Lecture - 10
Hierarchical Structure of Proteins: Secondary, Tertiary and Quaternary Structure
Welcome back to this new session. In the last session, we have discussed about the
peptide synthesis and the related issues. Now, we will go on to theof the peptide We
have already covered the primary structure of a peptide or a proteins (large peptides are
proteins). We have done the primary structure which means that how the amino acids are
linked to one another; that is called the amino acid sequence.
Now, proteins have a hierarchical structure; that means, that beyond the primary
structure, there are other kinds of structure. So, the first layer of structural analysis is the
primary structure; that means, the amino acid sequence, then there comes the second
(higher) order and that is called the second degree structure.
(Refer Slide Time: 01:31)
If you look at the slides, see this is the primary structure of a protein and; these small
blocks are representing one amino acid each. So, that is the protein chain. So this is
primary structure. Then what happens, as the protein is synthesized? It tries to fold at
different locations and at different local points, you will see different types of
geometries. That means, the geometry or the three dimensional structure of the protein at
different locations of the protein are not same.
There are distinct features of structural patterns at different locations. And some of these
distinct features are known, you have already heard of them possibly that there may be
something which is called a helical structure like some portions of the protein may look
like a helix as is shown here; and this is called α helix; or some portion may look like a β
sheet; or some portion may look like a loop which means kind of a turn shape; u turn; or
some portion maybe completely random. So, all these possibilities are there.
Now, these different geometries at different locations together make up what is called a
secondary structure. And here the definition is already given; what is a secondary
structure of a protein? Itt is the local conformation; that means, the local geometries
which are present at different locations of the protein throughout the entire protein
molecule. And remember the primary structure is obtained by connection of the amino
acids through peptide bonds.
So, this in a primary structure, the covalent bonds are the important ones and they are the
only ones to be considered (the peptide bonds). In a secondary structure, now you are not
connecting any amino acids with each other through covalent bonds. What you are doing
is that the protein is taking different shapes at different locations ok.
So, what are those forces that stabilize the different geometries at different locations?.
The primary stabilizing forces of this α helix, β sheet or β turn etc, are the hydrogen
bondings; this is occurring within the same molecule, hence the hydrogen bonding that is
taking place here is intramolecular hydrogen bonding. And also it is intrastrand hydrogen
bonding; if you take the α helix the hydrogen bonding is present within this strand itself.
β sheet has a little bit different connotation about the hydrogen bond, but the most
important point is that the force that plays a dominant role in forcing the protein to take
up different geometries at different locations are the hydrogen bondings. The hydrogen
bonding between the NH and the CO; NH because it is the hydrogen bonding donor and
the acceptor is the carbonyl; that means, the amide bond. Those are the points of interest
for considering the hydrogen bond.
Now, after we analyze the local conformations, we will consider the overall geometry of
the molecule. This overall geometry may take different shapes, but here we are not
talking about local conformations. It is the molecule in total; that means, the whole
molecule itself; how the 3D geometry of the molecule look like. Taking everything
together, the β sheet the α helix, everything together. Thus, it is a complete three
dimensional conformation of the molecule and that is what is called the tertiary structure.
There is one more higher order stucture that is called the quaternary structure.
Sometimes some proteins can exist in more than one monomeric form. That means, say
one monomer can combine with another monomer to make a dimer or it could be a
tetramer; all sorts of possibilities are there. So, when a single molecular protein, attaches
through weak interactions to another protein molecule and remain present as an
ensemble, that is called the quaternary structure of the protein.
So, quaternary structure means combination of multiple polypeptide chains; each
polypeptide chain represents a protein. Now this quaternary structure can be between the
same polypeptide chains; in that case if it is a called a homodimer. If that is between
different polypeptide chains that will be called a heterodimer.
So, now we have all these four layers of structures; primary is the first one if you isolate
a protein the first thing, you need to determine is its sequence. Once you know the
sequence, the second layer comes which addresses that what are the local conformations
at different positions. After that is analysed, then finally, that is still not final. For some
proteins, which are only monomeric in nature, upto tertiary structure, that is a final. But
for proteins which are multimeric in nature, you have to go for the quaternary structure.
