Flexibility of Protein Structure
• Proteins show varying degree of conformational
flexibility
• Due to movements of atoms in molecules
• vibration in bond length and angles
• Reflects the existence of populations of
alternative conformations
• Severe constraints on the extent of
conformational variations
• Integral proteins are less flexible than watersoluble proteins
Hydrogen Exchange
• The best evidence for extensive mobility of protein
structure
• Hydrogen atoms bonded covalently to various atoms
exchange with solvent at different intrinsic rates,
depending on the tendency of that atom to ionize
– H atoms on oxygen, nitrogen, or sulfur exchange rapidly
– H atoms on carbon change at slow rate
• Intrinsic rate of exchange is temperature dependent
– 3 fold increase with 10 C increase
– Also, influenced by the environment and by inductive and charge
effects on the amide
Hydrogen Exchange
• Local unfolding by hydrogen exchange in interior
group atoms
– By diffusion of solvent molecules to various sites
• Rates are correlated with the solvent
accessibility of the residue and of its neighbors,
not distance
• Rate is decreased by increased pressure
• Wide variations in processes depending on the
proteins and conditions
Stability of the Folded
Conformation
• The folded conformations of proteins are only
marginally stable even under the best conditions
• Can be easily denatured by changes in
environmental conditions
–
–
–
–
Temperature increase
pH changes
Pressure increase
Addition of denaturants
Reversible folding & unfolding
• Anfinsen (1950’s) –
showed reversibility
of denaturation with
urea for RNAse A
– amino acid
sequence encodes
structure:
thermodynamic
hypothesis
– Folding is
“cooperative”
S-S bonds in brown
Denaturation-studied
by various methods
is reversible
Stability of Proteins
Stability of the folded structure of a globular protein
depends on the interplay of three factors.
1. The unfavorable conformational entropy change, which
favors random chains instead.
2. The favorable enthalpy contribution arising from
intramolecular side group interactions.
3. The favorable entropy change arising from the burying of
hydrophobic groups within the molecule.
• Factor 1 works against folding, whereas 2 and 3 help
stabilize folding.
Denaturation
• Do need not changes in covalent structure
• Usually reversible if it is due to unfolding
• Proteins can be unfolded by
–
–
–
–
Breaking any disulfide bonds present
Removing any essential cofactors
Mutation in certain crucial residues
Deleting residues from the primary structure
Reversible Unfolding Transition
• No decrease in biological activity
• Unfolding is a two-state phenomenon, with only fully
folded (F) and fully folded (U) states
• Partially folded structures are unstable relative to U or F
• Under conditions that favor unfolding,
–
–
–
–
The folded conformation changes very little if at all
Increase in flexibility
Localized conformational alterations
Average structure not changed
• Then protein unfolds completely within a limited range of
conditions
Nature of the Unfolded State
• Unfolded states produced under various
unfolding conditions have different physical
properties
• Unfolded proteins do not contain cooperative
folded structure
• But, unfolded and folded proteins are
indistinguishable thermodynamically
Molten Globule State
• Conformation that are neither fully folded nor
fully unfolded
– The overall dimensions of the polypeptide chain are
much less than those of a random coil
– Average content of secondary structure is similar to
folded
– The side chains are in homogeneous surroundings
– Many amide groups exchange hydrogen atoms with
the solvent much rapidly than the folded
– The enthalpy is nearly the sale as the folded state
– Interconversions of the molten globule state with the
fully folded state are rapid
Physical Basis for Protein
Denaturation
• Extreme pH
– Non-ionized His and Tyr buried in folded structure can
be ionized
– Disruption of salt bridges
• Denaturants: Urea and Guanidinium salts
– Break H bonds
– Have hydrogen bonding capabilities comparable to
that of water, but different geometries
– Increase the solubilities of both polar and nonpolar
molecules
Thermodynamics of Unfolding
• Elevated temperature increase unfolding
– Proteins from thermophiles are more heat stable
– Unfolded state has a greater heat capacity than the
folded state
– Large temperature dependence of the changes in
enthalpy and entrophy
– The more hydrophobic the interior of a protein, the
lower its net stability
Forces that hold protein structure
together
•
•
•
•
Dispersion (van der Waals) forces
Electrostatic interactions
Hydrogen bonds
Hydrophobic interactions
• What pulls it apart?
