MICR5831 L8: Protein Transport and Folding 8/6/25 NEEDS SAQ

How do proteins self-fold? (slide 4)

1. Spontaneous Folding:

2. Driving Forces (hydrogen bonds, VDW, hydrophobic)

3. Role of Molecular Chaperones

4. Folding Pathways

Which chaperone is required to associate with a ribosome to initiate self-folding? How does it work? (slide 6)

-Trigger Factor (TF)

-Binds to the ribosome's exit tunnel

-Interacts with the nascent polypeptide chain as it emerges

-Helps to prevent aggregation and promote proper folding.

What are the chaperones and steps in the protein disaggregation pathway? (slide 7)

SAQ

What macromolecular machine is required for protein re-folding? What are the components of this machine? (slide 8)

SAQ

How does the GroES/EL chaperone result in protein refolding? (slide 9)

SAQ

What are the common characteristics of integral membrane proteins? (slide 11)

SAQ

Describe the steps in the signal recognition particle pathway for protein transport. (slide 12)

SAQ

Describe the steps in the Sec-dependent protein transport pathway for bacterial proteins. (slide 13)

SAQ

Describe the steps in the twin arginine transport pathway for bacterial proteins (slide 14)

SAQ

What is the role of oxidoreductases in bacterial protein folding. How do they operate? (slide 15)

SAQ

How are outer membrane proteins exported to the outer membrane of Gram-negative bacteria? (slide 16)

SAQ

Describe the components of the protein degradation pathway and how the pathway works (slide 20)

SAQ

True or False: Many proteins do not require extra energy input to fold by themselves

True

True or False: The intracellular environment of the cytoplasm is mostly empty space with a few proteins

False, it is very crowded (300-400 mg/mL protein)

True or False: When they are synthesized, most proteins will be unfolded/inactive and made incorrect contacts with other proteins

True

What determines the 3D structure of a protein?

-Linear sequence of amino acid residues

-Hierarchical folding pattern

What is the first step of a hierarchical protein folding pattern?

Hydrophobic Collapse:

-Snapping" of protein into a compact globular structure

-Get hydrophobic residues away from water

-Formation of secondary structural elements

What happens after this step during protein folding?

1) Hydrophobic collapse to get away from water

-Secondary structures form in milliseconds

-Foci for flexible sections to naturally fold around

-Molten globule forms

What happens after this step during protein folding?

2) Secondary structures form foci for flexible sections to fold around and create molten globules

3) Side chains undergo movement to attain the final "native conformation"

True or False: Protein folding does not require any extra energy in the form of ATP from the cell

True

True or False: Native conformation of most proteins requires the most energy to maintain and therefore is unstable

False

True or False: Being in a misfolded conformation is a high energy state yet proteins can become stuck in it

True

What do you use to rescue misfolded proteins?

Chaperones

What is this?

-Chaperones

-Associate with target proteins then dissociate after target protein is completely folded

-Originally identified as proteins produced in response to cellular stress

What are some examples of cellular stress that might cause proteins to unfold?

-Temperature/pH changes

-Exposes hydrophobic amino acids which clump together and form aggregates

What happens to 70% of proteins after they are released from a ribosome?

Fold themselves into low energy native conformation state

What is this?

-Trigger Factor (TF)

-Peptidyl-prolyl cis-trans isomerase (PPlase)

-Binds to proteins emerging from the ribosome

-Speeds up proline isomerization from trans to the cis

True or False: Using Trigger Factor (TF) to speed up proline isomerization (trans to cis) requires ATP for energy

False

True or False: If a protein unfolds due to heat stress, a chaperone is required to refold it

True

What are some chaperones that exist in the cytoplasm and are used by 30% of proteins to reach native conformation?

DnaK/DnaJ/GroES/EL complex

What are some chaperones that exist in the periplasm and are used by 30% of proteins to reach native conformation?