Now, for all these secondary, tertiary and quaternary structures, weak forces take the role
to induce particular geometries for the proteins and even to have multimeric nature of the
proteins. So, the covalent bond formation is basically involved in the amide bond;
However, there is one more covalent bond we should be aware of; and that is by the
cysteine molecule which has got S-H, you know that it is a sulphur containing amino
acid with the free thiol group that can be easily oxidized to a disulphide. And if two
cysteines happen to be very close by, then what happens? They can be oxidized and form
a disulphide bond (S-S). So, the disulphide are the only other covalent bond that is
possible in a protein structure apart from the amide between the amino acids.
Other than that, the disulphide plays a role in the tertiary structure and in the quaternary
structure. Sometimes, the tertiary structures are stabilized by formation of disulphide
bonds; suppose there is a cysteine here at this location and another cysteine at that
location. They are close by. So, there may be a disulphide bond formation which holds
these two parts together in this shape. And the disulphide can also be there between the
different monomeric forms to make the multimeric ensemble; that is also there.
But apart from disulphide, leave aside disulphide, the others are all weak interactions,
specially hydrogen bonds. In the secondary structure, in the tertiary structure; it is
hydrophobic interactions, it is salt bridges and it could be π-stacking interactions; all
these things will be there. So, we may go to the next slide.
(Refer Slide Time: 09:59)
Let us talk about the secondary structure now. The secondary structure is made up of the
local conformations and these are distinct structural features that can be seen in a protein
if looked at different regions.
(Refer Slide Time: 10:23)
The first one that strikes our mind is the helical form of this protein chain. This is called
an α-αhelix and this is a right handed α-αhelix. Now, whenever you draw this helix, there
will be an N terminus somewhere and a C terminus at the other end. In this case, this
arrow is shown here; N going to C; that means, the N terminus is somewhere here and
the C terminus is here. So, this helix is going from this direction to that direction; from
the N to the C terminus.
Now, this right handed α-αhelix. Now I will show you some very simple models that
how does it really look. If you look from the top side or from different angles, and what
is the driving force that keeps it in this helical form? As I already said, hydrogen bond is
the driving force, but the question is which amino acid is forming hydrogen bond with
which other amino acid; because an amino acid here forms a hydrogen bond with an
amino acid just at the top of it.
Now, if you take a line, just at the top of it, it can make a hydrogen bond if the
orientations are favourable like a carbonyl pointing downwards and then NH pointing
upwards. That can form the hydrogen bond. Now let us consider the various questions
that come. First of all, why is it forming this right handed α-αhelix, why not the left
handed? that is number one. Number two is, if we look from this side it is right handed
helix whether it will be right handed, if we look from this side or not?That is the second
I have seen many students they face difficulty; they think that it is like a rotation of a
fan; that if the fan rotates in an anti clockwise direction from the bottom then from the
top, if we see that will become clockwise. But in cae of helix, interestingly it is not like
that; it appears right handed upon looking from the bottom side, it will be also right
handed if we look from the top side.
And then the question comes that what is the pitch of this helix? Helix is like a screw.
What is the pitch of the screw? Screw pitch means the distance that is travelled that if
there is a complete turn of a screw. So, here the distance is basically between a point here
and a point there; that what is pitch. And then the next question is how many amino acids
are there in a complete turn? So, these are some of the issues.
(Refer Slide Time: 13:39)
Now, let usshow you the model. We can make a model of these very simple things like
aluminum foil. I can make an α helix. αIf you see from the middle, you can see that there
is an axis. So, the helix is basically perpendicular to the helical axis.
Now, the question is how do you know this is right handed or left handed helix? In the
right handed screw, what happens? You push it in a clockwise direction. So, if you do
that, you also see here that as I do it, it is going in this direction. So, this is a right handed
helix and you will see from both sides it will be the same; if you look from this side or if
you look from the other side, it will look like the same. So, it is a right handed helix.