• Conformational entrophy
Stabilizing Forces of Folded
Conformations
• Hydrogen bonds
– The major stabilizing force for folded conformation
– H-bonds in the folded conformation is stronger than those in
unfolded ones
– H-bonds with water and a protein are present a fraction of a time
while that within protein present are present all of the time (more
negative enthalpy)
• Electrostatic interactions
– More important van der Waals interactions
• Van der Waals interactions
• Hydrophobic interactions
– Unique in folded structure
– Driving force for folding
Contributions to protein stability
type of interaction
hydrophobic group burial
hydrogen bonding
ion pairs/salt bridges
disulfide bonds
total contribution*
~200 kcal/mol
small??
<15 kcal/mol
4 kcal/mol per link
*For globular protein of 150 residues hydrophobic burial
is the chief interaction favoring protein stability, but
this is balanced by a huge loss of conformational
entropy that opposes folding.
Consequently typical net protein stabilities are 5-20
kcal/mol--> so even minor interactions can make a
difference!
Hydrophobicity and the Stability of Folded
Conformation
• Hydrophobicity is not the Primary Force to the Stability of
Folded Conformation. Why?
• Although the interior of proteins are densely packed,
they are not closely packed with all adjacent atoms in
Van der Waals contact simultaneously
• Imperfect Van der Waals interactions
• Van der Waals interactions in proteins are in less than
optimal conditions and weaker than those occur in liquid
Stability of the Folded conformation
• Folded state has only marginal net stability
because of the large conformational entrophy of
the unfolded state
• Depends on its primary structure
• Single amino acid replacement can alter the
stability of a folded protein quite drastically
Alanine scanning
• A way of assessing the importance of amino-acid side chains for
structure/stability etc.
• “Remove” each residue one by one by replacement with Ala
• Many Ala mutations have no effect on stability--> about half!
• A large group also cause significant effects-->several kcal/mol
• Occasional a mutant will stabilize the protein--> natural proteins not
maximally stable!
The interior is more important for stability
than the exterior
side chains with stability-neutral Ala
mutations
side chains with destabilizing Ala
mutations
Side-chain packing in the hydrophobic core
• Protein interiors have a “jigsaw
puzzle”-like aspect.
• Their packing densities are similar
to those of crystals of organic
molecules.
• This dense packing are importance
for stability and a precise threedimensional structure
• Over or under filling can be
detrimental to stability
• However, not all cores with equal
volume are equally stable.
Effect of nondisruptive hydrophobic core
mutations
nondisruptive
means not causing
any steric clashes
or uncompensated
buried charges, H-bonds
etc
leucine
in core
mutation to
alanine
difference in water-octanol transfer
free energies of leucine and alanine
is ~2 kcal/mol. Effects of Leu-->Ala
mutations are typically larger than
this. Why?
Not all buried Leu-->Ala mutations
give the same destabilization. Why?
The cost of cavity formation in protein cores
• A Leu-->Ala core mutation leaves a cavity in the hydrophobic
core.
• In addition to the ~2 kcal/mol transfer free energy difference
between Leu and Ala, there is a penalty of 20 cal-mol/Å2 for
forming this cavity.
• This is due to loss of van der Waals interactions with the mutated
side chain.
• This increases the cost of a Leu-->Ala mutation from 2 to about 5
kcal/mol!
• Since proteins are only stable toward unfolding to the extent of 5
to 15 kcal/mol, such mutations are potentially devastating
• This suggests that having good packing in terms of not having
cavities is important to stability.
Disruptive mutations in hydrophobic cores
Three kinds:
steric mutants
extreme volume mutants
polar mutants
change in shape, not volume
increase core volume
put polar/charged residue in core
• Polar/charged core mutants are almost invariably very destabilizing,
for obvious reasons.
• Charged groups or groups that can form hydrogen bonds that are
isolated within a protein interior are bad for stability
• Energetic effects of increased volume are often hard to predict-subtle backbone shifts often occur to accommodate the extra
volume
Mutations of surface (solvent-exposed)
residues
• Although the surface of proteins are very polar overall, individual surface
positions can usually be replaced by many other residues including
hydrophobics without much effect on stability.
• average effect on stability of surface mutations is small
• little stability penalty for change of individual surface polars to
hydrophobics
However:
• too many hydrophobics on a protein’s surface will reduce solubility and
promote aggregation
• surface polar-to-hydrophobic mutations can reduce structural specificity by
favoring alternative conformations in which the introduced hydrophobic
side chain becomes buried.
