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02. LEVELS OF PROTEIN STRUCTURE

As previously mentioned, this is an artificial classification: proteins do not think by themselves
which level of structure are they folding. This classification was artificially created to understand
better how proteins fold. Summing up, the four structural levels are:
 Primary: Sequence of amino acids linked by peptide bonds. Maintained by covalent bonds.
 Secondary: Structural patterns in which adjacent aa’s are arranged. Kept by hydrogen
bonds between atoms of the peptide bond. They are the so-called fibrous proteins.
 Tertiary: Three-dimensional conformation of the protein: globular proteins. There are
different types of interactions that keep it together (electrostatic, hydrophobic, van der
Waals or H/S bonds. Disulfide bonds of cistine, despite being covalent bonds, are normally
considered part of the tertiary structure.
 Quaternary: Several subunits join together (globules) in order to become functional.
All proteins have primary structure, and normally secondary structures as well. However, there
are proteins that contain no tertiary structure. These are fibrous proteins (keratin, colagen and
firboin). Most enzymes are tertiary proteins and the classic example of quaternary complex is
haemoglobin.

02.1. Primary structure

The first primary structure in being discovered was that for insuline, which was the first protein
to be sequenced.
Proteins evolution from one another. Therefore, we can distinguish several types of relationships
between proteins:
Firstly, homologue proteins come from a common ancestor. There are two subgroups of
homologue proteins:
 Orthologue proteins: proteins in different species that evolved from a common ansector
and perform the same function. An example: bovine and human ribonucleases.
 Paralogue proteins: Paralogues are genes related by duplication within the genome. They
acquire new functions from their original one, even if they are related to the first one. For
example, human angiogenine evolved from ribonuclease but their physiological functions
are quite different.
Secondly, analogue proteins are those which evolved separately but perform the same function
in two different species. The most remarkable example is haemoglobin (in vertebrates) and
haemocyanin (in invertebrates).
This relationships between proteins are determined by the primary structure, and they are very
useful. When working with a non-model organism which peptidic structure has not been
sequenced, it can help looking for the orthologue.

02.2. Secondary structure

The secondary structure is maintained by hydrogen bonds between carbonylic oxygen and the
amine hydrogen. There are two main ways the peptide chain organizes itself:

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02.2.1. Alpha-helix
This structure can either be righ-hand or left-hand.
Alpha-helix is maintained by H---O hydrogen interactions
between amino acids in relative positions 1 and 4 (1-4, 2-5, 3-6,
4-7…). Since all the amino acids present these interactions, the
alpha-helix is very consistent and difficult to denaturalize.
The N-terminus will have a possitive partial density charge
whereas the C-terminus will present negative partial density
charge. This is important in order to analyse the structure: amino
acids in relative positions 1 and 4 have very closely located their
radical chains. Therefore, they will have to be likely to interact
(both hydrophobic, both polar, one possitively charged and the other negatively charged…).
The alpha-helix stability is determined by some factors:
 Attraction/repulsion forces between charged residues (Glu (-) and Lys/Arg (+)). If there is a
long block of one of these, there will be no helix.
 Size of the Radical groups: Asn, Ser, Thr, Leu/Cys residues can also destabilize the helix if
they are located closely.
 Interaction between radicals 3 residues apart: possitive and negative charges are usually
located 3 aa’ apart, permitting the formation of ionic pairs.
 Proline cannot stablish hydrogen bonds and glycine permits more conformational stability,
so they are rarely found in helical domains.
 Identity of the aa’ found near the ends: possitively charged close to the C-terminus and
negatively charged found close to the N-terminus
The classical example of α-Helix is keratin, which can complete its function in this form. Keratin is
found, for example, in the hair, and its conformation is determined by disulfide bonds in cistine
complexes.
There is an exception: collagen is a protein formed of left-hand helixes grouped together 3 by 3.
Unlike other helical structures, collagen contains high proportions of proline and glycine, being
specially common the following sequences:
 Gly-X-Pro
 Gly-X-4Hyp (4Hyp = 4-Hydroxyproline)
 Gly-Pro-4Hyp
When ingested, we are only ingesting these sequences, since the protein will have been hydrolized
by the time it reached the stomach.

02.2.2. Beta-sheet
The beta-sheet is formed by the same hydrogen interactions.
However, they are located antiparalelly. If the first sequence has
a CN nature, the second sequence will be NC and like this
succesively.

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There are other sheets which have parallel conformation. On them, the structures are parallely
disposed since the hydrogen interactions are different.
Turns are common in proteins’ secondary structure. Turns are a special secondary structure which
involve glycine and proline.
The most classic protein with β-sheet-like structure is silk fibroin. This protein has artificially been
imitated in order to create nylon (polyamide).