-PPlases

-Oxidoreductases

Describe the Disaggregation pathway that occurs with DnaK, DnaJ and ClpB (20% of proteins)

1) DnaK (Hsp70) clamps onto insoluble aggregates, recruits DnaJ

2) DnaK and DnaJ use ATP to refold short nascent peptide

3) Nascent peptide is recognised by ClpB foldase

4) ClpB binds the protein and DnaK/DnaJ complex is released

5) ClpB uses ATP to continue re-folding

6) Native protein is released from ClpB

7) Medium proteins with a complex structure

What are the first 3 steps of the Disaggregation pathway with DnaK, DnaJ and ClpB?

1) DnaK (Hsp70) clamps onto insoluble aggregates, recruits DnaJ

2) DnaK and DnaJ use ATP to refold short nascent peptide

3) Nascent peptide is recognised by ClpB foldase

What are the last 4 steps of the Disaggregation pathway with DnaK, DnaJ and ClpB?

4) ClpB binds the protein and DnaK/DnaJ complex is released

5) ClpB uses ATP to continue re-folding

6) Native protein is released from ClpB

7) Medium proteins with a complex structure

What is this?

-ClpB (Hsp100)

-Foldase that uses ATP to refold proteins

-Recognizes nascent peptipe and binds to it

-Releases DNaK/DnaJ complex before refolding

What is this?

-DnaK (Hsp70)

-Found in cytoplasm

-Clamps onto insoluble aggregates

-Recruits DnaJ (Hsp40)

-Uses ATP to refold short nascent peptide

-Released when ClpB binds to complex

What is this?

-DnaJ

-Found in cytoplasm

-Recruited by DnaK (Hsp70)

-Uses ATP to refold short nascent peptide

-Released when ClpB binds to complex

What type of proteins can use the DnaK, DnaJ, and ClpB Disaggregation pathway?

Medium length proteins w/ complex structure

What is different about the Disaggregation pathway with ClpB for very long proteins with complex tertiary structures?

-ClpB release partially folded protein

-Protein enters GroES/EL complex to complete folding

Describe the Refolding pathway that occurs with the GroES and GroEL complex

1) Misfolded proteins bind to the exposed hydrophobic face of the GroEL ring

2) GroES cap is attached to the top

3) Cylinder conformational change with hydrophilic face towards the center

4) Binding of 7 ATP molecules to the GroEL ring 1 triggers conformational change that results in slight twist and tilt in the subunits and in exposure of hydrophobic patches that interact with and help to unfold misfolded protein.

5) 14 ADP and GroES is released from ring 2 and ATP and GroES bind to ring 1- and the shifts are repeated

6) Thus, at any given time one of the rings has ATP and GroES bound and the other is empty

What are the first 3 steps of the Refolding pathway with GrosEL/GrosEL complex?

1) Misfolded proteins bind to the exposed hydrophobic face of the GroEL ring

2) GroES cap is attached to the top

3) Cylinder conformational change with hydrophilic face towards the center

What are the last 3 steps of the Refolding pathway with GrosEL/GrosEL complex?

4) Binding of 7 ATP molecules to the GroEL ring 1 triggers conformational change that results in slight twist and tilt in the subunits and in exposure of hydrophobic patches that interact with and help to unfold misfolded protein.

5) 14 ADP and GroES is released from ring 2 and ATP and GroES bind to ring 1- and the shifts are repeated

6) Thus, at any given time one of the rings has ATP and GroES bound and the other is empty

What is this?

-GroEL-GroES Chaperonin Machine

-Nano-cage for protein folding

-Partially folded proteins will leave ClpB to enter it if they are too long and complex for the disaggregation pathway

What is this?

-GroEL

-Hydrophobic cylindrical structure

-7 subunits in 2 stacked rings

-Misfolded proteins attach to exposed face

-7 ATP bind to it after conformational change

-Exposes more hydrophobic patches to fix misfolded protein

What is this?

-GroES

-7 subunits that form a cap attached to top of GroEL ring

-Binds to GroEL ring, causing conformational change

-Released with 14 ADP from ring 2, then binds with ATP to Ring 1 repatedly

How does the GroEL ring 1 help unfold a misfolded protein after 7 ATP bind to it?

