Principles that Govern
Protein Structure and Stability
Understanding Biology through structures
Course work 2009
Forces that stabilize protein structure
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Interactions between atoms within the protein chain
Interactions between the protein and the solvent
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Bond types in proteins
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Covalent bonds
Hydrogen bonds
Metal ligands
Ionic interactions
Disulfide bonds
Non-bond interactions
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Favourable conformations in polypeptides
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Covalent interactions establish the structural framework of the
protein molecule, the chemical expression of primary structure
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Backbone conformation constrained by steric restrictions on 
and  torsions
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Sidechain conformations are also constrained
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Favourable sidechain conformations depend on the sidechain
and also on its neighbours.
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S-S Bonds between cysteine residues can form within proteins
Understanding Biology through structures
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Understanding Biology through structures
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Electrostatic Interactions
• Charged side chains in protein can interact favorably with
an opposing charge of another side chain according to
Coulomb’s law:
q1q 2
F
Dr 2
• Examples of favorable electrostatic interaction include
that between positively charged lysine and negatively
charged glutamic acid.
• Salts have the ability to shield electrostatic interactions.
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Charge-charge interactions
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Coulomb interaction between two ions
At close range, Coulomb interactions are as strong as covalent
bonds
Their energy decreases with 1/r and fall off to less than kT at
about 56 nm separation between charges
In practice, charge-charge interactions have been shown to be
chemically significant at up to 15 Å in proteins
Small charged metal ions can act as positive charge in an ion
pair
O
+
NH3
NH 2
C CH2
O
O
-
O
+
NH3
O P
O
(CH2)4
N
Mg2+
O
O
P
O
O
O
P
O
N
O
N
N
O
OH OH
H2C C
Salt Bridge
O
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Mg-ATP
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Understanding Biology through structures
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Hydrogen bonds
C
O
H
N
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Noncovalent chemical bond in which an electronegative atom
(a hydrogen-bond acceptor) shares a hydrogen atom with an
electronegative atom with a bound hydrogen
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Energy : 10-40 kJ/mol
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Approximately 1.7-3 Å in length
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Strength varies with angle of hydrogen-bond interaction
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Individually, not very strong, but the large numbers of
hydrogen bonds in regular secondary structures stabilize the
framework of the protein
Understanding Biology through structures
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Hydrogen bonds in protein structure
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van der Waals forces
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van der Waals forces are also known as London forces.
They are weak interactions caused by momentary changes in electron density in a molecule.
• They are the only attractive forces present in nonpolar compounds.
Even though CH has no net dipole, at
4 electron density may
any one instant its
not be completely symmetrical, resulting
in a temporary dipole. This can induce a
temporary dipole in another molecule.
The weak interaction of these temporary
dipoles constituents van der Waals
forces.
The surface area of a molecule
determines the strength of the van der
Waals interactions between molecules.
The larger the surface area, the larger
the attractive force between two
molecules, and the stronger the
intermolecular forces.
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Dispersion
A neutral atom: argon. It is like a large spherical jelly
with a golf ball at the centre. The golf ball is the nucleus
carrying a large positive charge and the jelly
represents the clouds of electrons.
At a point external to the atom the net average field will be zero because
the positively-charged nucleus' field will be exactly balanced by the
electron clouds.
However, atoms vibrate (even at 0K)
and so that at any instant the cloud
is likely to be slightly off centre.
This disparity creates an "instantaneous
dipole":
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Dispersion
Suppose that we have another argon atom close to the first. This atom will
see the electric field resulting from the instantaneous dipole.
This field will effect the the electron and induce a dipole
The two dipoles attract one another - producing an attractive interaction.
The Dispersion interaction can be shown to vary according to the inverse
sixth power of the distance between the two atoms:
B depends on the polarizability of the atoms
r is the distance between them
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Repulsion
When two atoms are brought increasing close together there is a large
energetic cost as the orbitals start to overlap.
The Pauli exclusion principle states that no two electrons can share the
same state so that in effect half the electrons of the system would have
to go into orbitals with an energy higher than the valence state.
