Protein Folding I and II

Sepideh Khorasanizadeh
September 2008
khorasan@virginia.edu

Material adapted from text books and journal articles

Protein folding
g is cooperative
p
The thermal denaturation of Ribonuclease. Solution viscosity increase (open square), near-UV CD at
365 nm (open circle), UV absorbance at 287 nm (open triangles). Filled triangle is a second
denaturation after cooling to prove reversibility.
reversibility
pH 2.1, if physiological pH then Tm = 75 degrees C
• Amino acid sequence determines secondary and tertiary structure.

• The decrease in conformational entropy when a protein folds disfavors folding this is
compensated in part by energy stabilization through internal noncovalent bonding.

• Details of H-bonding in a typical protein is shown.

Covalend and noncovalent bond energies.

Noncovalent bonds have one to two orders of

magnitude weaker energies

Types of noncovalent interactions. q- and q+ represent a fraction
of an electron or proton charge.
 Molecules may attract one another by noncovalent forces but can not interpenetrate:
van der Waals radii determine molecular surfaces.

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.

The free energy change for a process at

constant pressure is:

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

Use of the linear relationship in the unfolding transition region:

ΔG (denaturant concentration) = ΔG (zero denaturant) – m x denaturant concentration

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.

Pathway Model - The "pathway" model of protein

folding is depicted at the left
left. Nucleation is critical
because it is much more difficult to begin an helix than
to extend it. Nucleation may start at a number of points
and all of these partially folded structures can be
"funneled" by energy minimizations toward the final
state. Thus, Levinthal's paradox is averted.

Matthews, Ahern, and van Holde, 3rd ed.

 Energy surface for protein conformations
Funnell with
F ith various
i paths
th iis
Levinthal “golf course” landscape more realistic.
 Refolding and S-S bond formation in BPTI. The intermediate structure may contain both

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

A factor of 4:1 occurrence in Proline vs. 1000:1 in

other
th amino
i acids
id

Common Errors - One of the most common folding

errors occurs via cis-trans isomerization of the
amide bond adjacent
j to a p
proline residue. Proline is
the only amino acid in proteins that forms peptide
bonds in which the trans isomer is only slightly
favored (4 to 1 versus 1000 to 1 for other residues).
Thus, during folding, there is a significant chance
that the wrong proline isomer will form first
first. It
appears that cells have enzymes to catalyze the cis-
trans isomerization necessary to speed correct
folding.
Time scale of protein motions
The Levinthal 'golf-course' landscape. N is
the native conformation. The chain searches
for N randomly, that is, on a level playing field
of energies
energies.
The 'pathway' solution to the random search
problem of Fig. 1. A pathway is assumed to lead
from a denatured conformation A to the native
conformation N,
N so conformational searching is
more directed and folding is faster than for random
searching.
An idealized funnel landscape.
As the chain forms increasing
numbers
b off iintrachain
t h i contacts,
t t
and lowers its internal free
energy, its conformational
freedom is also reduced.
Dill and Chan, (1997) From Levinthal to pathways to
funnels, Nature Structural Biology, 4:10-19
Differentt folding
Diff f ldi scenarios.i Th vertical
The ti l axis
i is
i internal
i t l free
f energy. Each
E h conformation
f ti iis
represented as a point on the landscape. The two horizontal axes represent the many chain degrees
of freedom. (a) shows a rugged landscape with hills and traps, folding kinetics is likely multi-
exponential. (b) shows a landscape in which folding is faster than unfolding. A is a throughway folding
path,, whereas unfolding
p g chains (p
(path B)) must surmount a barrier in order to reach the most stable
denatured conformations. (c) shows a landscape in which folding is slower than unfolding. Most
folding paths (path A) pass through a kinetic trap, whereas some low-lying denatured conformations
are readily accessible from the native state during unfolding (path B).

Chan and Dill, (1998) Proteins 30:2-33.

 Funnelscape for a fast folding protein Folding is limited
by the rate of meandering downhill
downhill.

Champagne glass landscape, to illustrate how

conformational entropy can cause "free energy
barriers" to folding. The "bottleneck" or rate limit to
folding is the aimless wandering on the flat plateau
as the chain tries to find its way downhill (From
From Levinthal to pathways to funnels) (b) Serpin
scenario shows a landscape with a deep kinetic trap
on the left (A), which is easily accessible from the
open conformations. Chain trapped in this deep
local minima anneal to the global minimum (B, (B in
the middle) only very slowly. This corresponds to
the folding of some serpins such as PAI-1.

Chan and Dill, (1998) Proteins 30:2-33.

 Figure 4. A rugged energy landscape with kinetic Figure 5. Moat Landscape, to illustrate how a protein could
traps, energy barriers, and some narrow throughway have a fast-folding throughway process (A), in parallel with a
ppaths to native. Foldingg can be multi-state. slow-foldingg pprocess ((B)) involvingg a kinetic trap.
p

Figure 6. Champagne Glass Landscape, to illustrate how

conformational entropy can cause free energy barriers to
folding. The 'bottleneck' or rate limit to folding is the
aimless wandering on the flat plateau as the chain tries to
find its way downhill.

Dill and Chan, (1997) Nature Structural Biology, 4:10-19

Global distribution of conformations of a polypeptide chain in a
random-coil state, a partially collapsed denatured state and a
compact non-native state. The different species within each ensemble
interconvert rapidly. (b) Chemical structure of an alanine residue and
the Ramachandran diagram representing its free-energy
free energy surface in a
protein environment. The surface features result primarily from steric
repulsions between the various atoms and lead to a distribution of
conformations in the coil state that corresponds locally to an average
over the low-energy regions of the conformational space for each
amino acid. The diagram was obtained by taking the natural logarithm
of the observed frequency of each pair of main-chain dihedral angles
(φ,ψ) in a set of 1000 representative protein structures). The contours
are spaced 0.5 units apart, so that each level is a factor of e 0.5=1.6
times more probable than the previous (lower) one. Dinner et al. (2000) Trends Biochem Sci 25:331-339.
Atomic structures of amyloid cross- spines reveal varied steric zippers

Nature 447, 453-457 (2007)

Sawaya et al.
http://www ncbi nlm nih gov/entrez/query fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list uids=17468747
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17468747

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.

Nature 447, 1021-1025 (2007)

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17522630

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

Interaction between the pKID domain of the gene

transcription factor CREB and the KIX domain of
the CREB-binding protein occurs in the cell
nucleus to regulate gene expression.

By elucidating the three-step binding reaction

between pKID and KIX using NMR spectroscopy,
Sugase et al. identified four states along the
reaction pathway.

Initially, the highly disordered, free state of pKID partially

populates
l t helix
h li A (A).
)

In the encounter complex with KIX, pKID is

tethered by nonspecific hydrophobic contacts
in its helix B region(
g (B)).

The intermediate state is characterized by a specifically

bound and largely configured helix A.

Finally, in the high

Finally high-affinity,
affinity bound conformation
conformation,
both helices are fully structured.
Enzymes decrease the activation energy and facilitate the formation of the transition state

Related to the Chaperonin-assisted Protein Folding

 Possible functional cycle of the GroEL-GroES Chaperonin
An unfoled protein molecule binds to one end of the GroEL
GroEL-ADP
ADP complex
with bound GroES at the other end. Folding can occur inside the cavity or
The cavity may provide an unfolding opportunity to allow refolding.

Further Reading: The Two Families of Chaperonin: Physiology and Mechanism

Annu. Rev. Cell Dev. Biol. 23, 115-45 (2007)
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=17489689