Last Update: 2 November 2017

ZOO
Part I
Protein structure M - 08
Proteins
M
Proteins are very complicated molecules. With 20 different amino acids that can be arranged in any
order to make a polypeptide of up to thousands of amino acids long, their potential for variety is
extraordinary. This variety allows proteins to function as exquisitely specific enzymes that compose a cell's
metabolism. An E. coli bacterium , one of the most simple biological organisms, has over a 1000 different
proteins working at various times to catalyze the necessary reactions to sustain life.

Amino Acids Diagram

All amino acids have the same general formula:

The twenty amino acids found in biological systems are:

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All proteins are linear chains composed of these 20 amino acids.
Polypeptides
Polypeptides are chains of amino acids. Proteins are
made up of one or more polypeptide molecules.
The amino acids are linked covalently by peptide bonds.
The image shows how three amino acids linked by peptide bonds
into a tripeptide.
One end of every polypeptide, called the amino terminal
or N-terminal, has a free amino group. The other end, with its
free carboxyl group, is called the carboxyl terminal or C-
terminal.
The sequence of amino acids in a polypeptide is dictated
by the codons in the messenger RNA (mRNA) molecules from
which the polypeptide was translated. The sequence of codons in
the mRNA was, in turn, dictated by the sequence of codons in
the DNA from which the mRNA was transcribed.
The schematic below shows the N-terminal at the upper
left and the C-terminal at the lower right.

Primary Structure
The primary structure of peptides and proteins refers to the linear number and order of the amino
acids present. The convention for the designation of the order of amino
acids is that the N-terminal end (i.e. the end bearing the residue with the free
a-amino group) is to the left (and the number 1 amino acid) and the C-
terminal end (i.e. the end with the residue containing a free a-carboxyl
group) is to the right.

Secondary Structure
Protein secondary structure refers to certain common repeating structures found in proteins. There
are two types of secondary structures: alpha-helix and beta-pleated sheet.
An alpha-helix is a tight helix formed out of the polypeptide chain. The polypeptide main chain
makes up the central structure, and the side chains extend out and away from the helix.
The CO group of one amino acid (n) is hydrogen bonded to the NH group of the amino
acid four residues away (n +4). In this way every CO and NH group of the backbone is
hydrogen bonded.
Here are three models of an alpha-helix. The first shows only the alpha-carbon
of each amino acid. The second shows all of the atoms that make up the backbone of
the polypeptide. The third shows all of the hydrogen bonds that hold alpha-helices
together. The third, and most complete, model is also shown here.

Alpha Helix

the R groups of the amino acids all extend to the outside

the helix makes a complete turn every 3.6 amino acids
the helix is right-handed; it twists in a clockwise direction
the carbonyl group (-C=O) of each peptide bond extends parallel to the axis of
the helix and points directly at the -N-H group of the peptide bond 4 amino acids below it in the
helix. A hydrogen bond forms between them
[-N-HO=C-]

Beta Conformation

consists of pairs of chains lying side-by-side

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 stabilized by hydrogen bonds between the carbonyl oxygen atom on one chain and the -NH group on
the adjacent chain.
the chains are often "anti-parallel"; the N-terminal to C-terminal direction of one being the reverse of
the other where amino acid n hydrogen bonded to amino acid (n +3) in a hairpin turn.

There is a special type of molecular model

used to highlight protein secondary structure. Follow
this link to view an example. This common type of
protein model represents segments of beta-pleated
sheets as ribbon arrows, and it
represents a-helices as helical
ribbons. The remainder of the
polypeptide chain is referred to
as random coil, and is
represented as a thin line.
Please note that random coil is somewhat of a
misnomer; the protein is definitely highly organized,
but the random coil regions do not show any easily
categorized secondary structure components.

Figure - Peptide Bonds Are Planar. In a pair of linked Figure - Typical Bond Lengths Within a Peptide
amino acids, six atoms (C a , C, O, N, H, and C a ) lie in Unit. The peptide unit is shown in the trans
a plane. Side chains are shown as green balls. configuration.

Figure - Trans and Cis Peptide Bonds. The trans form is strongly favored because of steric clashes that
occur in the cis form.

