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Lecture notes MCB2021F
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This module discusses secondary, tertiary and quaternary structure of proteins.
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➰Protein Secondary Structure
How do we know that the three-dimensional structure of a protein that confers its
function is determined only by its amino acid sequence, its primary structure?
This was demonstrated by Christian Anfinsen in a series of elegant experiments
with the protein called RNase A, an enzyme that cleaves single-stranded RNA
molecules.
RNase A consists of a single polypeptide chain that contains several
intramolecular disulphide bonds.
Anfinsen treated RNase A with beta-mercaptoethanol to break the disulphide
bonds by reductive cleavage and with a high concentration of urea, a chaotropic
reagent that disrupts the weak, non-covalent forces that drive folding of the
protein into an active three-dimensional structure.
The result of this treatment is that the polypeptide chain of RNase A completely
unfolds. The unfolding of RNase A can be monitored by following its enzymatic
activity.
Because proteins must be folded into their correct three-dimensional structure
on order to function, unfolding leads to a loss of function.
The process by which proteins lose their three-dimensional structure is called
denaturation and unfolded proteins are called denatured proteins.
The important observation was that this process is reversible. The inactive,
unfolded RNase A spontaneously refolds into its native three-dimensional
structure and becomes active again if one slowly removes the beta-
mercaptoethanol and urea by dialysis.
Based on these experiments, Anfinsen postulated that, at least for small globular
proteins, the characteristic and functional three-dimensional structure of proteins
is determined only by their amino acid sequence and the environment in which
protein folding occurs.
That means that at physiological conditions in the cell, the correctly folded
protein structure must correspond to a Gibbs free energy minimum.
Protein Secondary Structure 1
, Gibbs free energy
The Gibbs free energy of a system at any moment in time is defined as the
enthalpy of the system minus the product of the temperature times the entropy of
the system.
Any reaction for which the change in the Gibbs free energy is negative, i.e., the
free energy of the system decreases during the reaction, is energetically
favourable and proceeds spontaneously in the written direction.
Since the folding of proteins occurs spontaneously, it follows that the folded
protein state must be at a lower energy state than the unfolded state.
Because the activity of the protein requires a specific three-dimensional
structure, this structure must represent an energy minimum when compared to
other possible protein folding conformations.
Protein folding is driven by weak, non-covalent interactions, including
electrostatic interactions (involve ions or dipoles), hydrogen bonds(electrostatic
interactions between a hydrogen attached to an electronegative atom, the
hydrogen bond donor, and a second electronegative atom, the hydrogen bond
acceptor.), Van der Waals interactions and the hydrophobic effect.
The final structure may also be stabilised by covalent bonds, by disulphide
bonds that we already discussed.
As I already mentioned, the non-covalent interactions stabilising the native
protein structure can be disrupted by different means, including high
temperature, extreme pH and denaturing agents such as urea and SDS.
Van der Waals forces occur between closely approaching atoms. This is
essentially an electrostatic interaction resulting in a small, distance-dependent
attractive force. However, if atoms get too close, the electron clouds start to
clash and that results in a strong repulsive force.
The most important driving force for protein folding is the hydrophobic effect.
Where non-polar substances are dissolved in water, water molecules form cage-
like structures around them called clathrates.
Because these structures are more restricted than free water molecules,
clathrate formation causes a loss in entropy, which is energetically unfavourable.
Protein Secondary Structure 2
, The energy of the system is lowered by spontaneous clustering of the non-polar
substances, which minimises exposure of the non-polar substance to the
surrounding water and which reduces the extent of clathrate formation.
Since the hydrophobic effect minimises the interaction of non-polar residues with
the polar water, the majority of non-polar amino acid sidechains of proteins are
generally buried in the interior of the protein structure.
An exception to this rule are of course membrane proteins that, because they
exist in a non-polar environment, present non-polar amino acid sidechains on
the protein surface.
Free energy change for protein folding
Let’s have a look at the overall free energy change for protein folding. As you
can see from the schematic, protein folding results in a loss in entropy, which is
energetically unfavourable.
The loss in entropy cannot be fully compensated by energetically favourable
interactions between functional groups and amino acid sidechains and in the
polypeptide backbone, such as hydrogen bonding, Van der Waals interactions
and electrostatic interactions.
What tips the balance in favour of protein folding is the contribution of the
hydrophobic effect.
There are four levels of protein structure:
1. Primary structure refers to the amino acid sequence of the polypeptide chain or
polypeptide chains making up the protein.
2. Secondary structures are regular, local structures formed spontaneously through
hydrogen bonding.
3. Tertiary structure refers to the fully folded 3D conformation of the polypeptide
chain.
4. Quaternary structure refers to the structural organisation of proteins that consist
of more than one polypeptide chain.
Part B
Protein Secondary Structure 3
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