Now, what is holding this helix? You see these different bonds shown here which are
marked as red these are those hydrogen bonds. So, the hydrogen bonds are actually
maintaining the helical shape of this polypeptide chain. So, these are the hydrogen
bonds. The hydrogen bond contains hydrogen and then nitrogen and a carbonyl which is
So, if this is your first amino acid; then as you go here, that will be the fifth amino acid
whose carbonyl will be coordinating with the NH. So, it is basically the hydrogen bond
between the first amino acid and the fifth amino acid. So, after each four amino acids,
you will have a hydrogen bond with the bottom one. I hope this is clear and then the
distance between this and that; that means, the pitch of the screw, the pitch of the helix is
5.4 Å. And another thing you have to consider is that when you start from the nitrogen
suppose, then come to the α carbon of the amino acid, then come to the carbonyl,
there is a rise in the helix because it is not on a horizontal plane, it is actually a spiral
thing which is going up slowly. So, if you start from the nitrogen of an amino acid and
go to the carbonyl, how much rise it is? how much you are elevating the surface? The
rise is 1.5 Å. So, now, we know that the pitch of the helix is 5.4 Å and the rise per amino
acid is one 1.5 Å.
So, then what will be the number of amino acids in one complete turn? That will be
obtained by just 5.4 divided by 1.5, that is 3.6, roughly 3.6 amino acids per complete
turn. Again I just go back to the questions; we have mentioned first of all that it is a right
handed helix. Why it is right handed and not left handed? Because if it assumes this type
of helical form, then the one thing that is not shown here, that is the substituents of the
helix; the amino acid side chains. The substituents can actually project out.
They cannot project inside because then there will be too much steric crowding. Since
they are in the L configuration, so the stereochemistry comes into play because they are
in L configuration; that means, the side chains are projected outwards of the helix. So,
that is why this is a right handed helix.
Now, if it is only glycine which does not have any substituent, then we have to
remember that glycine is much more flexible and it is not constrained to interrupt this
type of helical form. And there is one more amino acid which is called proline. Proline
because of the secondary nature of the amine, it cannot participate in hydrogen bond
because when it forms the amide bond with another amino acid; the amide here is a
tertiary amide where the N doesn’t have any H; So, it is no longer a hydrogen doner.
So, proline and glycine are exceptional amino acids; glycine does not have any
substituent. So, it is much more flexible. So, whenever you want to bring flexibility in a
in a polypeptide chain, you incorporate glycine; you will see that at flexible regions,
there are glycines. And if you want to bring in rigidity; means if you want to disrupt this
kind of system which is stabilized by intramolecular hydrogen bond, then incorporate
proline since proline cannot participate in hydrogen bonding.
So, proline is basically is also called a helix breaker; it breaks the helix. So, if some
protein is going in a helical shape and suddenly changes into another shape; at that
changeover point, proline may be present because proline is the one which does not
allow any helix formation. Now, apart from this αright handed α helix, there is another
one which is also very interesting, that is called the β sheet structure.
(Refer Slide Time: 20:07)
What is the β sheet structure? These are the amino acid planes these are the amino acid
side chains, these are pointing upwards and then downwards; we have not put any
substituent here. So, when the ridge is on the top, then the R group is going to the top
side. Ridge means the point where these two surfaces are meeting, that meeting point. If
the ridge is going downwards, then the substituent will be on the left side.
So, this is the β sheet. Now these two β sheets are actually interconnected by hydrogen
bonds between the two sheets. Basically now I have shown the two sheets, now what is
actually connecting these two sheets? The two sheets are connected by hydrogen bonds.
So, this is the sheet structure where the hydrogen bonds are like this; see this is the
extended form of the polypeptide and NH is pointing downward, CO is pointing
What did I say that when this is pointing upwards this carbon α carbon containing the R
group is going upwards. And in the next amino acid, theR group is going downwards.,
thus it just alternates. If you have a very similar chain like this that there is a CO here;
NH here and then alternating CONH is like that which are present in amino acids by the
So, then this chain and this adjacent chain can be interlocked by hydrogen bond which
can be formed between this NH and CO. Now this is, by the way, suppose this is your N
terminus say, and this is your C terminus so; that means, the direction of peptide bond
peptide is generally as we write it from N to C. So, this is going from N to C and here
also, you see the amine at this position and the carboxy at this position; that means, this
is the N terminus and this is the C terminus.
