? Why or why not?

Answer 4

1. The GET/Trc40-system (1)
2. No SRPp, because it recognizes N-terminal signal sequences (2)

Question 5

1. Topic Chaperones: (3 points)
   
   1. Describe the differences between Hsp70 (DnaK) and GroEL in terms of:
      
      1. Substance binding
      2. Consequence of ATP hydrolysis and
      3. Cochaperone action

Answer 5

1. DnaK
   
   1. DnaK binds extended polypeptide stretches with hydrophobic amino acids,
   2. ATP hydrolysis switches DnaK from the low affinity state to the high affinity state,
   3. the cochaperone DnaJ stimulates the ATPase of DnaK
2. GroEL
   
   1. GroEL binds substrates at the apical domain and engulfs them in a cavity, substrate size is limited to 60 kDa,
   2. ATP hydrolysis is the timer for the time the substrate spends inside the cavity,
   3. the cochaperone GroES closes the cavity and induces ADP release from the trans ring.

Question 6

What is the precursor for N-glycosylation? Where is it synthesized and which enzyme (full name) transfers the sugar moiety where in the cell onto which amino acid? (3 points)

Answer 6

1. Dolichol-P-P-polysugar, precursor is synthesized on the ER membrane (flipase), OST: oligosaccharyl-transferase, next to translocon, Asn

Question 7

Please draw the basic reaction catalyzed by all proteases and name three processes in the mammalian cell where this reaction takes place. (3 points)

Answer 7

1. Many different examples, e.g.
2. To degrade proteins:
   
   1. To switch off the signals that peptides and proteins initiate by degrading either them or the proteins they bind to
   2. To recycle amino acids by degrading the proteins.
   3. To destroy potentially lethal or toxic proteins from parasites and pathogens
   4. To release antigenic peptides from parasites and pathogens.
   5. To obtain amino acids from food proteins.
3. To remove the initiating methionine from the newly synthesized, cytoplasmic proteins
4. To remove signal peptides from proteins targeted to the cell’s secretory pathway.
5. To remove targeting signals from proteins targeted to specific organelles such as the mitochondrion or chloroplast.
6. To remove propeptides from enzymes, hormones and receptors that are synthesized as precursors, so that these are activated.
7. To release individual proteins and peptides from polyproteins
8. To release bioactive peptides from protein precursors.
9. Pathogens and parasites also use proteolytic enzymes to invade their hosts, and to inactivate any host protein that could harm them or interfere with their reproduction.

Question 8

1. How does a weak base like Choloquine inhibit lysosomal proteases? (3 points)

Answer 8

1. Lysosomal proteases have an acidic pH optima and are largely inactive at cytosolic pH.
2. Uncharged form of the weak base enters cells and lysosomes.
3. Accumulates in charged form in lysosomes and increases pH, this inhibits the lysosomal protease.

Question 9

1. Draw the tripeptide Lys-Val-Asp (3 points)

Answer 9

Question 10

1. What type of proteases are caspases and what cellular program do the execute? How many active sites are found in an activated caspase? (3 points)

Answer 10

1. Cysteine protease (1)
2. Apoptosis (1)
3. 2 (1)

Question 11

Protein-Folding: Name three biophysical methods for folded proteins. How do they work?

Answer 11

1. hydrophobic collapse model
2. primary and secondary structure interactions

need to come back to this question, not entirely sure what it is asking

Question 12

1. Draw the active site of a Cysteinprotease; Amino acid residues

Answer 12

Question 13

1. GroEL: Function of ATP-binding; Which domain of GroEL binds ATP; What happens during ATP-Hydrolysis

Answer 13

1. ATP binding to the trans ring causes conformational change and
2. After ATP hydrolysis is GroES dissociation and substrate release from the trans ring
3. ATP hydrolysis serves as a timer for the time the substrate spends inside the cage

Question 14

1. 26S-Proteasome: Which subunit contains an ATPase; What is its function

Answer 14

1. 19S regulatory particle
2. Acts as a gatekeeper, recognizes substrate, deubiquitylates it, unfolds it, and translocation of unfolded protein into the core

Question 15

1. What kind of protease is Cathepsin B. Where does it occur in the cell? What kind of proteases are Caspases 3/7/8/9. In what pathway are they involved?

Answer 15

1. Apoptotic pathway
2. Caspases are cysteine proteases in apoptosis
3. and Cathepsin B as a cysteine protease found in the lysosome

Question 16

1. Draw the dipeptide KE at pH7.0

Answer 16

find good image for this

Question 17

1. Cotranslational protein transport in eukaryotes: Typical signal sequence/structure of the signal sequence; name two interaction partners of the signal sequence; how do they interact?

