Protein folding: the native and nonnative states

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Protein foldingthe native and non-native states
Any peptide (in theory) could adopt many
different secondary and tertiary structures But
in general ALL molecules of a given protein
species adopt the SAME 3D-conformation. This
structure is called the NATIVE STATE of the
protein - the native state is usually (but not
always) the most stable (lowest energy) state of
the folded protein

• - disruption of the native structure (by BREAKING
  the weak bonds responsible for 2 and 3
  structures) is called denaturation
• - the result is a protein in a non- native or
  denatured state
• Denaturation can be caused by
• - raising (or more rarely lowering) the
  temperature
• - extremes of pH
• chaotropes (such as 8M urea or guanidine
• hydrochloride)
• - detergents etc.

Most denatured proteins precipitate
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Denatured proteins will spontaneously refold in
vitro (in the test tube) e.g. folding of RNAse A
denaturation
renaturation
Incubate protein in guanidine hydrochloride (GuHCl
) or urea
100-fold dilution of protein into
physiological buffer
- the amino acid sequence of a polypeptide is
sufficient to specify its three-dimensional
conformation Thus protein folding is a
spontaneous process that does not require the
assistance of extraneous factors
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Protein Folding the Levinthal paradox
in vitro
in vivo
denatured protein random coil- a very large
number of possible extended conformations
folding
folding
native protein 1 stable conformation
t seconds
t seconds or less
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Levinthal paradox a new folding view is needed
Consider a protein with 100 amino acids - using a
very simplified model where there are only 3
possible orientations per residue
- assume 1 conformation can form every 10-13
seconds (100 picoseconds) - then 5 x 1047
x 10-13 s 1.6 x 1027 years to correctly fold a
protein Obviously NOT ALL conformations can be
sampled during folding!
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Resolving the paradox
a. there are a limited number of secondary
structural elements b. these elements tend to
form spontaneously during the co-translational
folding of a protein c. proteins fold via
so-called folding landscapes, where the
proteins follow pathways of folding that lead
to the correct three-dimensional structure d.
folding intermediates may be important in such
folding landscapes/pathways
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Protein folding theory
limited number of secondary structure elements
helices, sheets and turns
folding can be thought to occur along energy
surfaces or landscapes
Dobson, CM (2001) Phil Trans R Soc Lond 356,
133-145
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A simplified view A folding funnel
unfolded (non-native) states (two of many
different conformations are shown)
Energy landscape - descent towards Free energy
minimum state - A- rapid folding B-
secondary energy minima
Native state (N)
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Thermodynamics of protein folding
Usually, ?G for folding is negative i.e.
favourable but this is due to a balance of
several thermodynamic factors -Conformational
entropy works against folding (contributes
positively to ? G), since unfolded condition
random cycling between many possible states, it
involves higher entropy than the single folded
state. -Enthalpy contribution works in favour
of folding (contributes negatively to ?G) i.e.
reduction in Enthalpy due to formation of
energetically favourable interactions (e.g. salt
bridges, H bonds, van der Waals etc.) between
chemical groups in folded state. -Entropy
contribution from hydrophobic effect works in
favour of folding (contributes negatively to ?G)
i.e. the burying of hydrophobic R groups in
protein increases entropy of whole system
(protein water).
balance -ve ? G
?G ?H - T?S
overall free energy change for folding
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Proteins fold in stages

• Local folding through nucleation of small
  clusters of residues
• A General Order of Folding
• Short regions rapidly form small stable secondary
  elements like alpha-helices and beta-sheets etc.
• These small structure elements interact with
  other local elements and interact folding into
  globular units on an intermediate time scale.
  Associations are through various weak chemical
  interactions.
• These globular domains may be a complete small
  protein or a number of these in larger proteins
  can interact more slowly to fold into the final
  folded structure.