02.3. Tertiary structure

The tertiary structure of a protein determines longer-range aspects of amino acid sequences
which conform globular proteins. It is determined by the amino acid sequence.
The scientist Christian Anfinsen, in 1950, performed an experiment which enabled the
renaturalization of unfolded and denaturalized ribonuclease using urea and mercaptoethanol. This
was done in order to reduce the presence of cistines to yield 8 Cys residues. Renaturalization
involves the correct re-establishment of these broken disulfide bonds.
One example of a protein with tertiary structure is myoglobin. It is a globular protein which
participates in oxygen-binding to muscle cells (its functions are both storing and diffuse oxygen in
rapidly-contracting muscle tissues). 80% of its structure are α-helical domains.
Other examples of tertiary proteins are:
 Cytochrome C: 40% of α-helixes. Vital component of the electronic transport chain.
 Lysozyme: Abundant globular protein present in egg white and human tears. It catalyzes
the hydrolitic cleveage of polysaccharides in the protective cell walls of some bacterial
families, which conferes it with antimicrobial properties. Its structure contains around 40%
of each α-helical and β-sheet-like domains.
 Ribonuclease: Enzyme secreted by the pancreas into the small intestine. It catalyses the
hydrolisis of certain bonds in the ribonucleic acids present in ingested food. It contains both
secondary domains.
Motifs are supersecondary structures (also called simply foods) which are particularly stable
arrangements of several elements of secondary structure and the connections between them.
Domains are parts of a polypeptide chain that is independently stable or could undergo
movements as a single entity with respect to the entire protein. Polypeptides with more than a
few hundread aminoacids normally fold into two or more independent subunits (domains).

02.4. Quaternary structure

Quaternary proteins are those with two or more polypeptides. In other words, they are composed
by two or more globular subunits binded with non-covalent interactions.
One example of quaternary protein is RUBISCO (Ribulose 1,5-biphosphate carboxylase oxydase),
the key enzyme in the fixation of atmospheric CO2. It is considered to be the most abundant
protein on Earth. It is composed by small and large subunits. 8 of each subunit types fold together
in order to form the final enzyme.
Another example of quaternary protein is haemoglobin, formed by α and β globulines or subunits
(both formed mayoritally by helixes). It contains also heme groups, which carry the oxygen.

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Haemoglobin, when binding to oxygen, suffer conformational changes. This protein is classified
as a chromoprotein, it contains a peptidic part and a prosthetic group of another different nature.
In this case, the heme group contains a Fe (II) cathion, vital for the oxygen-binding.

03. MODIFICATIONS
The base structure of proteins can be modified once translated in order to create new
macromolecules.

03.1. Addition of a prosthetic group

For example, the heme group of haemoglobin, does not have a peptidic nature. These are called
holoproteins or chromoproteins, which contain a peptidic part (apoprotein) and a non-peptidic
part (prosthetic group). The structure of the heme group is basically 4 porphirine rings around a
Fe (II) cathion. Chlorofill, porphirinic as well, contains a Mg (II) cathions. Other chromoproteins,
such as haemocyanin (present in invertebrates’ blood), contain a Cu (II) cathion which does not
present porphirinic rings.

03.2. Covalent modifications

 Formation of disulfide bonds: inter or intramolecular
 Loss of an aminoacid (normally methionine)
 Loss of a small peptide at the beginning or the end of the polypeptide
 Processing the protein to make it active (insulin)
The posttranslational modification of insulin is a crucial process for this protein to acquire its
function: Proinsulin is synthesized in the ER. There, it is folded and disulfide bonds are oxydized.
Then it is transported to the GA, where it is packaged and processed by proteases to form mature
insulin.
Mature insulin has 35 amino acids less than proinsuline. 31 of them form the C-peptide, which is
removed from the center of the proinsuline. The two remaining ends (A and B subunits) continue
binded by disulfide bonds.
Prions can cause the Creutzfeldt-Jakob disease (the Crazy Cows disease),
resulting on an irremediable death. This is caused by the missfolding of the
proteins. When transmitted into the tissues, it interacts with the healthy
ones and transmitts this missfolding. Up to the day, it is still unknown how
this disease transmitts from one individue to another, since when the
missfolded proteins are ingested, they are destroyed in the stomach but the
disease is transmitted.

03.2.1. Denaturalization
Denaturalization consists of breaking every structure of the protein, leaving just the peptidic
chain. Therefore, denaturalized proteins are never functional. Several techniques to denaturalize
proteins are SDS (Dodecyl sodium sulphate), Mercaptoethanol (breaks cistines), urea (allows
renaturalization) or guanidium chloride.

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The native state of a protein is a more stable conformation inside cells. Denaturalized proteins
only retain the primary structure, losing their function and enabling the precipitation of the
proteins. Denaturalization can either be reversible or irreversible.
An example of non-reversible denaturalization we carry out several times is the denaturalization
of ovoalbumine by heat (when frying an egg).

03.3. Modifications of amino acids

The amino acids themselves can also suffer modifications:
 Proline and Lysine can hydroxilate. 4-hydroxyprolin (4Hyp) is contained in collagen.
 Acetylation of Lysine’s amine groups
 Methylation of Histidine
 Glycosilation of Asparagine, Threonine and Serine
 Union of lipids
 Reversible phosphorilation in Serine, Threonine and Tyrosine
 Ubiquination, which produces the death kiss.

BIOCHEMISTRY | Biomolecular Chemistry