Answer 17

1. Signal sequence (ER import) = N-terminal hydrophobic sequence
2. Signal sequence recognized by Signal recognition particle (SRP), stopping translation
3. SRP brings ribosome to the ER membrane, where it binds to the SRP receptor (SR) which it bound to the ER membrane.
4. Signal sequence is then transferred to the Sec-complex (the major conducting channel - translocon), leading to GTP hydrolysis and dissociation of the SRP from the SR.

Question 18

Properties of proteins bound by Calnexin/Calreticulin; How can this proteins be released?

Answer 18

1. Are lectins
2. Calnexin/calreticulin bind to incompletely folded proteins containing one terminal glucose on N-linked Oligosaccharrides -> traps them in the ER
3. Glucosidase removes terminal glucose -> protein released from calnexin

Question 19

In which transport pathways are they following GTPases involved: Ran/Rab/NSF/SRP/Arf1/SecA

Answer 19

1. Ran: involved in gated transport through the nuclear envelope
   
   1. import: dissociates cargo from import receptor
   2. export: Enables cargo binding to the export receptor
2. Rab: Vesicle transport pathway
   
   1. Coordinators of vesicle transport
   2. Rab-GTPases recruit Rab effector proteins
   3. These attract membrane specific carriers
3. NSF: Vesicle Transport from ER-Golgi
   
   1. NSF dissociates Cis-SNARE complex
4. SRP: Signal recognition particle involved in co-translational translocation through the ER membrane in the Sec Pathway.
5. Arf1: vesicular transport
   
   1. Arf1 localized to golgi and has central role in intra-Golgi transport
   2. Arf family plays role in vesicular trafficking as activators of phospholipase D
6. SecA: Posttranslation translocation in Secretory pathway in bacteria

Question 20

Amyloid structures: Different phases of the nucleation growth mechanism; What happens during the different phases?

Answer 20

1. Have a solution of monomers for amyloid
2. Trigger amyloid formation via shift in pH
3. Lag phase –
   
   1. very little is happening in this phase as monomers are very slowly coming together and nucleus is formed
   2. is energetically unfavorable
4. Fibril growth:
   
   1. Start growing by adding monomers until pool of monomers depleted
5. At higher concentrations of monomers, the lag time is shorter and maximimum fibril formation peak is higher
6. Nucleation is rate limiting step
7. Can by-pass nucleation phase by seeding with preformed aggregates

Question 21

Which biophysical methods are suitable to study protein folding at the single molecule level?

Answer 21

1. Single molecule force microscopy
   
   1. Optical tweezers or AFM
   2. Can be used to look at interactions and forces between individual molecules
2. smFRET – single molecule fluorescence resonance transfer
   
   1. normal FRET can be used to describe energy transfer between two light sensitive molecules
      
      1. and if 2 fluorophores are within certain distance of each other
   2. smFRET can be used to check the migration of SRP-SR-GTPase complex on SRP-RNA
   3. measuring high and low FRET states

Question 22

What are the hallmarks of cystic fibrosis: Cause of the disease, pathophysiology (molecular – cellular – organismal)? How are the new drugs VX-770 and VX-809 thought to work?

Answer 22

1. A loss of function and improper trafficking disease
2. Caused by mutations in gene for CTFR
   
   1. Is an ABC transporter-class transmembrane ion channel protein
      
      1. Transports chloride ions across epithelial membranes
   2. WT CFTR is present, and ΔF508 CFTR is absent from apical surface of ciliated airways cells
3. Disease causes difficulties breathing
4. The drugs:
   
   1. VX-770
      
      1. For CFTR mutation G551D
      2. Improves transport of chloride through the ion channel
   2. VX-809
      
      1. For CFTR mutation ΔF508

Question 23

1. Topic: Co-translational protein targeting at the endoplasmic reticulum (ER) membrane. Two GTPases coordinate translation of a secretory protein with its translocation through the ER-membrane.
   
   1. Give the name of these two GTPases.
   2. What triggers GTP-binding to these GTPases?
   3. What is the specific (biochemical) consequence of GTP binding to these GTPases?

Answer 23

1. Give the name of these two GTPases.
   
   1. SRP – signal recognition particle
   2. SR – SRP receptor
2. What triggers GTP-binding to these GTPases?
   
   1. The binding of the M domain of SRP to signal sequence increase affinity of the G domain of SRP for GTP
   2. Docking of targeting complex to the ER membrane causes allows signal sequence to be transferred to the SEC complex leading to GTP hydrolysis activity and dissociation of SRP and SR
3. What is the specific (biochemical) consequence of GTP binding to these GTPases?
   