So, here the direction of the peptide is N to C from this side. So, this is N to C from that
side and this is N to C from this side. So, they are basically called anti-parallel β sheets.
There may be other sheets or other chains that can also be linked to this chain; Because
here you see, there is this NH and then followed by a carbonyl. They are not coordinately
saturated means they are hydrogen bond capacity is still there that is not satisfied; only
this carbonyl and this NH are satisfied, then the next NH and CO are still available for
So, what can happen? Another chain can come on the top of it and then, they can
assemble βto form a β sheet. So, β sheet is nothing, but you have first an extended chain
of peptide, then there is some turn there and then another extended sheet of peptide and
then there will be interchain hydrogen bonding. And then it can again take a turn and
come to that side.
(Refer Slide Time: 24:13)
So, basically if I draw it here, what is happening is that there is something like this. So,
the β sheet can be like this. So, there is hydrogen bond; all this interlinked. Here I have
only two chains in this model; I have shown only two chains; I think you can see. So,
what I am saying is that at at the top, there has to be a turn, otherwise how can we get
this shape because if there is no turn, then that will go to the top of it and there will be no
hydrogen bond between the two chains. So, there should be a turn like this which is also
called the β turn; I will come to β turns slightly later.
And then again there can be another β turn which can form another layer of polypeptide
and this can continue. And this whole thing becomes a bundle of a large sheet structure.
This is the one which shows the β sheet. Here, one chain is going up and down. Then
another chain in the middle going up and down and these chains are connected by
hydrogen bonds and then there is another chain.
Now, what are the major differences between α and β sheet? One difference is
manifested through and Ψ angles that are there according to the Ramachandran plot; we
will come to that little bit later. And the second one is also very important; in α helix,
there is hydrogen bond within the helix within the same chain (intrachain).
But in β sheet, the hydrogen bond that is taking place is between the two chains
(interchain); that means, it is the same molecule, but this part or that part is not
individually stabilized by hydrogen bonds. These two are interlinked by hydrogen bonds
from here to there. So, there are hydrogen bonds in between these chains. So, that is the
major difference between these two and in fact, in this β sheet, the hydrogen bond
directions are very favourable. So, hydrogen bond strengths are more in β sheet than the
helix. So, the β sheet structure is more stable than the α structure.
However this delicacy of α and β; their equilibrium from one to the other is a major issue
in controlling the activity of the proteins that we have in our body. We will come to that
when we discuss medicinal chemistry.
So, we have discussed the secondary structure; then we talk about the bends say; what
are these bends? Now again I should say that there are parallel β sheets; parallel means
you have N to C in this direction and N to C in this direction. If you want to have parallel
sheet, then there should be two turns that you need; one is a turn on this side, the chain
goes like this and then you get another turn and then the two directions become same N
to C (same direction).
But in order to have anti-parallel chains, it will have just one turn and there is no cross
over like this and obviously, if you look at the structure of the two, here they are aligned
perfectly well interms of H and CO (orientation for hydrogen bonding), but here that
alignment is not there. So, the anti parallel sheet is more stable than the parallel β sheet.
(Refer Slide Time: 29:09)
Now, how to get this turn or what makes this turn? There are primarily two types of
turns. Say this is a turn comprised of amino acid 1 suppose, then amino acid 2 amino
acid 3, amino acid 4. So, basically the peptide is now starting from here, going like this;
this is the backbone and then it is taking a turn here.
Now, in order to induce turn again, you should have a hydrogen bond and that hydrogen
bond now is between the carbonyl of the first amino acid and the NH of the fourth amino
acid. So, if it is i then this is i + 3; because this is C number 1, but this may not be the
starting point of the peptide that is why it is better that you say that this is the ith amino
acid and this is the i + 3 amino acid.
So, there is the formation of intramolecular hydrogen bond and if you calculate the size
of this, that will come to be 10 membered ring. So, because of the presence of this 10
membered hydrogen bonded network, the chain takes a turn. It was going in this
direction and then ultimately comes in the opposite direction. So, that induces the turn;
and there are two types of turns and these are actually again based on Ramachandran plot
(Φ and Ψ angles), but the most important aspect that what are the amino acids which can
induce these turns; that means, the amino acids which does not give any stability to the α
helix or the amino acids which does not give any stability to the β sheet.
