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Protein Folding, Processing
and Degradation
Protein Folding
• Protein in native state is not static
– 2° structural elements and domains move
• Function of proteins often dependent on
large conformational changes, triggered
by ligand binding
2
Energy of Folding
• Difference in energy (free energy)
between folded (native) and unfolded
(denatured) state is small, 5-15 kcal/mol.
• Two major contributions to energy
difference between unfolded and folded
state, enthalpy and entropy
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Free energy of folding
• Enthalpy (H) is increased upon folding
•
– Noncovalent interactions are maximized
• Stronger and more frequent in native state
– Enthalpy difference can reach several hundred
kcal/mol
Entropy is decreased upon folding
– Folding causes one main conformation=highly
ordered structure
– Entropy difference can reach several hundred
kcal/mol
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Free energy of folding
• Enthalpy and entropy differences balance each
•
•
other, and DG is a small positive number.
Small DG is necessary because too large a free
energy change would mean a very stable
protein, one that would never change
However, structural flexibility is important to
protein function, and proteins need to be
degraded
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Protein folding
• For any given protein, there is one conformation
•
•
that has significantly lower free energy than any
other state
Achieved through kinetic pathway of unstable
intermediates (not all intermediates are
sampled)
Assisted by chaperones and protein disulfide
isomerases so intermediates are not trapped in a
local low energy state
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Pathways to folding
• There are multiple folding pathways for
some proteins, but a single main pathway
for other proteins
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Transition State Model
• Free energy barrier separates the D (denatured)
•
state from the N (native) state
Folding pathway can vary between proteins
– Can be a single energy barrier with one pathway or
– A single folding pathway that has sequential
transitional states that have limited flexibilities along
the pathway or
– Can have multiple transition states with similar energy
values and a variety of pathways to get to the final
folded state
8
Fig. 6-39,9p.189
10p.190
Fig. 6-40,
Pathways to folded state
• Molten globular is state in between unfolded
and final folded state
– Can be multiple molten globular states for each
protein
• Has helices and sheets, which may be positioned
•
correctly
Unfolded loops, less compaction, different
conformations of surface structures
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fast
Denatured
slow
Molten globular
Native
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13p.191
Fig. 6-41,
Disulfide bond formation
• Cellular environment is reducing
• Cysteines must be oxidized to form S-S
bonds
• Bacteria use Disulfide bridge-forming
protein, Dsb, for oxidation in periplasm
• In eucaryotes, protein disulfide isomerase,
PDI, used for oxidation in the
endoplasmic reticulum (ER)
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Dsb
• Crystal structure shows a thioredoxin-like domain
• Mechanism of thioredoxin is based on reversible
•
oxidation of two cysteine thiol groups to a disulfide-redox-active disulfide bridge is Cys-X-X-Cys (X any amino
acid)
Oxidized Dsb is less stable than reduced form-– S-S is very reactive, therefore a strong oxidizing agent
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Isomerization of prolines
• Most peptide bonds are trans (100x more stable than
cis)
• When second residue is proline, trans form is only 4x
•
•
more stable
In native proteins, cis-proline peptides are stabilized by
tertiary structure but in unfolded state there is an
equilibrium between cis and trans isomers
Cis-trans isomerization of proline peptides is the ratelimiting step in folding for some proteins
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Protein Folding
• Proteins are assisted in folding by molecular
chaperones
• Hsp60 (chaperonins) Hsp70 and Hsp90 are
three main classes
• Hsp70 recognizes exposed, unfolded regions of
new protein chains - especially hydrophobic
regions
• It binds to these regions, protecting them until
productive folding reactions can occur
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18
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Fig. 31-2b, p. 1026
The GroES-GroEL Complex
• The principal chaperonin in E. coli
• GroEL forms two stacked 7-membered rings of
60 kD subunits; GroES is a dome on the top
• Nascent protein apparently binds reversibly
many times to the walls of the donut structure,
each time driven by ATP hydrolysis, eventually
adopting its folded structure, then being
released from the GroES-GroEL complex
• Rhodanese (as one example) requires
hydrolysis of 130 ATP to reach fully folded state
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21
Fig. 31-3a, p. 1027
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Fig. 31-3b, p. 1027
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Fig. 31-3c, p. 1027
Eucaryotic Hsp90 Chaperones
• Hsp 90 is 1-2% of total cytosolic protein
• Signal transduction molecules such as
tyrosine kinase receptors, steroid hormone
receptors, non-receptor tyrosine kinases
are clients for Hsp90
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Fate of nascent proteins
• Association of nascent polypeptide with
various chaperone systems commits them
to folding pathways
• If proteins fail to fold, they are recognized
and targeted for degradation
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Protein processing
• Post-translational modifications are
covalent modifications that alter function
• Hundreds of amino acid variations have
been described
• Methylation, phosphorylation, lipid and
carbohydrate addition are a few
• Human proteins number around 300,000
from 30,000 genes
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Proteolytic cleavage
• Why remove amino acids?