For this reason the repulsive core is sometimes termed a
"Pauli exclusion interaction".
The Hard Sphere Model:
atoms have a characteristic radius
(below the van der Waals radius) and cannot overlap.
Can represent the energy costs of close approach using a term
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van der Waals forces
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Nonspecific forces between like or unlike atoms
Decrease with r6
approximately 1 kJ/mol
If r0 is the sum of van der Waals radii for the two atoms. Van
der Waals forces are attractive forces when r> r0 and repulsive
when r< r0.
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Approximate Strengths of
Interactions between atoms
Bond Type
Covalent Bond
kJ/mol
250
Electrostatic
5
van der Waals
5
Hydrogen bond
20
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The hydrophobic force
Observation:
• Hydrophobic residues are buried while hydrophilic residues are
on the outside.
• The exterior surface area of proteins can be up to 60% polar
atoms
• Proteins fold to maximize their effectiveness as hydrogen-bonding
partners to water
Explanation:
• When hydrophobic residues are exposed to solvent, the extended
hydrogen bonding structure of water is disrupted
• Breaking hydrogen bonds in water is energetically unfavourable
• Water molecules reorient around the hydrophobic molecule, so that
the least number of hydrogen bonds are sacrificed to accommodate
it
• Burying hydrophobic residues releases water and increases
entropy.
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Protein Interior and Exterior
Internal packing of atoms in a protein can be analysed by depicting every
atom in the protein as a sphere with the appropriate van der Waals radius.
Overlapping regions (regions of covalent bonds) are truncated. This
surface is called the van der Waals surface.
Cannot measure a van der Waals surface of a protein because any chemical
probe will have some dimension greater than zero.
A more realistic representation is the solvent accessible surface that is
defined by the center of a water molecule (sphere with radius 1.4 Å) as it
moves over the surface of a protein.
Understanding Biology through structures
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Protein Interior and Exterior
A more realistic representation is the solvent accessible surface that is
defined by the center of a water molecule (sphere with radius 1.4 Å) as it
moves over the surface of a protein.
Protein-protein interactions, which
form the basis for most cellular
processes, result in the formation
of protein interfaces.
The protein-protein docking
problem is the prediction of a
complex between two proteins
given the three-dimensional
structures of the individual proteins
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Packing of Globular Proteins
• Secondary structures pack closely to one another and also
intercalate with extended polypeptide chains
• Most polar residues face the outside of protein and interact with
solvent but may be buried if H-bonding and charge is satisfied
• Most hydrophobic residues face the interior of the protein and
interact with each other thereby minimizing contact with water
• van der Waal’s volume is about 72-77% of the total protein
volume; about 25% is not occupied by protein atoms. These
cavities provide flexibility in protein conformation and dynamics
• Random coil or loops maybe of importance in protein function
(interacting with other molecules, enzyme reactions)
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The Thermodynamics of Folding
• Folding of a globular protein is a thermodynamically favored
process, i.e. G must be negative.
G = H - TS
• The folding process involves going from a multitude of
random-coil conformations to a single folded structure.
• The folding process involves a decrease in randomness and
thus a decrease in entropy -S and an overall positive
contribution to G. This decrease in entropy is termed
“conformational entropy”.
• An overall negative G : a result of features that yield a large
negative H or some other increase in entropy on folding.
Understanding Biology through structures
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Protein Folding :
No Net Enthalpic Contribution
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Formation of secondary structure is an enthalpy driven process
– Energy derived from the formation of many van der waals and Hbonding
interactions as well as the alignment of dipoles
overcomes the loss of entropy associated with the formation of the
peptide backbone conformation.
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Formation of tertiary structure is enthalpically unfavorable
– Energy loss in the burying of ion-pairs (~1 kcal/mol) and the
breaking of shorter, stronger H2O bonds.
– Though some energy is gained from van der waals packing, very
little is gained from the formation of internal h-bonds because as
many h-bonds with water are broken as are formed in the process
of folding a protein.
