Structural organisation of proteins

• Two main levels can be distinguished in the protein structure:

1. The basic structure (primary) that represents the sequence of the polypeptide
chain of proteins, formed on the base of the chemical nature, proportion and
succession of the component amino acids.
2. The conformation, or the superior organisation, as a result of the
tridimentional orientation of the polypeptide chain.

Primary structure

Each protein has a unique sequence of amino acids which is determined by the
genes contained in DNA.
The primary structure is responsible for its superior structures and for its
function.
The modification of this sequence by the replacement, adding, elimination of one
or more amino acids produces modification of conformation, and implicit, of
the biological function of the respective protein.
In certain cases, these modifications produce pathological aspects
sickle cell (falciform) anemia.
• The sequence comparison is currently used in the evaluation of protein
similitude at the level of structure and functionality. These comparisons
consider the alignment of sequences, the presence of a maximal number of
identical amino acids.
• homologous sequences - if their sequences have a high grade of similitude.
• analogous proteins - proteins that have similar structures, but have no
evolutionary report between them.
• The differences can be given by the substitution of an amino acid with
another one. These substitutions can be:
• Conservatives, when an amino acid is replaced with another one having
similar polarity (Val → Ile). These substitutions appear to the same protein
but at different species. If an amino acid is found in the same position, it is
called invariable residue and it is considered as having an essential role in
the structure or the functions of the protein.
• Non-conservatives, when an amino acid is substituted with another one,
having different polarity
⇒ produces severe modifications in the protein properties. In the case of
amino acids substitution, polarity plays an important role, but also the
volume and the surface area of amino acids.
 Proteins conformation

The superior, spatial structure of proteins is the result of the bonds formed
between the side chains of the component amino acids.
Levels of organisation, function of their nature, there are several :
secondary, tertiary and quaternary structure
The passing to a spatial conformation is due to the protein tendency to have a
minimal ∆G in the particular conditions of the medium: pH, ionic strength,
temperature, solvent.
The conformation is dependent on the primary structure and is responsible
for the specific biological function of the protein.

Any modification of the conformation will affect the specific biological

function of the protein, this thing being an important aspect in the
aetiology of numerous diseases.

1. Primary structure
(amino acids sequence in the polypeptide chain)
 2. Secondary structure

α-helix β-pleated sheet domains

in the immunoglobulin molecule
 a complete proteic chain – Hemoglobin β-chain
3. Tertiary structure

hemoglobin σ and β tubuline molecules

(4 separated protein chains) in a microtubule
4. Quaternary structure
A. Secondary structure
• regular, repetitive organisation, achieved by hydrogen bonds
between the components of the peptide bond (between the
hydrogen of the NH- group and the oxygen of the carbonyl
group).
• The bonds can be intra- or interchains.
• Proteins with secondary structure present variants or models
of structure:
• α - helix
• β pleated sheet
• β turn and Ω loop
• superhelix (collagen)
• Although not periodic, these common turn or loop structures
are well defined and contribute with α helices and β sheets to
form the final protein structure
 H O
6
H
a. Alpha helix O 20 C
2
C
N
C
R
33
C N R
H38 H
Is a coiled structure stabilized by intrachain C HO
46

hydrogen bonds (parallel to the axe of R N C O

49
H
helix). H C C57 H
R N C62
R
N
Pauling and Corey considered which H

conformations of peptides were

sterically allowed and which most fully
exploited the hydrogen-bonding
capacity of the backbone NH and CO
groups. The first of their proposed
structures, the α helix, is a rod-like
structure

In schematic diagrams of proteins,

α helices are depicted as twisted ribbons or rods.
• A tightly coiled backbone forms the inner part of the rod and the side
chains extend outward in a helical array. The CO group of each amino acid
forms a hydrogen bond with the NH group of the amino acid that is situated
four residues ahead in the sequence.

• O H

C (NH CH C O) 3 N

• ⇒ all the main-chain CO and NH groups are hydrogen bonded. Thus,

amino acids spaced three and four apart in the sequence are spatially quite
close to one another in an α helix.
• Both the right-handed and the left-handed helices are among allowed
conformations.
• However, right-handed helices are energetically more favourable because
there is less steric clash between the side chains and the backbone.
Essentially all α helices found in proteins are right-handed.
• The α-helical content of proteins ranges widely, from nearly none to almost
100%.

• The model is destabilized by:

• the presence of proline residues (they do not have the –NH- group),
• the presence of bulky radicals (Val, Ile)
• Radicals with potential of forming hydrogen bonds between the side chains
(Ser, Asp).