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Fig. Rotation About Bonds in a Polypeptide. The structure of each amino acid in a polypeptide can be
adjusted by rotation about two single bonds. (A) Phi (f) is the angle of rotation about the bond between the
nitrogen and the a-carbon atoms, whereas psi (y) is the angle of rotation about the bond between the a-
carbon and the carbonyl carbon atoms. (B) A view down the bond between the nitrogen and the a-carbon
atoms, showing how f is measured. (C) A view down the bond between the a-carbon and the carbonyl
carbon atoms, showing how y is measured.

Figure - A Ramachandran Diagram Showing the Values of f and y. Not all f and y values are possible
without collisions between atoms. The most favorable regions are shown in dark green; borderline regions
are shown in light green. The structure on the right is disfavored because of steric clashes.

Ramachandran Plot:
A Ramachandran plot (also known as a Ramachandran map or a Ramachandran diagram), developed
by Gopalasamudram Narayana Ramachandran, is a way to visualize dihedral angles against of
amino acid residues in protein structure. It shows the possible conformations of and angles for a
polypeptide.

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Mathematically, the Ramachandran plot is the visualization of a function . The
domain of this function is the torus.
Hence, the conventional
Ramachandran plot is a projection of
the torus on the plane, resulting in a
distorted view and the presence of
discontinuities.
One would expect that larger side
chains would result in more
restrictions and consequently a
smaller allowable region in the
Ramachandran plot. In practice this
does not appear to be the case; only
the methylene group at the position
has an influence. Glycine has a
hydrogen atom, with a smaller van
der Waals radius, instead of a methyl
group at the position. Hence it is
least restricted and this is apparent in
the Ramachandran plot for Glycine
for which the allowable area is
considerably larger.
In contrast, the Ramachandran plot
for proline shows only a very limited
number of possible combinations of
and .
One can also plot the dihedral angles in polysaccharides and other polymers in this fashion. For the first two
protein side-chain dihedral angles a similar plot is the Janin Plot.

Super-secondary Structure
Some proteins contain an ordered organization of secondary structures that form distinct functional
domains or structural motifs. Examples include the helix-turn-helix domain of bacterial proteins that
regulate transcription and the leucine zipper, helix-loop-helix and zinc finger domains of eukaryotic
transcriptional regulators. These domains are termed super-secondary structures.

Tertiary Structure
Tertiary structure refers to the complete three-dimensional
structure of the polypeptide units of a given protein. Included in this
description is the spatial relationship of different secondary
structures to one another within a polypeptide chain and how these
secondary structures themselves fold into the three-dimensional
form of the protein. Secondary structures of proteins often constitute
distinct domains. Therefore, tertiary structure also describes the
relationship of different domains to one another within a protein.
The interactions of different domains is governed by several forces:
These include hydrogen bonding, hydrophobic interactions, electrostatic interactions and van der
Waals forces.

Tertiary structure is important!

The function of a protein (except as food) depends on its tertiary structure. If this is disrupted, the protein
is said to be denatured [Discussion], and it loses its activity. Examples:

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 denatured enzymes lose their catalytic power
denatured antibodies can no longer bind antigen

A mutation in the gene encoding a protein is a frequent cause of altered tertiary structure.

The mutant versions of proteins may fail to reach their proper destination in the cell and/or be
degraded.
Examples:
o Cystic fibrosis is caused failure of the mutant CFTR protein to reach its destination in the
plasma membrane [More].
o Diabetes insipidus is caused by improper folding of mutant versions of
V2 - the vasopressin (ADH) receptor or
aquaporin
o Familial hypercholesterolemia is caused by failure of mutant low-density lipoprotein
(LDL) receptors to reach the plasma membrane. [Discussion]
o Osteogenesis imperfecta is caused by failure of mutant Type I collagen molecules to
assemble correctly. [More]
Mutant proteins may aggregate forming insoluble, nonfunctional deposits. This is particularly likely
if the mutation causes hydrophobic R groups to be displayed at the surface of the molecule rather
than in its interior.
Examples:
o Alzheimer's disease is characterized by insoluble protein deposits - called amyloid - in the
brain.
o Bovine spongiform encephalopathy (BSE) ("mad cow") disease and the human version -
Creutzfeldt-Jakob disease (CJD) - are characterized by amyloid deposits in the brain of a
mutant version of the prion protein.