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Free energy associated with solvation of an ion is ~-60 kcal/mol
– An ion will NOT be buried in the hydrophobic interior of a protein.
Understanding Biology through structures
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Protein Folding :
Entropy Driven Process
• Upon protein folding
– hydrophobic residues move to the interior of the protein
– caged H2O molecules are released
– Enthalpy is gained : unfavorable (H +)
– entropy is also gained (S +) : extremely favorable
• Increase in entropy of water compensates for the loss of
conformational entropy of the protein and drives the
protein folding process
Understanding Biology through structures
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Free energy of folding
• Difference in energy (free energy) between folded (native) and
unfolded (denatured) state is small, 5-15 kcal/mol
• Enthalpy and entropy differences balance each other, and G is
a small positive number.
• Small G 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
Random Coil
Native conformation
• What are the forces that guide this process?
• What are the Steps Involved?
• How Fast Can this Happen?
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The Thermodynamic Hypothesis
“The native, folded structure of a protein, under optimal
conditions, is the most energetically stable conformation
possible”
Christian Anfinsen, 1972
•Most of the information for determining the three-dimensional
structure of a protein is carried in its amino acid sequence
Anfinsen, C.B. Principles that govern the folding of
protein chains. Science 181, 223-30 (1973).
Understanding Biology through structures
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Levinthal’s Paradox
Consider a protein of 100 amino acids. Assign 2
conformations to each amino acid. The total
conformations of the protein is 2100=1.27x1030. Allow 10-13
sec for the protein to sample through one conformation in
search for the overall energy minimum. The time it needs
to sample through all conformations is
(10-13)(1.27x1030)=1.27x1017sec = 4x109 years!
Levinthal’s paradox illustrates that proteins must only
sample through limited conformations, or fold by “specific
pathways”. Much research efforts are devoted in
searching for the principles of the “specific pathways”.
Understanding Biology through structures
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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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The Kinetic Theory of Protein Folding
-Folding proceeds through a definite series of steps or a
Pathway. A protein does not try out all possible rotations of
conformational angles, but only enough to find the pathway
- The final state may NOT be the most stable conformation
possible, but it could be the most stable conformation that is
accessible in a reasonable amount of time. This is also the
biologically important time frame
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Folding Pathways
• Protein folding is initiated by reversible and rapid formation
of local secondary structures
• Secondary structures then form domains through the
cooperative aggregation of folding nuclei
• Domains finally form the final protein through “Molten
globule” intermediates.
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Models for Protein Folding
1. Hydrophobic collapse. Formation of a 'molten globule'
2. Framework model. Secondary structure forms first,
perhaps including supersecondary structure.
3. Nucleation. Domains fold independently, and subdomains serve as 'structural kernels’.
Understanding Biology through structures
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Framework model
• Local interactions are the main determinants of protein
structures
• Interactions as the ones responsible for forming secondary
structural elements, a-helices and b-sheets
• Isolated helices/sheets form early in the protein folding
pathway, then assemble in the native tertiary structure
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What's really happening?
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Hydrophobic side chains are being buried
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Secondary structure formation insulates the polar
protein backbone from the nonpolar protein interior
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Hydrogen bonds, disulfide bonds and salt bridges begin
to form and stabilize structure
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van der Waals interactions bring protein substructures
into stable contact
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What's most important in folding?
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Nonspecific interactions (hydrophobic effect, van der
Waals) are required to bring the protein together into a
globular conformation
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Steric interactions (restraints on the backbone) limit the
ways in which the globular conformation can form
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Chemically specific interactions (hydrogen bonds, ionic
interactions, dipolar interactions) determine the fine detail
of the protein structure
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Enzymes that speed folding
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Protein disulphide isomerase Facilitates formation of
correct disulphide bridges
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Peptidyl proline isomerase
Catalyses cis-trans
isomerisation of peptide bonds involving proline
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Molecular chaperones Help folding, especially of large
proteins, by preventing interaction with other proteins
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What is the molecular basis of the
observation that proteins of
thermophiles are more stable than
their homologues in mesophiles?
Understanding Biology through structures
Course work 2009