Protein Folding I and I I
Protein Folding I and I I
Protein Folding I and I I
Sepideh Khorasanizadeh
September 2008
[email protected]
• The decrease in conformational entropy when a protein folds disfavors folding this is
compensated in part by energy stabilization through internal noncovalent bonding.
van der Waals radius is the effective radius for closest molecular packing total interaction
energy at any distance is the sum of the attraction and repulsion energies.
Van der
V d Waals
W l radiidii
relevant to Proteins
Hydrogen
y g bonds are the strongest
g and most specific noncovalent bonds
The internal energy of a system includes all forms of energy that can be exchanged via simple
physical process and chemical reactions.
First Law of Thermodynamics: The internal energy can change only by the exchange of heat or
workk with
ith th
the surrounding.
di Th
The h
heatt evolved
l d iin a reaction
ti att constant
t t pressure iis equall tto th
the
enthalpy, ΔH. The enthalpy change in a reaction is the energy change of most interest to biochemists.
Reversible processes occur always near a state of equilibrium; irreversible processes drive toward
equilibrium.
q
Entropy is a measure of the randomness or disorder in a system.
Second Law of Thermodynamics: The entropy of a system will tend to increase to a maximum
value.
ΔG = ΔH – T ΔS
An example of the interplayy of enthalpyy and entropyy
The signs of ΔH and ΔS determine the effect of temperature on
processes or reactions
The burying of hydrophobic groups within a folded protein molecule produces a
stabilizing entropy increase known as the hydrophobic effect.
effect
Favorable free energy of folding is a net result of
Th
Thermodynamic
d i FForces
Breaking disulfide bonds by 3 commonly used reagents
β-Mercaptoethanol
DTT
Denaturing chemicals
Disulfide bonds stabilize folding
off some proteins
t i
Amino acids have to sample and settle with acceptable
dihedral angles in native structure of a protein
Folding into an alpha helix requires concerted efforts of side chains and
backbone interactions
The relative frequency of every amino acid within
diff
different
t secondary
d structures
t t
Same chain can be found in two different
conformation
co o at o in tthe
e co
context
te t o
of a d
different
ee tp protein
ote
architecture
N U
[U] fU
KU = =
[N] 1 - fU
ΔG = - R T ln KU
ΔG (zero denaturant
denaturant, H2O) = m (Cmidpoint – C)
Folding Paradox - Levinthal's paradox states that
there are approximately 1050 possible conformations
for a protein, such as ribonuclease (124 residues). If
one new conformation could be attempted every 10-13
seconds, it would still take over 1030 years to randomly
test all of the possibilities, y
yet ribonuclease can
completely fold in about a minute. Thus, folding must
not be a completely random phenomenon.
native and nonnative S-S bonds. Interconversion within boxed regions is rapid.
Disulfide Bond Formation - Proteins with disulfide bonds have a built-in advantage if they are denatured with their
disulfide bonds intact
intact. The intact disulfide bonds eliminate many degrees of freedom associated with denaturation,
denaturation so
fewer events need to occur to bring about the correctly folded state. This can be verified by removing the disulfide
bonds of a protein and then denaturing it. Refolding of this polypeptide occurs, but at a slower rate than when the
disulfides are left intact. Interestingly, disulfide bonds not found in the native structure sometimes form during
intermediate stages of folding. Also, the folding process can be aided by enzymes that make disulfide bonds.
Cis versus Trans Conformation
Amyloid fibrils formed from different proteins, each associated with a particular disease,
contain a common cross- spine. The atomic architecture of a spine, from the fibril-forming
segment GNNQQNY of the yeast prion protein Sup35, was recently revealed by X-ray X ray
microcrystallography. It is a pair of -sheets, with the facing side chains of the two sheets
interdigitated in a dry 'steric zipper'. Here we report some 30 other segments from
fibril-forming proteins that form amyloid-like fibrils, microcrystals, or usually both.
These include segments from the Alzheimer's
Alzheimer s amyloid
amyloid- and tau proteins, the PrP
prion protein, insulin, islet amyloid polypeptide (IAPP), lysozyme, myoglobin, -synuclein
and 2-microglobulin, suggesting that common structural features are shared by amyloid
diseases at the molecular level. Structures of 13 of these microcrystals all reveal steric
zippers, but with variations that expand the range of atomic architectures for amyloid
amyloid-like
like
fibrils and offer an atomic-level hypothesis for the basis of prion strains.
Despite their fundamental similarity,
th reported
the t d structures
t t display
di l variations
i ti
of the basic steric-zipper structure and
thereby expand our understanding of
amyloid
y structure.
Th 8 classes
The l off steric
t i zippers
i
Mechanism of coupled folding and binding of an intrinsically disordered protein
Sugase
g et al.
Protein
P t i ffolding
ldi and d bi
binding
di are analogous
l processes, iin which
hi h th
the protein
t i ''searches'
h ' ffor favourable
f bl
intramolecular or intermolecular interactions on a funnelled energy landscape1, 2. Many eukaryotic
proteins are disordered under physiological conditions, and fold into ordered structures only on
binding to their cellular targets. The mechanism by which folding is coupled to binding is
poorly
l understood,
d t d b butt it h
has b
been h
hypothesized
th i d on ththeoretical
ti l grounds
d th
thatt th
the bi
binding
di ki kinetics
ti
may be enhanced by a 'fly-casting' effect, where the disordered protein binds weakly and
non-specifically to its target and folds as it approaches the cognate binding site7. Here we show,
using NMR titrations and 15N relaxation dispersion, that the phosphorylated kinase inducible
acti ation domain (pKID) of the transcription factor CREB forms an ensemble of transient enco
activation encounter
nter
complexes on binding to the KIX domain of the CREB binding protein. The encounter complexes
are stabilized primarily by non-specific hydrophobic contacts, and evolve by way of an intermediate
to the fully bound state without dissociation from KIX. The carboxy-terminal helix of pKID is only
partially folded in the intermediate
intermediate, and becomes stabilized by intermolecular interactions formed
in the final bound state. Future applications of our method will provide new understanding of the
molecular mechanisms by which intrinsically disordered proteins perform their diverse biological
functions.
Coupled Folding and Binding