Protein Folding and Quality Control

Do secondary protein structures arise spontaneously or require assistance?

Spontaneously

Do tertiary protein structures arise spontaneously or require assistance?

Often require assistance

In a cytoplasmic or periplasmic protein, would you expect hydrophobic amino acid residues (like leucine, valine, tryptophan, etc.) to be exposed on the surface or bruied in the interior of the mature folded protein?

Buried in the interior

Proteins acquire their tertiary structure through…

folding intermediates such as molten globules

Some molten globules can be trapped in

“off-pathway” structures

What do proteins need if they don’t get their folding right the first time?

need help to fold into their correct conformation

• folding/unfolding

• enhancing nucleation

Chaperones can act as

“holdases” to stabilize non-native protein conformations

“foldases” to assist proteins in folding to the native state

“unfoldases” to unfold misfolded proteins or extract proteins from aggregates

Substrate holding is generally energy-independent or dependent?

energy-independent

Active assistance of protein folding is energy-independent or dependent?

Active assistance of protein folding often requires energy (typically ATP hydrolysis)

What is Trigger Factor (TF)?

a ribosome-associated chaperone in bacteria that engages the nascent polypeptide as it exits the ribosome and assists in its initial folding

After release from TF, polyptide either…

folds spontaneously (~70% of cytosolic proteins in E. coli under normal growth conditions) or requires further folding-assistance by downstream chaperones - DnaJ-DnaK-GrpE (KJE system) and/or GroEL-GroES (ELS system)

Why is TF not essential?

Its loss is compensated by induction of the heat shock response with enhanced KJE and ELS action

The absence of both TF and DnaK…

is lethal at temperatures >30 deg C, but can be partially suppressed by overexpression of the ELS systems of SacB

What is L23 and SRP?

The ribosomal protein L23 is the binding site for both TF and the signal recognition particle (SRP)

TF and SRP can co-exist at the ribosome where they likely compete for nascent polypeptide chain

What are two more essential enzymes that act on polypeptide chains as they exit the ribosome?

Peptide deformylase (PDF) and methionine aminopeptidase (MAP)

What do PDFand MAP do?

PDF: removes the formyl group from the f-Met at the N-terminus of the nascent polypeptide

MAP: removes methionine residue

What is DnaK?

ATPase that unfolds proteins

Hsp70 homolog

What is DnaJ?

co-chaperone for DnaK

Hsp40 homolog

What is GrpE?

Nucleotide exchange factor for DnaK

The KJE system can function in either…

co-translational or post-translational mode

What is GroEL?

cage-like structure, requires GroES for activty

Double-ring barrel structure

Hsp60 homolog

What is GroES?

“lid” for GroEL

Hsp10 homolog

What are examples of chaperonins?

GroEL and GroES

The ELS system functions only in a…

post-translational mode

First step in the KJE system

DnaJ binds to the unfolded or partially folded protein and then to DnaK

Second step in the KJE system

DnaJ stimulates ATP hydrolysis by DnaK. DnaK-ADP binds tightly to the unfolded protein

Third step in the KJE system

In bacteria, the nucleotide-exchange factor GrpE stimulates release of ADP

EF-Ts is another place where we have seen a nt-exchange factor before

Fourth step in the KJE system

ATP binds to DnaK and the complex dissociates from the protein

First step in the GroEL-GroES reaction cycle

Non-native substrate protein enters GroEL reaction cycle on the open trans heptameric GroEL ring

Second step in the GroEL-GroES reaction cycle

Substrate protein binding stimulates release of ADP from the trans ring and ATP hydrolysis in the opposite cis ring

Third step in the GroEL-GroES reaction cycle

Binding of the substrate protein with the GroEL cavity results in unfolding of the substrate protein. Cavity is lined with hydrophobic amino acids that interact with surface exposed hydrophobic residues on substrate protein

Fourth step in the GroEL-GroES reaction cycle

GroES binding causes conformational change in the trans ring (forms a folding cavity) that is coupled with disassembly of folding cavity on the opposite ring - release of GroES and folded substrate protein

Fifth step in the GroEL-GroES reaction cycle

Many of the hydrophobic amino acids that lined the cavity of the trans ring are buried in the folding cavity (the cavity is more hydrophilic), which forces the substrate protein into native, folded conformation. The opposite GroEL ring is now ready to receive non-native substrate protein

Structure of GroEL ring in the trans conformation

Helix H and Helix I expose multiple hydrophobic amino acids towards the center of the ring and form a surface for the binding of non-native substrate proteins

Structure of GroEL ring in the cis confomation

Helix H and Helix I are no longer exposed to the central cavity and contact the mobile loops of GroES

What is the protease complex?