• The model α-helix is common both to

• fibrous proteins (myosin, tropomyosin, α-keratin)
• globular proteins (globins in haemoglobin).

• α-Keratin – major component of hair, skin, nails – is formed almost entirely

of α-helix structures.

• The cytoskeleton (internal scaffolding) of cells is rich in so-called

intermediate filaments, which are two stranded α-helical coiled coils.

• Many proteins that span biological membranes also contain α-helices.

b. Beta pleated sheets Pauling and Corey

• stabilized by hydrogen bonding between polypeptide strands (perpendicular on

peptide chain).

• The β-pleated sheet (or, more simply, the β sheet) differs markedly from the rodlike
α-helix.

• A polypeptide chain, called a β strand, in a β sheet is almost fully extended rather

than being tightly coiled as in the α helix.

• The side chains of adjacent amino acids point in opposite directions. A sheet is
formed by linking two or more β strands by hydrogen bonds.

• Adjacent chains in a β sheet can run in opposite directions (antiparallel β sheet) or

in the same direction (parallel β sheet).

• Many strands, typically 4 or 5 but as many as 10 or more, can come together in β

sheets. Such β sheets can be purely antiparallel, purely parallel, or mixed.

• β sheets can be relatively flat but most adopt a somewhat twisted shape.
 H C2 R R 2C H R 13C H
R 24C H
H N C3 O C14 O
C23 O
H N H N
H N C4 O
H CR H C16 R
H C21 R R 5C H 5

O 20C O 6C O 17C
N H
O N H N H
N H
R 8C H R C H
19
R 18C H H C8 R
C9 O C20 O
C17 O H N
H N H N
H N C10 O

Antiparallel (left) and parallel (right) β sheets

• The β sheet is an important structural element in many
proteins.
• Ex. fatty acid-binding proteins, important for lipid metabolism,
are built almost entirely from β sheets
• The β sheet model is found both in fibrous and globular
proteins,
• Characteristic example: β-keratin from the silk fibres, where a
repetitive sequence Ser-Gli-Ala-Gli forms an antiparallel β
sheet structure.
• An example that shows the dependence of the conformation
type on the surrounding medium is the transformation:
heat, moisture, traction
α-keratin β-keratin
c. Turns and Ω loops

• Polypeptide chains can change direction

by making reverse turns and loops. Most
proteins have compact, globular shapes,
requiring reversals in the direction of
their polypeptide chains. Many of these
reversals are accomplished by a common
structural element called the reverse
turn (also known as the β turn or hairpin
bend)
• In many reverse turns, the CO group of
residue i of a polypeptide is hydrogen
bonded to the NH group of residue i+3.
This interaction stabilizes abrupt changes
in direction of the polypeptide chain. In
other cases, more elaborate structures
are responsible for chain reversals.
• Unlike α helices and β
strands, loops do not have
regular, periodic structures.
• Nonetheless, loop structures
are often rigid and well
defined.
• Turns and loops invariably lie
on the surfaces of proteins
and thus often participate in
interactions between
proteins and other
molecules.
• The distribution of α helices,
β strands, and turns along a
protein chain is often
referred to as its secondary
structure.
d. Superhelix model = collagen model

• characteristic protein of this model is

collagen, the most spread protein in the
human organism.
• It is found in all tissues and organs where
it constitutes the backbone, giving to
these tissues their form and strength.
• Collagen represents 74% in the skin, 23%
in bones, 64% in cornea, 50% in cartilage,
12-24% in aortas, 10% in lungs, 4% in liver.
• The fibrous structure, extremely
compact, is the result of its
special amino acid composition
that is different comparing with
globular proteins. Collagen has a
high content of :
• glycine (33%), proline (13%),
• derived amino acids (10%): 4-
hydroxyproline and 5-
hydroxylizine,
• comparing with globular proteins
that contain glycine (3%), proline
(4%), derived amino acids (0%).
• The presence of derived amino
acids permits the formation of O-
glycosidic bonds with different
carbohydrates.
• The mature collagen molecule is formed of three polypeptide
chains, coiled together over a common axe in a superhelix (left
handed). Each molecule has, also, its own α helix structure. The
general aspect is that of a woven rope.

• There are different types of collagen, and they appear as a

consequence of the sequence difference between the three
polypeptide chains.