“Hydrogen bond inventory”
• Definition: Although hydrogen bonds probably do not
stabilize proteins per se, it is nonetheless important that all
potential hydrogen bond donors and acceptors be hydrogen
bonded to something - solvent, protein backbone, or protein
side chains.
• Removal of one partner of a hydrogen bonded pair by
mutation can be quite destabilizing if the remaining partner
is not able to satisfy its hydrogen bond potential by
interacting with solvent.
• Also applicable to ion pairing/salt bridge interactions.
• Even though ion pairs don’t contribute much to stability,
charged groups which are neither paired with oppositely
charged groups nor solvated by water can be very
destabilizing!
Role of solvent-exposed salt bridges
• Typical mutations of surface salt bridges are destabilizing by less than 1
kcal/mol, but there are cases where larger effects are observed.
• Surface salt bridges are thus not large contributors to protein stability.
• However, some salt bridges may be important at the level of specifying a
particular precise structure, much in the way that hydrophobic packing
interactions are.
• Buried salt bridges (and buried polar interactions in general) not important
for stability per se, but removal of individual partners can be hugely
destabilizing.
• Buried polar interactions serve more to impart specificity to the structure
rather than stability, due to the strict requirement for satisfaction of H-bond
potential (H-bond inventory) and compensation of charge.
Stability-activity trade-offs?
• Proteins have not evolved to maximize stability.
• They generally evolve to preserve adequate stability.
• However, sometimes stability and activity are selected at the
expense of the other.
• Some mutations in the active sites are more stable but less
active.
• For instance, the active site of barnase is highly positively
charged because it has to bind a negatively charged
pentacoordinate phosphate at the transition state.
• When substrate is not bound, the positively charged side
chains repel each other, reducing stability.
Mutation of catalytic residues in T4 lysozyme
Asp 20
protein
wild-type
E11F
E11M
E11A
E11H
E11N
D20N
D20T
D20S
D20A
ΔTm
, °C
0
4.3
4.1
2.6
0.1
-0.6
3.1
2.2
1.6
-0.8
ΔΔGf,
kcal mol-1
0
1.7
1.6
1.1
0.1
-0.1
1.3
0.9
0.7
-0.3
active site cleft
activity
(relative)
1
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
<0.0001
0.0005
Glu 11
Replacement with some amino acids increases stability but strongly diminishes
activity. This same phenomenon was found to occur for residues involved in
substrate binding. Glu11 and Asp20 are examples of what has been referred to
as “electrostatic strain” in enzyme active sites. However, not all mutations which
remove the charge stabilize the protein, emphasizing that the situation is complex.
Shoichet et al. PNAS 92, 452 (1995)
Factors Determining
Secondary and Tertiary
Structure
29
The Information for Protein Folding
• The information for determining the 3-dimensional
structure of a protein is carried entirely in the amino
acid sequence of that protein.
• Native structure is the natural 3-dimensional structure
of a protein.
• Denaturation is a process for the lost of natural
structure of a protein, along with many of its specific
properties.
• In a cell, a newly synthesized polypeptide chain will
spontaneously fold into the proper conformation.
30
Protein Folding
• Proteins normally want to fold into their “native,” or
lowest-energy, conformation – the most stable
• Christian B. Anfinsen won the Nobel Prize in 1973 for
finding that this folding is based on “directions” from the
amino acid sequence and from its hydrophobic/phillic
nature
• He started by unfolding proteins into random coils, and
found the proteins folded back into their native shape
spontaneously, without any other catalysts
Thermodynamics of Folding
The overall free energy change on folding must be
negative.
• Conformational Entropy
– ΔG = ΔH – TΔS
• Charge-Charge Interactions
– Salt bridges
• Internal Hydrogen Bonds
• van der Waals Interactions
• The Hydrophobic Effect
32
Gibbs Free Energy (ΔG)
• A thermodynamic potential that measures the "useful"
or process-initiating work obtainable from an
isothermal, isobaric thermodynamic system
• The maximum amount of non-expansion work that
can be extracted from a closed system
• Gibbs free energy (ΔG) or available energy is the
chemical potential that is minimized when a system
reaches equilibrium at constant pressure and
temperature.
Contributions to ΔG of folding
What features of protein folding produce large, negative ΔH
or large positive ΔS changes, to compensate for the
conformational entropy loss ?