The normal protein has lots of alpha helical regions and is soluble. In the mutant version, the
alpha helix is converted into beta conformation and the protein becomes insoluble.

Curiously, tiny amounts of the mutant version can trigger the alpha-to-beta conversion in the
normal protein. Thus the mutant version can be infectious. There have been several cases in
Europe of people ill with Creutzfeldt-Jakob disease that may have acquired it from ingesting
tiny amounts of the mutant protein in their beef.

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Quaternary Structure
Many proteins contain 2
or more different polypeptide
chains that are held in association
by the same non-covalent forces
that stabilize the tertiary
structures of proteins. Proteins
with multiple polypeptide chains
are termed oligomeric proteins.
The structure formed by
monomer-monomer interaction in
an oligomeric protein is known as
quaternary structure.
Oligomeric proteins can
be composed of multiple identical
polypeptide chains or multiple
distinct polypeptide chains.
Proteins with identical subunits
are termed homooligomers.
Proteins containing several distinct polypeptide chains are termed heterooligomers.
Hemoglobin, the oxygen carrying protein of the blood, contains two a and two b subunits arranged
with a quaternary structure in the form, a2b2. Hemoglobin is, therefore, a hetero-oligomeric protein.

Fig. Amino acid sequence of bovine Insulin

Complex Protein Structures

Proteins also are found to be covalently conjugated with carbohydrates. These modifications occur
following the synthesis (translation) of proteins and are, therefore, termed post-translational modifications.
These forms of modification impart specialized functions upon the resultant proteins. Proteins covalently
associated with carbohydrates are termed glycoproteins. Glycoproteins are of two classes, N-linked and O-
linked, referring to the site of covalent attachment of the sugar moieties. N-linked sugars are attached to the
amide nitrogen of the R-group of asparagine; O-linked sugars are attached to the hydroxyl groups of either
serine or threonine and occasionally to the hydroxyl group of the modified amino acid, hydroxylysine.
There are extremely important glycoproteins found on the surface of erythrocytes. It is the variability
in the composition of the carbohydrate portions of many glycoproteins and glycolipids of erythrocytes that
determines blood group specificities. There are at least 100 blood group determinants, most of which are due
to carbohydrate differences. The most common blood groups, A, B, and O, are specified by the activity of
specific gene products whose activities are to incorporate distinct sugar groups onto RBC membrane
glycoshpingolipids as well as secreted glycoproteins.
Structural complexes involving protein associated with lipid via noncovalent interactions are termed
lipoproteins. Their major function in the body is to aid in the storage transport of lipid and cholesterol.

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Forces Controlling Protein Structure
Hydrogen Bonding:
Polypeptides contain numerous proton donors and acceptors both in their backbone and in the R-
groups of the amino acids. The environment in which proteins are found also contains the ample H-bond
donors and acceptors of the water molecule. H-bonding, therefore, occurs not only within and between
polypeptide chains but with the surrounding aqueous medium.

Hydrophobic Forces:
Proteins are composed of amino acids
that contain either hydrophilic or hydrophobic
R-groups. It is the nature of the interaction of
the different R-groups with the aqueous
environment that plays the major role in
shaping protein structure. The spontaneous
folded state of globular proteins is a reflection
of a balance between the opposing energetics
of H-bonding between hydrophilic R-groups
and the aqueous environment and the
repulsion from the aqueous environment by
the hydrophobic R-groups. The
hydrophobicity of certain amino acid R-
groups tends to drive them away from the
exterior of proteins and into the interior. This
driving force restricts the available
conformations into which a protein may fold.