Barrel-shaped protease complex that catalyzes the proteolysis of proteins targeted for degradation

In bacteria, the proteasome recognizes egradation signals that are:

normally buried inside proteins and indicate an incorrectly folded protein if exposed on surface

amino acid sequences at the N- or C-terminus of protein (“degrons” - regulatory signals)

In eukaryotes, proteins are target for degradation through a specific posttranslational modification by a small protein called

ubiquitin

What is sampylation

archaea use a ubiquitin-like modification system (attach a small archaeal modifier protein [SAMP] to target protein)

Actinobacteria target some proteins for degradation by attaching a

prokaryotic ubiquitin-like protein (Pup - pupylation)

Bacterial proteasome consists of a…

chaperone (ClpX and others)

• ATPase, unfolds protein and delivers protein to

ClpP (or ClpQ) protease

• does the dirty work (peptidase)

• barrel-shaped structure

ClpP Proteasome Structure

• Central barrel formed by two heptameric rings of ClpP or ClpQ (protease domain)

• A hexameric ATPase cap that recognizes, binds, and unforlds target protein substrates for entry into the protease chamber

Binding of the cap hexamer to the ClpP ring is stabilized by…

docking of the hydrophobic ClpP-loops on the cap proteins into hydrophobic clefts on the surface of the ClpP ring

The different cap hexamers have different substrate binding properties

The various cap hexamers recognize different sequences (degrons or degradation tags) in the N- or C-terminus of proteins targed for degradation (often inherently short-lived proteins)

SspB assists in…

delivering ssrA-tagged proteins to ClpXP for degradation

What is the purpose of targeted degradation of specific proteins?

• Degrade incomplete or damaged proteins

• Cell stress response proteins are often tagged with degrons

• Turnover of proteins under conditions where they are not needed (ex. ClpXP degrades Fnr repressor under aerobic growth conditions

• Toxin-antitoxin modules - operon that encodes a stable toxin and an unstable antitoxin - addiction modules on certain plasmids that ensure their retention in a population

Acyldepsipeptide antibiotics stimulate unregulated proteolysis by the Clp protease system

ClpP is unable to degrade proteins by itself

Clp-ATPase recognizes protein substrates marked for degradation and then dock with ClpP. Clp-ATPase unfolds protein substrate and threads it into catalytic chamber of ClpP

ADEP bind ClpP, which results in opening of axial pores. Also prevents binding of Clp_ATPase to ClpP, which prevents degradation of the natural protein susbtrates. In addition, some nascent polypeptide chaines are not degraded (degradation tags are not required for ADEP-mediated protein degradation)

What is the Lon cytoplasmic protease?

• Funcations as a multimer of a single subunit (unlike Clp)

• Involved primarily in “quality control” and degradation of proteins unfolded by heat shock or other stresses, but also selectively degrades some short-lived regulatory proteins

• Recognize degrons

Lon structure

Lon forms homohexamers that can degrade both large and small substrates, and a dodecamer (12 subunits) that is active only on small substrates - may be a mechanism to regulate the stubstrate specificity of Lon under different conditions

Requires ATP hydrolysis for function

Protein folding and degradation in the periplasm

• Periplasmic proteins may be transported by one of two transport systems (Sec for unfolded and Tat for folded state)

• need different systems for protein folding (chaperones) and degradation (proteases) in the periplasmic space

In almost all peptide bonds, which conformation is favored and why?

the trans confomration is favored because the steric hinderance between the R-groups of adjacent amino acids is lowest in this conformation

In peptidyl-prolyl bonds, the difference in free energy for the trans and cis conformations is…

much smaller. Thus ~5-6% of peptidyl-prolyl bonds are in the cis confomation but is determined by contextual determinants