   1. GTP binding to SRP stops translation and SRP brings ribosome to ER membrane and SR
   2. Binding to SR allows translocation of nascent polypeptide through translocon into ER and dissociation of SRP and SR.
      
      1. Not sure if this answer is what they are looking for or not.

Question 24

1. Topic: Membrane protein insertion into the ER membrane How do tail-anchored membrane proteins distinguish from type I membrane proteins concerning
   
   1. Topology?
   2. Targeting pathway to the ER membrane?
   3. Machinery required for insertion into the ER membrane?

Answer 24

1. Topology?
   
   1. Tail anchored proteins
      
      1. Transmembrane protein
      2. Long N terminal tail in cytosol, C-terminal in ER lumen
      3. Transmembrane domain in ER membrane
   2. Type I
      
      1. Transmembrane protein
      2. N terminal in ER lumen, C terminal in cytsosol
      3. Transmembrane domain in ER-membrane
2. Targeting pathway to the ER membrane?
   
   1. Tail anchored
      
      1. Post translational Get-pathway
      2. Pre-targeting complex captures the transmembrane domain (TMD)
      3. TMD is loaded onto main targeting factor (TRC40 in mammals, Get3 in yeast) and targeted to ER-membrane
   2. Type I
      
      1. Co-translational – SRP-pathway
      2. SRP stops translation after binding to signal sequence
      3. Brings ribosome and nascent polypeptide to ER membrane
      4. TMD exits ribosome (has already had chance to pre-fold) and enters cytoplasmic vestibule of Sec61 where it samples orientation
      5. Becomes type I or II depending on hydrophobicity, charges, helicity
      6. Type I is non-loop inser, type II is loop insert
3. Machinery required for insertion into the ER membrane?
   
   1. Tail anchored:
      
      1. Interaction with Get1-Get2 receptor and ATP hydrolysis -> release into bilayer and Get3 recycling
   2. Type I:
      
      1. Cotranslational
      2. Uses SRP and SR receptor
      3. Orientation sampled at sec61

Question 25

1. Please describe the catalytic mechanism of threonine proteases. Why does it need an N-terminal threonine?

Answer 25

1. Use the secondary alcohol of their N-terminal threonine as a nucleophile to perform catalysis
2. Threonine must be N-terminal since the terminal amine of the same residue acts as a general bae by polarizing an ordered water which deprotonates the alcohol to increase its reactivity as a nucleophile
   
   1. So there must be a free terminal amino, so there can’t be another residue linked on that side

Question 26

1. How can a cell actively manage protein aggregation once it occurred?

Answer 26

1. Disaggregation via chaperones
2. Ubitiqutylation and autophagy via the ubiquitin proteasome system

Question 27

1. Topic: General protein translocation Name three protein translocation/insertion machineries located in the bacterial inner membrane. Which of these proteins has a homolog in mitochondria? What is its name in mitochondria?

Answer 27

1. Sec
2. Tat – Twin arginine transport system
3. YidC – homolog of mitochondrial Oxa1

Question 28

1. Topic: Structure of the Sec-complex Name three prominent features in the structure of the Sec complex and briefly outline their proposed functional role.

Answer 28

1. Conserved across kingdoms, the Sec complex is the major protein conducting channel for translocation of periplasmic proteins and insertion of inner membrane proteins: translocates unfolded proteins
2. Consists of α, β, and γ components (sec YEG in E. coli)
3. Binds signal sequence, ribosome, and/or SecA as well as additional factors such as signal peptidase, OST, and chaperones (Hsp70, BiP)
4. Can do verticle gating for secretory proteins
5. Lateral gating for transmembrane segments
6. Maintains ion permeability berrier
7. Structure:
   
   1. Central “plug” serves as seal to prevent diffusion of proteins and ions
   2. Hourglass-shpaed aqueous funnel with central constriction (“pore-ring”)
   3. Opening on front side called “lateral gate”
8. Model: binding of signal sequence could induce a movement of the plug, leaving a narrow pore ring for passage of nascent polypeptide chain.

Question 29

1. Topic: Membrane proteins Briefly describe a method to reconstitute a purified membrane protein (e.g. the SecYEG complex) in vitro into an artificial lipid bilayer.

Answer 29

1. Proteoliposomes formation
2. Destabilize liposomes with detergent
3. Add purified membrane protein
4. Remove detergent with something like biobeads
5. Will result in a proteoliposome