• A common characteristic is the presence of glycine every three

amino acids, and the repeating hundreds of times, some time in
tandem, of the sequences Gly-Pro-Y, Gly-X-HO-Pro (X and Y
being any amino acid). The intrachain bonds of the collagenic α-
helix are steric repulsions of the pyrrolidyne rings of Pro and
HO-Pro residues; the hydrogen bonds within a strand are absen

• Glycine does not present a side chain ⇒ allows chains to be

compacted to maximum in the protein structure.
In collagen - covalences between lysine and modified lysine (called allysine)
Allysine is formed from lysine under the action of an enzyme that transform
the ε-amino group of lysine into a ε-aldehyde group. That aldehyde group
suffers immediately a nucleophylic addition of the ε-amino group of lysine
residues, or of the ε-aldehyde group of another allysine residue.
A. Secondary structure
• regular, repetitive organisation, achieved by hydrogen bonds
between the components of the peptide bond (between the
hydrogen of the NH- group and the oxygen of the carbonyl
group).
• The bonds can be intra- or interchains.
• Proteins with secondary structure present variants or models
of structure:
• α - helix
• β pleated sheet
• β turn and Ω loop
• superhelix (collagen)
• Although not periodic, these common turn or loop structures
are well defined and contribute with α helices and β sheets to
form the final protein structure
The analysis of the amino acids frequency in different types of structure had
revealed some general preferences, as:
• α-helix structures - Long side chains (Leu, Met, Gln and Glu) - probably due
to the fact that extended side ramifications can be projected outside of the
central region of the helix; Branching at the α-carbon atom (Val, Thr and
Ile) tends to destabilize α-helices because of steric clashes. Ser, Asp and Asn
tend to disrupt α-helices because their side chains contain hydrogen-bond
donors or acceptors in close proximity to the main chain, where they
compete for main-chain NH and CO groups.
• β pleated sheet – AA with ramifications at carbon β (Val, Ile and Phe)
• turns - Gly, Asn and Pro
• Proline tends to disrupt both α-helices and β-strands because it lacks an NH
group and because its ring structure restricts its angle value.
• Glycine readily fits into all structures and for that reason does not favour
helix formation in particular.
• the local effects are those that predominate in the establishment process
of the type of structure that a part of the protein can adopt: α-helix, β-
strand, β-turn, loop or an unregulated conformation.
 Supersecondary structures (domain organisation)

= structural motifs = folds = particularly stable arrangements of several

elements of secondary structure and the connections between them
• helix-turn-helix
• leucine zipper
• calcium binding turn, found between two helices
• zinc finger
• Greek key
• β-α-β

Greek key motif

β-α-β motif β hair pin motif αα motif

 Leucine zipper Zinc finger

The „motif“ term is used in the structural biology under two aspects:
1. The term defines a particular sequence of amino acids, characteristic
for a specific biological function. Such an example is the so called motif “zinc
finger”, found in a family of various proteins, which bind DNA.
 2. “motif“ means a group of adjacent elements of the secondary
structure that has a particular functional significance, or it defines a portion
of a domain, independently folded.
Examples:
• the motif of the 2-helix bundle, found to proteins that bind DNA.
• the motif of the 4-helix bundle (found at many hormones and other types of
proteins);
• the “Rossmann folding”, an α/β fold that binds NAD+ cofactors and has clear
functional implications.
• the “catalytic triad” of serine-proteases, made of Asp, His and Ser residues,
which interact between them and which can be situated in different positions,
as a function of the protease family in which they appear.

• These amino acid residues form a catalytic unity with identical geometry
and with the same biochemical function.

• In each class, different arrangements of these elements are possible, and

each distinct arrangement is a structural motif.
α Motifs
• The motif of the 4-helix bundle – is found in a large variety of α domains
and has different functions: oxygen transport, binding nucleic acids, and
electron transport. In humans is found mostly in the growth hormone.
• The motif - “globin fold”, found in myoglobin, and which contains 8 α-
helices forming inside of the domain a hydrophobic “pocket” where organic
and organo-metallic groups can bind. The 8 helices form a single domain
that wraps up the heme. This motif appears in a different form in
cytochroms too, proteins involved in electron transport.

β Motifs
• formed only of antiparallel β structures, connected by β turns and large
bends. Proteins that are formed of β motives are: immunoglobulins, certain
enzymes as superoxid dismutase, and proteins that bind carbohydrates at
the cell surface.
• I.e. the enzyme neuraminidase of the influenza virus is formed of a
repetitive motif of 4 antiparallel chains.
B. Tertiary Structure

• refers to the spatial arrangement of amino acid

residues that are far apart in the sequence and
to the pattern of disulfide bonds. It includes
the arrangement of different secondary
structures. β-pleated sheet

• Secondary and tertiary structures induce

stereospecificity, responsible of the chemical
and biological reactivity of proteins.

