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Transcript
1
Lecture 2:
4. SECONDARY STRUCTURE
4.1 Polypeptide main chain conformations
Many of the important features of protein three-dimensional structure arise from the
regular repeating nature of a polypeptide chain.
First, the amino acids are all of the same enantiomer; they are L-amino acids. If the
amino acid is viewed along the hydrogen to C bond - with the hydrogen towards the
observer, the groups read clockwise CO-R-N.
Figure 4.1.1. Trans and cis peptide groups for alanine and proline.
R
R
O
O
C
C
C

O
L-Amino
acid
C
N

N
O
D-Amino
acid
Second, the peptide bond is planar, due to the delocalisation of the  electrons over
the N-C-O of the peptide. It is almost always trans with respect to the polypeptide
backbone (see Figure 4.1.1); the trans arrangement is normally favoured over cis by
103-fold. However, the cis arrangement is more favourable for Xaa-Pro peptide bonds
(Figure 4.1.2), where Xaa is any amino acid and Pro is proline, the only amino acid
with a carbon of the sidechain bound to the N instead of a hydrogen atom. In that
case, the trans: cis ratio is only 4:1. The energy barrier to rotation between cis and
trans is 15-20 kcal/mol (63-84 kJ/mol).
2
In polypeptide chains the conformation of the backbone at each planar peptide unit is
specified by two torsion angles:  (psi) refers to rotation about the C-C single bond;
 (phi) refers to rotation about the C-N bond. These angles are both close to 180
for the fully extended polypeptide chain as shown in Figure 4.1.2.
C

C
O

O
C
N
C
C
N
C
R
N
 = 180
 = 180
R
N
C
O
C
 = 0
 = 0
C
C
Figure 4.1.2. Definition of torsion angles between peptide planes.
Certain combinations of  (psi) and  (phi) are not allowed, because of steric
hindrance of the peptide group with the side chain. For glycine, where there is no
sidechain, more conformations are available. This can be visualised using a
Ramachandran plot or conformational map, which shows all possible combinations
of  and  and divides them into allowed regions, representing conformations where
no steric hindrance exists, and disallowed regions where steric hindrance occurs.
Figure 4.1.3 shows the
asymmetric plot for amino
The
acids with a sidechain. Steric
Ramachandran
p
hindrance occurs as a result

