Transcript Lecture 1. Introduction to Biochemistry

Protein Folding & Biospectroscopy
Lecture 3
F14PFB
David Robinson
Protein Folding
2.
Introduction
Protein Structure
3.
Interactions
1.
4.
5.
6.
Protein Folding Models
Biomolecular Modelling
Bioinformatics
Tertiary Structure
determined by weak interactions
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Steric
Hydrogen bonds
Electrostatic interactions (ionic bonds)
Hydrophobic interactions
Van der Waals interactions
Backbone Torsion Angles
• ω tends to be planar (0º - cis, or 180 º - trans)
- due to delocalization of carbonyl p electrons
and nitrogen lone pair
• φ and ψ are flexible  rotation
• but limited rotation -- due to steric hindrance
• Only 10% of the {φ, ψ} combinations are
generally observed
• First noticed by G.N. Ramachandran
Ramachandran Plot
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Used computer models of small
polypeptides to vary φ and ψ
systematically, to find stable
conformations
Atoms treated as hard spheres,
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with radius = van der Waals radius
φ and ψ angles which cause spheres to
collide correspond to sterically disallowed
conformations
Computed Ramachandran Plot
White = sterically
disallowed
conformations (atoms
come closer than sum of
van der Waals radii)
Blue = sterically
allowed conformations
Experimental Ramachandran
Plot
φ, ψ distribution in 42 high-resolution
protein structures (x-ray crystallography)
Ramachandran Plot
& Secondary Structure
• Repeating values of φ and ψ along the chain 
regular structure
• Example:
repeating values of φ ~ -57° and ψ ~ -47°
 a-helix
Cytochrome C: many segments of helix.
Ramachandran plot - tight grouping of φ, ψ angles near
-60°, -40°
a-helix
cytochrome C Ramachandran plot
Repetitive values at φ = -110° to –140° and ψ = +110°
to +135°  b strands.
Plastocyanin: mostly b sheets; Ramachandran plot
shows values in the –110°, +130° region.
b-sheet
plastocyanin Ramachandran plot
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White = sterically
disallowed
conformations
Red = sterically
allowed regions
(right-handed a helix
and b sheet) with
strict (greater) radii
Yellow = sterically
allowed if shorter
radii are used (i.e.
atoms can be closer
together; brings out
left-handed helix)
Non-bonding Forces Influence Protein Structure
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Amino acids of a protein are joined by covalent
bonding interactions. The polypeptide is folded
in three dimensions by non-bonding
interactions. These interactions can easily be
disrupted by extreme pH, temperature,
denaturants, reducing reagents.
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H-bond interactions (12-30 kJ/mol)
Hydrophobic Interactions (<40 kJ/mol)
Electrostatic Interactions (20 kJ/mol)
Van der Waals Interactions (0.4-4 kJ/mol)
The total inter-atomic force acting between
two atoms is the sum of all the forces they
exert on each other.
Hydrogen bonds
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H-bond: a favourable interaction between a
proton bonded to an electronegative atom and
an atom carrying a lone pair of electrons.
D-H + A
Acceptors (A):
Donors (D):
D H A
C
O
N
O
H
O
H
O
H
N
Important for maintaining backbone interactions
Hydrogen bonds are dependent on geometry
Hydrogen bonds
Example: hydrogen bonds
(white dash lines) hold a small
molecule in place at the active site
of an enzyme.
H-bonds in helix
Peptide
bond
Thermodynamics
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First law: total energy of a system and its
surroundings is constant. Energy released from
formation of chemical bonds must be used to
generate heat or to form other new chemical bonds
or both.
Second law: the total entropy of a system and its
surroundings always increases for a spontaneous
process.
The First Law of Thermodynamics
The 1st law can be expressed as:
“The internal energy of an isolated system is constant”
Essentially this means that energy is neither created nor destroyed,
but only transformed from one form to another.
For processes occurring with no volume change, w=0 and the internal
energy change is equal to the heat transferred between the system
and its surroundings
U  qV
However, very many processes take place under conditions of
constant pressure rather than constant volume when
U  qP  w
For constant pressure processes it is convenient to refer to changes in
the enthalpy of a system.
qP  H
A heat change at constant pressure equals the enthalpy change.