Now, there are two ways to do that on the basis of conformational flexibility of the
amino acids; one is by incorporatingamino acid is very flexible; that is one possibility.
The other is to have amino acid that is very rigid and devoid of hydrogen bonding
capability and that is proline. So, you see in type I, the number 2 amino acid; that means,
the i + 1 amino acid is proline and in type II turn, the i + 2 amino acid is glycine. You see
this flexible amino acid makes the formation of the β turn and the hydrogen bonding
inability of proline makes the possibility of the type I β turns. So, these are the major
Other β turns are there like type III, then type II a prime; all these are there; but for our
level, I think it is sufficient to know that there are primarily these two types: I and type II
(Refer Slide Time: 32:31)
So, these are sometimes also called β bends, β turns or Ω loop. It is also said that this is
the β sheet; the sheet structure that was going in this direction and then it takes a turn and
then the sheet structure goes in the opposite direction. So, that is anti-parallel. So, that is
how the different sheets are connected.
So, what are the characteristics of β bends? First of all, it permits the change of direction
of the peptide chain to get a folded structure. It gives the protein globularity; that means,
rather than linearity; now it can be ircular when you have a turn; that means, it is going
through a kind of a circular direction. So, a globule is kind of a circular thing. So, the
proteins generally try to adapt a globular shape and these turn structures assist the protein
to adapt the globular state.
Hydrogen bond stabilizes the β-bends; again hydrogen bond plays the dominant role,
proline and glycine are frequently found in β turns. β turns often promote the formation
of anti-parallel β sheets as shown here, it usually occurs at protein surfaces and not at
inside and involve four successive amino acid residues; i and up to i + 3. So, that
completes the discussion on secondary structure of protein.
(Refer Slide Time: 34:01)
The tertiary structure is a little bit easier; tertiary structure, as I said it is the complete
three dimensional structure of a single monomeric protein molecule and ultimately it can
be a combination of α helix, some portion may be β sheet, some portion may have loops.
And incidentally now another class of proteins are there where some portion may be
intrinsically disordered; that means, they do not have any order, they are vibrating from
one geometry to another geometry. So, they are called intrinsically disordered, but they
are much less in number; a major class of proteins actually have a well defined 3D
(Refer Slide Time: 34:59)
These are the interactions that take place when a protein adopts the tertiary structure.
Remember again the primary structure is stabilized by covalent bonds, the secondary
structure is stabilized by hydrogen bond. It could be in the same chain; it could be in the
same locality; that means, α helix have intrachain or it could be interchain as found in β-
sheets. And then the tertiary structure is stabilized by hydrogen bonding; there are lots of
hydrophobic interactions; that means, water repellent groups or it could be salt bridges
like if you have an aspartate or glutamate and if you have a lysine, then lysine will be
present at the biological pH as NH3 plus this will be CO2 minus.
So, there can be electrostatic interaction that is sometimes called the salt bridge
formation; water plays a dominant role that in some of the pockets water may be there
which also can participate in hydrogen bonds with the R groups.
(Refer Slide Time: 36:13)
So, these are the stabilizing factor of for the tertiary structure.
(Refer Slide Time: 36:21)
And quaternary structure are very similar; quaternary structures are stabilized by
hydrogen bonds and salt bridges.
(Refer Slide Time: 36:25)
And disulphide; disulphide it is not mentioned here; disulphides are very important. They
play a very important role; to give a well defined shape to the protein; like a very simple
example, that some people have curly hair and some people have straight hair. The
people who have curly hair have a lot of cysteines in their keratin and cysteines are
actually present as disulphide (oxidized dimeric form of cysteine containing the S-S
linkage is known as cystine). Now when they are present in disulphide, you get a bend
shape. So, the hair becomes very curly.
So, now there is a tradition that curly hairs can be straightened and how to do that? You
break those disulphide bonds with reducing agents and you break that bond and then
make the hair straight. So, basically these disulphide bonds also play their role in
controlling the geometry of the tertiary as well as quaternary structure. Like in many
antibodies which are also proteins, there are a lot of disulfide bonds that stabilize the
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