plot
of the sidechain and the most

favourable conformations
Those
have torsion angles with
conformations

negative  (phi) values. An
where there are
no steric
equivalent plot for glycine is
clashes
symmetrical around the
i.e. they are
centre, as there is no side
allowed
chain.
L
R
Figure 4.1.3. Ramachandran plot for residues with sidechains.
3
Ramachandran plots for real proteins show a variety of  /  values, mainly with
negatice  (phi) values as expected from the presence of L-amino acids. A few
asparagines, aspartic acids and some other amino acids, have positive  (phi) values,
in addition to many of the glycines. This is because the sidechains of these amino
acids can interact with the backbones atoms to stabilise conformations involving
positive  (phi) under some circumstances.
4.2 The major secondary structures
In the Ramachandran plot in Figure 4.1.3 the two allowed regions on the left side
(with negative  values) labelled  and /p correspond to the conformations of the
amino acid residues in the common secondary structures. R corresponds to the helix (also known as the 3.613 helix), This has 3.6 residues per turn. It is a righthanded helix, with the carbonyls pointing towards the C-terminus of the helix, and the
sidechains and NH groups towards the N-terminus (see figure 4.2.1). A left handed
helix, with all residues having the conformations L in the Ramachandran plot in
Figure 4.1.3, would have the sidechains clashing with the carbonyls and is therefore
rarely found, except in short regions with glycines. The -helix is further stabilised
by hydrogen bonds between the CO of residue i and the NH of residue i+4, thereby
closing a ring of 13 atoms: H-N-C-C-N-C-C-N-C-C-N -C-O.. This is the 13 of
the 3.613. Such helices are found widely in globular proteins and in fibrous proteins
such as keratin. Tighter helices with three residues per turn (310-helix) or looser ones
with 4.4 residues per turn are also possible, but have either less linear H-bonds or a
hole in the middle and are consequently less stable. Only short helices of these kinds
are found in globular proteins, and then often at the termini of the more stable helices.
A large number of amino acid residues fall into the R region in haemoglobin. These
residues are mainly be in the -helices which make up most of the structure, but they
are also found in irregular regions that allow the chain to turn between the secondary
structures. Some of the helices in haemoglobin tighten into 310-helices or loosen into
helices with 4.4 residues per turn as the chain moves into these irregular loop regions.
All peptide CO groups point to the C-terminus of the -helix, while the NH groups
point towards the N-terminus. Thus, all peptide dipoles are aligned, giving rise to a
helix dipole. This probably explains why the negatively charged residues, such as
aspartate and glutamate, tend to ‘cap’ the helix at the N-terminus and positively
charged residues, such as arginine, lysine and
histidine, do so at the C-terminus. Also helices in
enzyme active sites that bind highly negatively
charged groups, like a phosphate for example, tend
to have helix dipoles with positive poles close to the
phosphate.
Figure 4.2.1 The right-handed -helix found in
proteins. The C, CO and NH atoms of backbone
and the C atom of the sidechain are shown
4
The p and the  allowed regions of the Ramachandran plot correspond to much more
extended chains, in which intra-chain H-bond formation is not possible. Instead the
NH and carbonyl groups of the backbone are involved in inter-chain H-bonds. The 
conformation leads to a slightly twisted, extended strand, actually a helix with slightly
more than two (2.2) residues per turn. This can contribute to two kinds of slightly
twisted sheet, one in which the strands run antiparallel and the other in which they run
parallel (Figure 4.2.2); in both the side-chains project alternately above and below the
plane of the sheet. The antiparallel -sheet is found in fibres such as silk and probably
in amyloid, a structure that most proteins seem to form under extreme acid conditions
but which some proteins form easily, giving rise to amyloid fibrils of Alzheimer’s,
prion diseases and amyloidosis, a genetic disease usually caused by mutations in
normal proteins.
Both parallel and antiparallel sheets have the same twist. They would only be flat if
the  values were both 180o. In fact , values are usually in the region -120o, 140o
for both kinds of sheets. This twist leads easily to the formation of barrels and open
pores.
Both parallel and antiparallel -sheets are found in globular proteins. In all -proteins
like the FGF or FGFR antiparallel -sheets are found and in the absence of helices
seem to be more stable. Where there are alternating  structures, parallel sheets can be formed as the chain can travel back to the other end of the sheet through
an -helix; for these the sequences are usually hydrophobic and stabilised by packing
of the helices on either side. Such parallel -strands with associated helices are found
both in the N-domain of the FGFR kinase and in the HIV-proteinase, although in
neither case are they pure parallel -sheets as found in the Rossman fold and 
barrels. Inspection of the relative frequency of occurrence of amino acid residues in
these regular secondary structures (Figure 4.2.3) shows that the preferences are not
absolute. Leucines, lysines and glutamates tend to favour -helices, while valines and