Definition of entropy change
When a quantity of heat is transferred to a system at constant temperature
and under reversible conditions the change in the entropy of the system is:
qrev
S 
T
The Second Law of thermodynamics:
“spontaneous processes are those which increase the entropy of
the universe”.
The Third Law of thermodynamics:
“the entropy of perfect crystals at zero Kelvin (absolute zero) is
zero”.
Gibbs energy defined
J.Willard Gibbs
1839-1903
Gibbs introduced a thermodynamic function G which specifies the state of a
system in terms of its enthalpy, entropy and temperature:
G = H – TS
At constant temperature
G = H - TS
and the criterion for a spontaneous change at constant temperature and
pressure can be expressed as:
G < 0
i.e. spontaneous changes decrease the Gibbs energy of a system.
G is also known as the change in ‘Gibbs free energy’ because it gives the
amount of energy which is free to do useful work.
Protein folding represents a
significant decrease in entropy,
but it occurs spontaneously.
This is due to an increase in
entropy of the water molecules
surrounding the protein.
The increase in entropy of the water molecules is due to
the hydrophobic nature of proteins and is termed the
hydrophobic effect. The water molecules around a non
polar molecule are extremely low in entropy (well
ordered). “Oil and water don’t mix.”
Hydrophobic Interactions
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Hydrophobic interactions minimize
interactions of non-polar residues with solvent.
Thus, nonpolar regions of proteins are often
buried in the interior, to exclude them from
the aqueous milieu.
However, non-polar residues can also be found
on the surface of a protein - to participate
protein-protein interactions.
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This type of interaction is entropy driven.
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poor solubility of non-polar groups in water is due to
the ordering of the surrounding water molecules
Electrostatic Interactions
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Charged side chains can interact (dis)favourably
with a charge of another side chain. According
to Coulomb’s law, the electrostatic force:
q1 q 2
F
2
Dr
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Favourable electrostatic interactions include that
between positively charged lysine and
negatively charged glutamic acid.
Salts shield electrostatic interactions.
Examples of Electrostatic Interactions
Intramolecular ionic
bonds between charged
amino acid residues
in a protein:
Magnesium ATP
NH2
N
Mg2+
O
+
NH3
O
C CH2
O
O P
O
O
O P
O
O
O
P
O
N
O
O
OH OH
-
O
H2C C
O
+
NH3
(CH2)4
N
N
van der Waals Interactions
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van der Waals interaction between two atoms is a result of electron
charge distributions of the two atoms.
For atoms that have permanent dipoles:
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Dipole-dipole interactions (potential energy ~r-3)
Dipole-induced dipole interactions (potential energy ~r-5)
For atoms that have no permanent dipoles:
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Transient charge distribution induces complementary charge distribution
(also called dispersion or London dispersion force) (potential energy ~r-6)
d+
transient
dipole
d-
d+
transient
dipole
d-
d+
d-
Repulsion between two atoms when they approach each other due to
overlapping of electron clouds (potential energy ~r-12)
van der Waals Interactions
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In general, the permanent dipole contribution is much less than the
dispersion and repulsion forces. Thus the van der Waals potential can
be expressed as 1/r12-1/r6.
r0 is the sum of van der Waals radii for the two atoms. Van der Waals forces are
attractive when r> r0 and repulsive when r< r0.
r0
H 0.1 nm
C 0.17 nm
N 0.15 nm
O 0.14 nm
P 0.19 nm
S 0.185 nm
Van der Waals potential
Van der Waals Force
Van der waals radii
of common atoms:
r0
r
Role of Sequence in Protein Structure
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Weak forces operate both within the
protein structure, between proteins and the
water solvent;
All of the information necessary for folding
the peptide chain into its “native” structure
is contained in the amino acid sequence;
Certain loci along the peptide chain act as
nucleation points;
Folded proteins avoid local energy minima.
Denaturation
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The loss of structural order in biomolecules is
called denaturation;
The central role of weak forces in biomolecular
interactions restricts the folding (and thus
function) of proteins to a narrow range of
physical conditions, such as temperature, ionic
strength, and relative acidity;
Extremes of these conditions disrupt the weak
forces essential to maintaining the intricate
structure and will lead to loss of their
biological functions.