folded protein
polypeptide
secondary structures
domains
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Co-translational protein folding
definition co-translational is a process which
occurs during the translation (synthesis) of a
protein on the ribosome
- initially, the first 30 amino acids of the
polypeptide chain present within the ribosome are
constrained and cannot fold until they exit from
the ribosome (the amino, or N-terminus emerges
first, and the C-terminus emerges last). - as
soon as the first part of the nascent chain is
extruded, it will start to fold
co-translationally (i.e., acquire secondary
structures, domains etc.) as the complete
polypeptide is produced and extruded, it will
fold in a similar fashion and then the final
tertiary structure will be established, followed
(in some cases) by assembly of subunits to form
the quaternary structure.
NH3
folding
assembly
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Molecular chaperones
- the great majority of proteins can fold without
assistance, in a co-translational manner - some
proteins, which may have difficulties reaching
their native states, must be stabilized by
molecular chaperones (or chaperonins) by assisted
folding - these bind to nascent (emerging)
polypeptides and stabilize them (usually by
associating hydrophobic residues). - otherwise
these hydrophobic residues tend to associate with
other hydrophobic residues, leading to intra- or
inter-molecular associations with other proteins
that prevent proper folding - there are dozens
of different types of molecular chaperones, and
some accomplish functions different from helping
protein folding - e.g., some help protein
assembly, some help to transport proteins to
various parts of the cell, some help
damaged proteins from refolding
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Most extensively-studied of all chaperonins the
GroEL-GroES complex of E. coli
EL
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Protein misfolding can cause serious human
diseases e.g. the prion-based, Creutzfeld-Jacob
disease (CJD)-basic mechanism for many
neurodegenerative diseases is similar
the formation of protein aggregates that kill
nerve cells.
(see pp362-363 of Alberts et al.)
Prion diseases are self-infectious misfolded
version of the prion protein, PrP, can induce
the normal PrP protein to misfold into a more ?
strand-based structure, resulting in damaging
aggregation via formation of cross-? filaments.
these filaments are visualized cytologically as
amyloid stacks
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depiction of cross ?-filament structure resulting
from extensive stacking of misformed ? sheets
this type of structure is resistant to proteases
Model for conversion of PrP tp PrP shows the
change of two ?-helices into four ? strands
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Protein quaternary structure
Association of multiple polypeptides into a
functional unit - many, although not all
proteins engage in this - individual proteins in
the quaternary structure are calledsubunits Ex
ample prefoldin (a so-called molecular
chaperone that assists the co-translational
stabilization of proteins during their folding
in vivo (in the cell)
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Prefoldin quaternary structure
two types of proteins (subunits) assemble into
a hexamer (6 subunits)
- structure of prefoldin hexamer -
oligomerization (assembly) domain is a double
beta-barrel structure composed of beta-strands -
coiled coils consist of two helices winding
around each other
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Symmetries of ProteinQuaternary Structure
Figure 6.30
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Protein modificationsrequirements for activity
- cleavage and covalent modifications of proteins
(often after synthesis) but may also be
co-translational
I. CLEAVAGE some proteins require sections of the
polypeptide chain to be removed for correct
maturation.
A zymogen is a catalytically inactive protein
precursor that must be cleaved proteolytically to
be activated
INSULIN Synthesized as PREPROINSULIN - 1st
cleavage removes signal sequence (PRE) - 2nd and
3rd cleavages remove joining (PRO) peptide
sequences - di-sulphide bonds hold the two
peptides together
H2N-
H2N-
disulfide bonds
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Zymogens in action
- the pancreatic proteases (such as trypsin,
chymotrypsin, elastase, and carboxypeptidase) ?
covalent enzyme activation by proteolytic
cleavage - synthesized in the pancreas in an
inactive form because if they were active in the
pancreas, they would digest the pancreatic
tissue. Rather, they are made as slightly longer,
catalytically inactive molecules called zymogens
(trypsinogen, chymotrypsinogen, proelastase, and
procarboxypeptidase, respectively)
represents active enzyme





cleavage event
Figure 11.39
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Chymotrypsin activation
Activation of Chymotrypsinogen (No Enzymatic
Activity) Chymotrypsin Serine Protease One of
the most complex examples of proteolytic
activation 1st cut is stabilized by S-S bond p
chymotrypsin Series of modifications. Each
triggering the next Final state a chymotrypsin
(by Trypsin)
(Ile)
Figure 11.40
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II. Covalent Modifications (p. 403-408)

• Sometimes proteins are covalently modified after
  synthesis
• These modifications can be
• Required to obtain the active conformation (e.g..
  collagen)
• Used to control the activity of a protein
• (e.g. histones, signal transducing proteins, etc.)

Examples Collagen Proline ? Hydroxyproline
(hydroxylation) This requires Vitamin C No
Vitamin C ? No Hydroxyproline ? Scurvy Due to
weakening of collagen fibres- hydroxylation of
prolines somehow stabilizes structure
OH
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Prothrombin, histones
Prothrombin Glutamate ? gamma-Carboxy Glutamate
(carboxylation)
This requires Vitamin K No vitamin K ? No Blood
Clotting
Histones Histones are proteins involved in the
folding/compacting of nuclear DNA. They are
often modified in regions of active
transcription. Acetylation of Lysine is the MOST
common- (decreases net positive charge of
histones).
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Phosphorylation- an important kind of protein
modification.
Signal Transduction Proteins Phosphorylation of
Hydroxyls (-OH) - these proteins become
transiently phosphorylated which either activates
or inhibits their activity - phosphorylation can
be on one of a 3 different amino acids - a
particular protein will only have specific
modification sites Serine ? phosphoserine Threo
nine ? phosphothreonine Tyrosine ?
phosphotyrosine
kinases are the cellular proteins that
phosphorylate these residues
phosphatases are the cellular proteins that
remove the phosphate groups
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More protein modifications