-Triggers conformational change

-Subunits twist and tilt

-Expose hydrophobic patches that can unfold the misfolded protein

True or False: At any given time during the refolding pathway, one of the GroEL rings will have ATP and GrosES bound to it but the other will be completely empty

True

What are the 3 pathways for protein selection and transport to the inner membrane/periplasm?

1) Signal Recognition Particle (SRP) co-translational pathway

2) Sec-dependent general secretion pathway

3) Twin arginine transport (TAT) pathway

What are these selection/transportation pathways best used for?

1) Signal Recognition Particle (SRP)

2) Sec-dependent general secretion

3) Twin arginine transport (TAT) pathway

1) Integral membrane proteins

2) Unfolded proteins into periplasm

3) Folded proteins into the periplasm

What are these selection/transportation pathway do these proteins use?

1) Integral membrane proteins

2) Unfolded proteins into periplasm

3) Folded proteins into the periplasm

1) Signal Recognition Particle (SRP)

2) Sec-dependent general secretion

3) Twin arginine transport (TAT) pathway

What enzymes are responsible for folding incomplete proteins that use the Sec-dependent general secretion to enter the periplasm?

-PPlases

-Oxidoreductases

What special structural features could these integral membrane proteins have?

1) Integral membrane proteins

2) Integral monotropic proteins

1) Hydrophobic domains that sit inside the phospholipid bilayer (alpha helices)- single, multipass

2) Hydrophobic domain that strongly associates with the lipids but does not cross the membrane

What special structural features could these integral membrane proteins have?

1) Peripheral membrane proteins

2) Lipid anchored proteins

1) Hydrophilic surface that binds to the hydrophilic headgroups of phospholipids

2) Tagged w/ lipid anchor (isoprenol) at one end

What happens during SRP Co-translational pathway?

1) Proteins that are destined for the inner membrane have a 15-30 amino acid hydrophobic sequence at the Nterminus

2) Signal sequences emerge from the ribosome

3) Ffh protein complexed with sRNA binds to signal sequence

4) FtsY protein binds to complex in the cytoplasmic membrane

5) Protein is either directly inserted/translated into membrane or sent to Sec system via FtsY

How do you tell if proteins are destined for the Inner Membrane?

-Signal sequence of 15-30 amino acids

-Hydrophobic sequence at the N-terminus

What binds to signal sequences as they emerge from the ribosome?

-Ffh protein

-Complexed with sRNA

What is this?

-Ffh protein

-Forms complex with sRNA

-Binds to signal sequences for proteins emerging from ribosome

What happens to the Ffh and sRNA protein complex that binds to a signal sequence emerging from a ribosome?

Binds to FtsY protein in cytoplasmic membrane

What is this?

-Protein found in cytoplasmic membrane

-Binds to the Ffh/sRNA protein complex for signal sequences

-Can carry protein to Sec system if protein is completely synthesized

FtsY

What happens after a signal sequence complex binds to FtsY in the cytoplasmic membrane?

1) Protein is directly inserted into the membrane as it is translated

2) Protein is completely synthesized and FtsY carries it to the Sec system for secretion

True or False: Protein translation occurs completely in the cytoplasm

True

What happens during Sec-dependent export to the periplasm?

1) 1. Trigger Factor allows ribosome to complete synthesizing the presecretion protein.

2) Protein wraps around the pilot protein SecB.

3) The protein is delivered to SecA and SecYEG.

4) The protein is pushed through the SecYEG channel to the periplasm. ATP hydrolysis is required.

5) Sec A repeatedly releases the protein, withdraws, and pushes more of the protein through SecYEG.

6) LepB cleaves the signal sequence. Periplasmic chaperones fold the protein.

What does Trigger Factor do in the Sec-dependent export to the periplasm?

Allows ribosome to complete presecretion protein synthesis

What is this?

-Piloting protein

-Captures unfolded protein after it is completely translated

-Delivers unfolded protein to SecA

SecB

What is this?

-ATPase that uses ATP to push unfolded protein through pore

-SecB delivers unfolded protein to it

-Forms inner membrane channel complex with SecYEG

-Pushes protein into the periplasm

SecA

What is this?

-Forms channel complex with SecA across inner membrane

-Allows SecA to push unfolded proteins into periplasm

SecYEG

Where does SecA get the energy to push an unfolded protein through a pore into the periplasm?