Electrostatic Forces:
Electrostatic forces are mainly of
three types; charge-charge, charge-dipole
and dipole-dipole. Typical charge-charge
interactions that favor protein folding are
those between oppositely charged R-groups
such as K or R and D or E. A substantial component of the energy involved in protein folding is charge-
dipole interactions. This refers to the interaction of ionized R-groups of amino acids with the dipole of the
water molecule. The slight dipole moment that exist in the polar R-groups of amino acid also influences
their interaction with water. It is, therefore, understandable that the majority of the amino acids found on the
exterior surfaces of globular proteins contain charged or polar R-groups.

van der Waals Forces:

There are both attractive and repulsive van der Waals forces that control protein folding. Attractive
van der Waals forces involve the interactions among induced dipoles that arise from fluctuations in the
charge densities that occur between adjacent uncharged non-bonded atoms. Repulsive van der Waals forces
involve the interactions that occur when uncharged non-bonded atoms come very close together but do not
induce dipoles. The repulsion is the result of the electron-electron repulsion that occurs as two clouds of
electrons begin to overlap.
Although van der Waals forces are extremely weak, relative to other forces governing conformation,
it is the huge number of such interactions that occur in large protein molecules that make them significant to
the folding of proteins.

The Rules of Protein Structure

The function of a protein is determined by its shape.

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 The shape of a protein is determined by its primary structure (sequence of amino acids).
The sequence of amino acids in a protein is determined by the sequence of nucleotides in the gene
(DNA) encoding it.

The function of a protein (except when it is serving as food) is absolutely dependent on its three-
dimensional structure. A number of agents can disrupt this structure thus denaturing the protein.

changes in pH (alters electrostatic interactions between charged amino acids)

changes in salt concentration (does the same)
changes in temperature (higher temperatures reduce the strength of hydrogen bonds)
presence of reducing agents (break S-S bonds between cysteines)

None of these agents breaks peptide bonds, so the primary structure of a protein remains intact when it
is denatured.
When a protein is denatured, it loses its function.
Examples:

A denatured enzyme ceases to function.

A denatured antibody no longer can bind its antigen.

Often when a protein has been gently denatured and then is returned to normal physiological conditions
of temperature, pH, salt concentration, etc., it spontaneously regains its function (e.g. enzymatic activity or
ability to bind its antigen).
This tells us

The protein has spontaneously resumed its native three-dimensional shape.

Its ability to do so is intrinsic; no outside agent was needed to get it to refold properly.

However, there are proteins, called molecular chaperones, that may enable a newly-synthesized protein
to acquire its final shape faster and more reliably than it otherwise would.
Chaperones
Although the three-dimensional (tertiary) structure of a protein is determined by its primary structure, it may
need assistance in achieving its final shape.

As a polypeptide is being synthesized, it emerges (N-terminal first) from the ribosome and the
folding process begins.
However, the emerging polypeptide finds itself surrounded by the watery cytosol and many other
proteins.
As hydrophobic amino acids appear, they must find other hydrophobic amino acids to associate with.
Ideally, these should be their own, but there is the danger that they could associate with nearby
proteins instead - leading to aggregation and a failure to form the proper tertiary structure.

To avoid this problem, the cells of all organisms contain molecular chaperones that stabilize newly-formed
polypeptides while they fold into their proper structure.
Most (~80%) newly-synthesized proteins are stabilized by molecular chaperones that bind briefly to their
surface until they have folded properly. The chaperones use the energy of ATP to do this work.
Chaperonins
However, some proteins (~20%) are so complex that a different group of chaperones - called
chaperonins - are needed.
Chaperonins are hollow cylinders into which the newly-synthesized protein fits while it folds. The
inner wall of the cylinder is lined with hydrophobic amino acids which stabilize the hydrophobic regions of
the polypeptide chain while it folds safely away from the

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 watery cytosol and
other proteins outside.

Chaperonins also use ATP as the energy source to drive the folding process.
As mentioned above, high temperatures can denature proteins, and when a cell is exposed to high
temperatures, several types of chaperonins swing into action. For this reason, these chaperonins are also
called heat-shock proteins (HSPs).
Protein aggregation is the cause of some disorders such as Alzheimer's disease, Huntington's disease,
and prion diseases (e.g., "mad-cow" disease). Perhaps, a failure of chaperones is involved. If so, perhaps
ways can be found to treat these diseases by increasing the efficiency of chaperones.
Despite the importance of chaperones, the rule still holds; the final shape of a protein is determined by
only one thing: the precise sequence of amino acids in the protein.
And the sequence of amino acids in every protein is dictated by the sequence of nucleotides in the gene
encoding that protein. So the function of each of the thousands of proteins in an organism is specified by one
or more genes.

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