isoleucines favour -strands. Proline tends to disfavour both as it can not form
hydrogen bonds through an NH function, but nevertheless it is accommodated with
small distortions particularly in long secondary structures and of course at the Ntermini of helices.
Studies on synthetic peptides have shown some of the reasons for the preferences for
certain secondary structures: thus, poly(valine) cannot easily adopt an -helical
structure because of branching at the  carbon of the side chain. Poly-(glutamic acid)
is helical at pH 3, but not at pH 7, where the side chains are ionised. Subtler effects
have also been revealed by such experiments. Thus, at pH 7.5, N-[Glu]20-[Ala]20-C
has a high helical content for the alanine region, whereas N-[Ala]20-[Glu]20-C has
very little -helical character. The explanation again probably lies with the -helix
dipole; the negatively charged glutamate residues at the N-terminus should stabilise
the helix.
5
Figure 4.2.2. -sheet with antiparallel and parallel ß-strands.
The p conformation, close to that of the -sheet but with  of ~-60, leads to an
extended helix with about three residues per turn. If every third residue is a glycine,
three strands can hydrogen bond together to give a triple helix which is found in
collagen. It also occurs C1q component of complement (Stryer III, p. 899). But this
conformation is also quite common in single strands in globular proteins, especially in
regions where prolines occur, presumably because the cyclic sidechain of proline
prevents conformations that are stabilised by intra-chain or inter-chain hydrogen
bonding.
Figure 4.2.3. The relative frequency of occurrence of amino acid residues in regular
secondary structures
Amino
acid
Ala
Cys
Leu
Met
Glu
Gln
His
Lys
Val
Ile
-helix
-sheet
-turn
1.29
1.11
1.30
1.47
1.44
1.27
1.22
1.23
0.91
0.97
0.90
0.74
1.02
0.97
0.75
0.80
1.08
0.77
1.49
1.45
0.78
0.80
0.59
0.39
1.00
0.97
0.69
0.96
0.47
0.51
Amino
acid
Phe
Tyr
Trp
Thr
Gly
Ser
Asp
Asn
Pro
Arg
-helix
-sheet
-turn
1.07
0.72
0.99
0.82
0.56
0.82
1.04
0.90
0.52
0.96
1.32
1.25
1.14
1.21
0.92
0.95
0.72
0.76
0.64
0.99
0.58
1.05
0.75
1.03
1.64
1.33
1.41
1.28
1.91
0.88
Secondary structure can be predicted more accurately if we bring information about
the amino acid patterns expected in the sequence. Thus, in an -helix which is packed
against the core of a globular protein or against another helix in some fibrous proteins,
we expect to see repeats of residues conserved as hydrophobic or hydrophilic at i, i+3,
i+4, i+7, as they would all be on the same side of the helix. This can be seen in the
haemoglobin sequences. In -sheet proteins the residues protrude alternately on
different sides of the sheet, so for an all -protein like FGFR where the strands are on
the surface of the protein, residues would be expected to repeat at i, i+2, i+4 and so
6
on. For proteins with poly-proline sequences the repeat is i, i+3, i+6 and so on. This is
of course seen in collagen triple helices.
4.3 Reverse turns
When torsion angles differ in three or four consecutive amino acid residues, the
polypeptide chains can change direction. Some combinations of torsion angles
reoccur throughout globular proteins, and probably in some fibrous proteins like
amyloid, giving rise to right angle bends (half turns) or reverses in chain direction
(reverse turns). For the common reverse turns, known as -turns because they often
occur between two antiparallel strands in a -sheet, there is a hydrogen bond between
the CO of residue i and the NH of residues i+3. Type II turns fit particularly well to
the twist of the antiparallel sheet, but one side chain is usually glycine to allow a
conformation with a positive  torsion angle. Type III turns resemble a half turn of
the 310 helix described above; in fact they have amino acid residues i+1 and i+2 at the
turn with similar torsion angles to those of the 310 helix.
Reverse turns are a common feature of proteins with all -structures. A good example
is the immunoglobulin domains D2 and D3 of the FGFR structure, where the chain
needs to reverse between each strand, but they are also found in the irregular regions
between helices, for example in haemoglobin. Glycines are often found in turns, so
that they can change direction sharply. Prolines are also quite common, as they
prevent H-bonding and so ‘cap’ helices; this is a very common feature of the structure
of haemoglobin. Reverse turns are usually found exposed to solvent, and often form
H-bonds to water. Larger loop regions at surfaces are critically important in many
recognition processes, e.g. at enzyme active sites and in antibody-antigen interactions
(see later).
4.4 Quantitation of secondary structure in polypeptides: circular dichroism
The spectral properties of polypeptide
chains in different secondary structures
be used to distinguish them. Because they
constructed from L-amino acids, natural
peptides absorb left- and right-circularly
polarised light unequally. This
measurable difference, termed ellipticity
constituting circular dichroism (CD), is
highly sensitive to the conformation of the
backbone. The magnitude of the effect,
quoted as the molar ellipticity , is plotted
against the wavelength of the incident
light. The CD spectrum of a protein will
the weighted average of the contributions
from the individual spectra for various
secondary structure types found in the protein.
Figure 4.4.1. The circular dichroism of poly-lysine in three different states:
can
are
and
be