– Create diversity
• Met-amidopeptidase removes Met from peptide so
that not all amino termini are Met
– Serves as activation mechanism
• Metabolically active enzymes are pro-proteins or
zymogens such as digestive enzymes
– Involves targeting of proteins to proper
destinations
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Protein Translocation
Common features in all organisms
• Proteins are made as preproteins containing domains
•
•
•
that act as sorting signals
Signal recognition particles (SRPs) recognize the sorting
signals as they emerge from the ribosome
Membranes involved in protein translocation have
specific signal receptors
Translocons (selectively permeable protein-conducting
channels) catalyze the movement of the proteins across
the membrane with metabolic energy (ATP, GTP, ion
gradients) essential
– Stop-transfer signals allow diffusion of protein into membrane
• Preproteins bind to chaperones to stay loosely folded
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Prokaryotic Protein Translocation
An essential process for membrane proteins and
secretory proteins
• Such proteins are synthesized with a leader
peptide, or signal sequence of about 16-26
amino acids
• The signal sequence has a basic N-terminus, a
central domain of 7-13 hydrophobic residues,
and a nonhelical C-terminus
• The signal sequence directs the newly
synthesized protein to its proper destination
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Prokaryotic Protein Transport
All non-cytoplasmic proteins must be
translocated
• The leader peptide retards the folding of the
•
•
protein so that molecular chaperone proteins
can interact with it and direct its folding
The leader peptide also provides recognition
signals for the translocation machinery
A leader peptidase removes the leader
sequence when folding and targeting are
assured
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Eukaryotic Protein Sorting
Eukaryotic cells contain many membrane-bounded
compartments
• Most (but not all) targeting sequences are N-
terminal, cleaveable presequences
• Charge distribution, polarity and secondary
structure of the signal sequence, rather than a
particular sequence, appears to target to
particular organelles and membranes
• Synthesis of secretory and membrane proteins is
coupled to translocation across ER membrane
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Events at the ER Membrane
• As the signal sequence emerges from the
ribosome, a signal recognition particle (SRP)
finds it and escorts it to the ER membrane
• There it docks with a docking protein or signal
receptor (SR) - see Figure 31.5
• SRP dissociates in a GTP-dependent process
• Protein synthesis resumes and protein passes
into ER or into ER membrane; signal is cleaved
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encoded signal sequence
mRNA
signal sequence at end of protein
emerging from ribosome
Signal recognition particle (SRP)
GTP
signal sequence at end of protein
emerging from ribosome
Binding site for SRP
receptor
SRP receptor
Signal peptidase
GDP + Pi
Signal peptidase
Signal peptidase
Ribosome dissociates
Signal peptidase cleaves
signal peptide
Completed polypeptide chain
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Signal Recognition Particle
• Complex of six polypepides and one 300-
base RNA molecule
• Signal peptide recognition depends on one
protein of 54,000 daltons-SRP54
• RNA (7S) is probably structural
• Removal of RNA disrupts SRP
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Mitochondrial protein import
• Mitochondria have two membranes, and
two spaces in between the membranes
• Signal sequences are N-terminal,
positively charged regions of 10-70 aa
• Form amphipathic a-helices, positive on
one side and uncharged, hydrophobic
on the other (Fig. 31.6)
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Mitochondrial protein import (Fig
31.7)
• Binding of preprotein to TOM (mitochondrial outer
•
•
•
membrane translocon)
For outer membrane proteins, transfer to SAM
(sorting and assembly complex)
Inner membrane proteins traverse TOM and are
taken up by TIM22 (inner mitochondrial membrane
translocon)
Matrix proteins are taken up by TIM23 and
transferred across the inner membrane into the
matrix
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Protein Degradation
• Some protein degradation pathways are
nonspecific - randomly cleaved proteins
seem to be rapidly degraded
• However, there is also a selective, ATPdependent pathway for degradation - the
ubiquitin-mediated pathway
• Ubiquitin is a highly-conserved, 76 residue
(8.5 kD) protein found widely in eukaryotes
• Proteins are committed to degradation by
conjugation with ubiquitin
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Ubiquitin and Degradation
Three proteins involved: E1, E2 and E3
• E1 is the ubiquitin-activating enzyme - it forms a
•
•
•
thioester bond with C-terminal Gly of ubiquitin
Ubiquitin is then transferred to a Cys-thiol of E2,
the ubiquitin-carrier protein
Ligase (E3) selects proteins for degradation. the
E2-S~ubiquitin complex transfers ubiquitin to
these selected proteins
More than one ubiquitin may be attached to a
protein target
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46
Fig. 31-8, p. 1034
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Fig. 31-8a, p. 1034
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Fig. 31-8b, p. 1034
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Fig. 31-8c, p. 1034
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Fig. 31-9, p. 1034
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Fig. 31-10a, p. 1035
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