The Role of Disulfide Bonds
• The formation of disulfide bonds between cysteine
residues further stabilize the 3-D structure of proteins.
• Formation of the disulfide bonds is depend on the protein
primary structure.
• Disulfide bonds are not essential for correct protein folding,
but for contribution to the stability of the structure once it is
folded.
• Site-directed mutagenesis, a powerful method for the test
of the effect of changing one or more amino acid residues
or adding or removing disulfide bonds.
35
Kinetics of Disulfide Bond Formation
• The process of refolding of a protein from a state in which
disulfide bonds have been cleaved is complicated and
slower.
• Some disulfide bonds that are not in the native structure are
formed in the intermediate stages of the folding.
• The protein can utilize a number of alternative pathways to
fold but ultimately finds both its proper tertiary structure and
the correct set of disulfide bonds.
• It is aided in vivo by enzymatic catalysis of -s-sbond rearrangement.
37
When Folding Goes Wrong
• Protein Misfolding: Proteins fold into conformations which are
local energy minima, but not the global minimum
• States reaching local, but not global, minima are sometimes
called “intermediate states”
• Misfolding is responsible for many diseases due to the
accumulation of “plaque” of misfolded proteins – Alzheimers,
Parkinsons, Mad Cow Disease, etc.
• “chaperone” proteins are present to help the target protein
reach the desired functional (native) conformation
Prions - Protein Folding and Mad Cow
Disease
• A class of diseases that is transmitted by a protein.
• Bovine spongiform encephalopathy or mad cow disease is
the best known of these diseases.
• The infectious agent is called prion, and the protein is
called prion-related protein, or PrP.
• PrP protein is normally present in many animals including
human in a nonpathological form of PrPc (prion-related
protein cellular).
• Under certain circumstances (not yet known), PrPc, can
change the conformation to a different structure called PrPsc
(or prion-related protein scrapie).
• The N-terminal portion of PrPsc is partially folded into a βsheet which wreaks havoc with the nervous system.
• It can induce conversion of PrPc in the recipient to PrPsc.
The Protein Folding Problem: Levinthal’s
paradox
How many conformations could a protein chain adopt?
there are about 3-5 combinations of φ/ψ for each amino acid that
would be acceptable to its neighbour.
• so its 3-5n conformations that a protein can adopt (n = number of
amino acids)
• 100 amino acids = 4100 i.e; about 1 x 1060 conformations
•
Levinthal paradox
• If a protein can sample 1014 conformations per second
(average frequency of bond rotation is about 10-14s, 0.01
ps)
• Then it would take our 100 residue protein 1 x 1045
seconds, or 5 x 1038 years to sample all its options.
• Folding cannot simply occur by the protein trying every
conformation until it finds the most stable one. There
isn't enough time.
• HOW DO PROTEINS EVER FOLD????
Time-scales for folding
•
•
•
•
•
cytochrome b562:
lambda repressor:
rat IFABP:
CRABP 1:
tryptophan synthase β2-subunit:
5 μs
0.67 ms
33 ms
24.5 sec
992 sec (396 aa)
Thermodynamic vs. Kinetic control?
• Folding is driven by thermodynamic considerations.
• The native state must be thermodynamically stable.
• But the kinetics of folding is also important.
• Folding must occur rapidly.
• Do folded structures represent true global energy
minimum, or just “kinetically accessible” local minima?
• What causes slow folding: a high transition-state barrier,
or just a large space to search?
Kinetics of Protein Folding
•
Folding takes place through a series of intermediate states,
unfolded protein U, nucleation protein, partially folded II, nearly
folded IN and the final rearrangement to become native protein F.
•
The funneling model for nucleation of proteins proposes that there
is not just one but many possible paths from the denatured state to
the folded state, and each path leads downhill in energy.
•
The molten globule, a compact structure in which much of the
secondary and tertiary folding has occurred, but the internalized
hydrophobic residues have not yet formed.
•
There is also off-path state in which some key element is
incorrectly folded, and the final folding time will be delayed.
Lowest energy state?
•
The forces of macromolecular interaction:
–
–
–
–
–
Covalent bonds
Electrostatics
Hydrogen bonds
van der Waals
Hydrophobic effect
– If not optimal then each of these forces contributes
an energy cost to the system.