• Domain represents the basic fundamental unit

of the three-dimensional structure;
• it is a distinct entity in terms of conformation
and function.

• Protein conformation depends on the protein

primary structure and is characterised by the
alternation of regulated and rigid structures (α
helix and β-pleated sheet) with disordered,
flexible parts, apparently randomly folded.
 Structural domains
• The structural motifs can associate to form supersecondary,
stable, three-dimentional structures, called domains (proteic
modules).

• Domains are distinct entities as conformation and biological

properties, being unitarily maintained in the protein structure by
the intermeddle of chain fragments with non-regular structure.

• Some proteins, as keratine in hair, are fibrous, but most of the

proteins are globular, their peptide chains wrapping to realise a
compact form.

• MW = 1,000 - 1,000,000 Da.

• MW < 20,000 ⇒ simple globular form, with the diameter 20-30

Å,

• bigger proteins form, usually, two or more structural domains.

 DOMAINS

• usually, but not always, are

formed of a continuous
segment of amino acid
sequences

• are capable to form a

structure, with own stability
in water solutions.

• These structural domains

often keep a part of the
biochemical function of the
big protein they derived
from.
 DOMAINS:

• Generally, a domain has 100-250

amino acids, having a
hydrophobic core and a
hydrophilic exterior.

• The hydrophobic cores are

essential for domains stability.

• So far, the highest number of

domains found in a protein is
13.

• Domains associate in a protein

by the same interactions that
stabilize the internal structure of
the protein.
• The folding process is the transition from primary structure to the final form
(native state) of a protein
• The folding takes place spontaneously due to the bonds that are formed
between the side chains of amino acid residues.
• These bonds are mainly hydrophobic, but in the case of certain proteins
hydrogen bonds, ionic, ester bonds, disulfide bridges –S-S- between Cys
residues can also be formed. The latter have a strong stabilized structure:
insulin, hair keratin, digestive enzymes.
• the structure of each protein is unique
• X-rays cristallography and NMR methods were used to reveal
the structure of thousands of proteins

MIOGLOBIN STRUCTURE : A. BALL AND STICK MODEL b. Ribbon model

MIOGLOBIN STRUCTURE : distribution of AA
• it has been found that different proteins (with different primary structure, different
functions or different species) present a high similitude concerning the
arrangement of the supersecondary structures. Such examples are:
• The model with all the domains having an α-helix structure, in which 7 or 8 α-
helix segments are bound by small peptide segments that allow their folding in
a globular form. The model is found to lysozime, myoglobin, hemoglobin
subunits.
• The model of domains α-β in which the domains with α structure bind to
domains with β structure which allows the adoption of a barrel type
conformations (triosephosphate isomerase) or twisted paper (lactate
dehydrogenase).
• The model with all domains having β structure, in which segments with β
strand structure are bound together by small peptide segments that allow their
folding in a globular form. The model is found to superoxide dismutase,
immunoglobulins, concanavalin A

immunoglobulin and Protein G

 HEMOGLOBIN

LYSOSYME
 LACTATE DEHYDROGENASE

TRIOSEPHOSPHATE
ISOMERASE
 IMMUNOGLOBULINS

SUPEROXIDE
DISMUTASE
• There are many proteins with two or more structural identical
domains. I.e. thioesterase from E. coli

• These proteins with a similar structure develop by gene

duplication. It is supposed that a single gene, which encodes a
domain of this kind of protein, is sequential copied so the two
genes will fusion, and their sequences will express a single
polypeptide.

• Sometime the gene duplication can appear in a single

structural domain, as it happens in the case of proteins γ-
crystalline in humans and β-crystalline in mouse.

• Conclusion = the protein formed of many structural domains

develops by gene fusion, genes that have previously encoded
distinct proteins.
According to the tertiary structure, proteins are divided into two categories:
• Globular, which have α helix and β sheet segments that alternate with
loops or β turns. They are divided into two subclasses:
• intracellular -soluble ones and
• Membrane- insoluble ones (intramembrane channels).
• I.e. ribonuclease has 124 amino acid residues, 4 disulfide bridges, an α helix
domain and an antiparallel β sheet.
• Fibrous, where a type of secondary structure is extended on a large portion
of the molecule. The high content in Gly, Pro, and HO-Pro makes them
insoluble.
• They have a structural localization.
Primary

Secondary

Tertiary

quaternary