Isomerization of proline residues is often…

the rate-limiting step in protein folding

What are peptidyl-prolyl cis-trans isomerase (PPIases)?

they convert proline trans and cis isomers to assist proteins to fold correctly

PPIases are also found in the cytoplasm as soluble proteins, and some are integral membrane protiens

Four periplasmic PPIases have been identified in E. coli

First step in disulfide bond formation and isomerization in E. coli periplasm

DsbA introduces disulfide bonds between cysteine residues in periplasmic proteins secreted by the Sec translocon

Second step in disulfide bond formation and isomerization in E. coli periplasm

Electrons from the oxidation of the cysteins are transferred from DsbA to DsbB, which then transfers the electrons to respiratory electron transport chain

Third step in disulfide bond formation and isomerization in E. coli periplasm

DsbC reduces incorrectly formed disulfide bonds in the protein substrate - recognizes exposed hydrophobic patches on protein substrate. Allows DsbA a second chance to get disulfide bond formation correct

Fourth step in disulfide bond formation and isomerization in E. coli periplasm

DsbC gets electrons for the disulfide bond reduction from DsbD (in inner membrane), which gets electrons from thioredoxin (a small redox protein) in the cytoplasm

What is DegP?

the major periplasmic protease in E. coli that is essential for survival at elevated temperatures

• has uncommon feature that it also functions as a chaperone - escorts unfolded proteins across the periplasmic space to the outer membrane for their incorporation into or secretion across the outer membrane

• DegP functions primarily as a chaperone at temperatures below 28 deg C, and the protease activity increases with increasing temperature

• Binding of misfolded protein substrate to DegP transforms it form protease inactive form (2 trimers) to protease active form (4 or 8 timers)

• DegP does not require ATP for either of its activities

What is the heat shock response?

• A rapid, transient protective mechanism triggered by stress (ex. heat, toxins) that increases levels of chaperones and proteases to protect the cell from protein aggregation - either refold or degrade the unfolded proteins

How is the heat shock response achieved?

Primarily by elevating the amount of sigma-32 (RpoH) to increase expression of heat shock proteins (ex. DnaK, DnaJ, GroEL/GroES, Clp proteins, Lon, etc)

Levels of sigma-32 are also controlled by DnaK/DnaJ , which binds sigma-32 and renders it sensitive to degradation by the protease FtsH

Under stress response, unfolded proteins accumulate in the cytoplasm to…

titrate DnaK/DnaJ and prevent them from promoting the degradation of sigma-32. Allows sigma-32-RNA polymerase holoenzyme to form and initate transcription of the heat shock genes - acts as a feedback loop

What helps prolong the heat shock response?

Adenyl dinucleotide alarmones synthesized by some of the aminoacyl-tRNA synthetases inhibit DnaK, which prevents DnaK/DnaJ from promoting the degration of sigma-32

Within the periplasm, DegP transitions from a…..

chaperone to a protease upon heat shock. Unlike the cytoplasmic chaperones and proteases though, the expression of degP is not sigma-32-dependent; but rather a different alternative sigma factor (sigma-24)

Ubiquitination of protein substrates involves…

three separate enzymes (E1, E2, and E3) - E1 (Ub activating enzme) uses ATP to activate Ub in a two-step process - adenylation (forms an Ub-AMP intermediate) followed by transfer of Ub to an actve site cysteine in E1 to form a thioester

What happens to the resulting activated Ub?

Is then transferred to E2 (conjugating enzyme), which functions with E3 (Ub ligase) to transfer Ub to target protein - either single Ub molecule or a chain of Ub molecules. Humans have 2 E1 enzymes, ~40 E2 enzymes, and >600 E3 enzymes

Monoubiquitination typically acts as a…

signal for non-protetolytic processes (protein trafficking)

Polyubiquitination is required for…

degradation by the Ub-proteasome pathway

Proteins are rapidly degraded by the 26S proteasome - composed by central barrel-shaped proteasome (ClpP homolog) and regulatory ATPases (ClpX-like protein) that unfold and inject the protein substrate into the central core of the proteasome

Deubiquitinating enymzes remove

Ub chains from susbtrate and break down into Ub monomers for recycling