– If optimal then the forces above are satisfied and the
energy is minimal
Balls roll downhill, and Levinthal is satisfied
• Levinthal’s problem is
removed if the folding
pathway is biased
towards the correct
structure.
• i.e. once you start moving
in the right direction,
you’ll get there
correctly folded state is the lowest energy state
Folding theory: Energy landscapes
•
formation of local structure
stabilizes
•
An intermediate called the
“transition state ensemble” is
thought to exist
– Metastable
– Key contacts
– Same topology as fully folded
structure
•
enables collapse to native fold
energy
Energy Landscapes in 2-D
E
Transition states
Local minima
Global minimum
•
In the process of folding from a random coil to the native state (at the
global minimum), proteins “pass through” intermediate states (at local
minima)
•
Sometimes the proteins can get “stuck” in these intermediate states and
cannot get over the “energy hump” needed (at transition states) to get to
the final native conformation
•
The study of protein folding from the denatured to the native state of vice
versa, over time, is kinetics
How does force modify the unfolding
landscape?
When you add FORCE (F):
Relative to the native state,
N,
TS
the barrier to unfolding
(∆GTS-N) is lowered by: Fxu
∆∆GTS-N
∆GTS
-N
D
xf
∆∆GD-N
N
xu
and the free energy of
unfolding (∆GD-N) is lowered
by: F(xu + xf)
The protein is less stable
and unfolds more rapidly the unfolding rate (ku) is a
measure of the height of the
barrier between N and TS
The Funnel (3-D) Landscape Model
• Protein Folding actually proceeds (as down a funnel)
through multiple kinetic pathways
• Unstable (High Energy) unfolded conformations at rim can
follow many energy gradients to get to the single global
minimum (Lowest Energy)
• The steepest path is the fastest folding trajectory, but other
slower trajectories to the minimum exist
• Note the diverse multitude of unfolded conformations, but
only one conformation for the native state
Energy Landscapes
How does an energy
landscape solve Levinthal’s
paradox?
a) A random walk to a level
energy surface has little
chance of finding N
b) The barriers surrounding
the minimum energy paths
that connect the different
local minima are very steep
c) A funnel lead to N via many
paths: some straight and
others with intermediates
d) Not a single conformation
of the chain which optimizes
all the interactions at the
same time
a) Golf course, b) Ant-trail, c) energy funnel,
d) realistic rugged funnel
Molten Globule
•
•
•
An intermediate state in the folding
of protein pathway of a protein that
has some secondary and tertiary
structure, but lacks the well packed
amino acid side chains that
characterize the native state of a
protein.
Observed for many protein under
both equilibrium and non-equilibrium
conditions.
By contrast, for fast folding proteins
without intermediates, the search for
a core or nucleus is likely to be the
rate-determine step; once the core is
formed, folding to the native state is
fast
Chaperonins
• A class of proteins which "chaperone" a protein to help
keep it properly folded and non-aggregated.
• Aggregation is a problem for unfolded proteins
• The hydrophobic residues, which normally are deep inside of a
protein, may be exposed when the protein is released from the
ribosome.
• If they are exposed to hydrophobic residues in other
strands, the two strands may associate with each other
hydrophobically (to aggregate) instead of folding properly.
• It provides a central cavity (shelter) in which new protein
chains can be "incubated" until they have folded properl
• ATP is required to drive the process in one direction.
24
Chaperonins
Enzymes that Speed Folding
•
Protein disulphide isomerase Facilitates
formation of correct disulphide bridges
•
Peptidyl proline isomerase Catalyses cistrans isomerisation of peptide bonds
involving proline
•
Molecular chaperones Help folding,
especially of large proteins, by preventing
interaction with other proteins
Prediction of Protein Structure
• Can we predict the protein structure?
• Yes, if we could obtain all the necessary
information, including the amino acid and
understanding the rules of folding.
• Secondary structure can be predicted fairly well
to-date, but not for the tertiary structure.
Problem of protein structure
prediction
• For over 30 years, there has been an ardent
search for methods to the predict threedimensional (3D) structure from the sequence
• Many methods were found which looked
initially very promising - but always the hope
has been dashed
Techniques of Structure Prediction
• Computer simulation based on energy calculation
– Based on physio-chemical principles
– Thermodynamic equilibrium with a minimum free energy
– Global minimum free energy of protein surface
• Knowledge Based approaches
– Homology Based Approach
– Threading Protein Sequence
– Hierarchical Methods