ATP

How can proteins that are destined for the periplasm be distinguished?

-Cleavable signal sequence

-LepB signal peptidase removes it before release

What is this?

-Signal peptidase that removes the signal sequence from proteins destined for the periplasm

LepB

What happens after the signal sequence for a protein destined for the periplasm is removed by signal peptidase LepB?

Proteins are released into periplasm to be folded by periplasmic chapterones

What chaperones are responsible for completion of protein folding in the periplasm?

1) Peptidyl prolyl cis trans isomerases

2) Oxidoreductases

What is this?

-Chaperone responsible for folding proteins in periplasm

-Includes SurA and Skp

-Similar function to cytoplasmic Trigger Factor

-Bind to Outer Membrane proteins and fold them

Peptidyl prolyl cis trans isomerases

What is this?

-Chaperone responsible for folding proteins in periplasm

-Normally has a disulfide bond with DsbB

-Introduces disulfide bonds between cysteines, is reduced

-Re-oxidized by DsbB

Oxidoreductases (DsbA)

What is this?

-Oxidizes DsBA in the inner membrane

-Re-oxidizes DsbB every time it becomes reduced from donating a disulfide bond

DsbB

What happens after an Oxidoreductase like DsbA introduces a disulfide bond between cysteines into the correct position?

-DsbA is reduced after donating the disulfide bond

-DsbA is reoxidized by DsbB

True or False: The periplasm is a strongly reductive environment

False

True or False: The periplasm is a strongly oxidizing environment, therefore cysteines will become easily form covalent disulfide bonds if close together

True

True or False: If disulfide bonds are not very stable, therefore if they form in the wrong place they can inactivate the protein

False, disulfide bonds can inactivate the protein in the wrong spot but are very stable

What feature do Outer Membrane proteins tend to have?

-Large beta barrel structures

-Huge protein that forms a pore

-Hydrophobic outside, hydrophilic core

What is the advantage of the OM beta barrel structure having a hydrophobic outside and hydrophilic core?

Hydrophobic Outside:

-Can sit in the phospholipid membrane

Hydrophilic Core:

-Can transport molecules in the aqueous phase

True or False: Outer Membranes are translated in a folded state in the cytoplasm

False

What happens after unfolded Outer Membrane proteins are translated in the cytoplasm?

Signal sequence is guided to Sec transporter

What happens after unfolded Outer Membrane proteins leave the Sec translocon?

-SurA and Skp bind to the protein and fold it

-Guide protein across periplasm to Bam complex

-Flip protein into Outer Membrane

What do these macromolecular secretion systems do?

1) RND pumps and ABC transporters remove toxins

2) Type III and Type IV systems Inject proteins into plant/animal cells

How do you keep a low level of certain proteins?

-Degradation signals (degrons) dictate protein stability

-Each protein has a given half-life

What is this?

-Degron

Degradation signal that dictates protein stability

What is this?

-N terminal rule

-N terminus (beginning amino acid of protein) is directly correlated to stability

-Arg, Lys, Phe: Short half-life (2 min)

-Asp, Cys, Gly: Long half-life (10 hours)

What half life would a protein beginning with these N-terminal amino acids have?

1) Arginine

2) Lysine

3) Phenylalanine

Short (Two minutes)

What half life would a protein beginning with these N-terminal amino acids have?

1) Aspartic Acid

2) Glutamic Acid

3) Cysteine

Long (10 hours)

What is this?

-Degrades abnormal proteins into smaller and smaller pieces

Proteases

What are the steps in the Proteasome degradation pathway?

1) DnaK and DnaJ chaperones bind to nascent protein.

2) Protein is folded directly to an active form.

3) If not folded, feed to GroEL for refolding.

4) If not folded, protein may be passed on to a protease for degradation.

What is this?

-Contains proteolytic core

-Made of two homoheptameric rings of protein ClpP

Clp

What is this?

-Used in Proteasome degradation pathway

-ATP dependent endoprotease

-Associates with hexamerica ATPase cap

-Uses ATP to unfold and digest the protein

ClpP