Properties of Amino Acids

G&G Chapter 4

Structure of amino acids

2
 Amino acids
building blocks of proteins

Each amino acid is built around a tetrahedral carbon atom, Cα

3
G&G Fig. 4-1, p. 78

Peptide bond links amino acids

The Cα is
always on the
N-terminal
side of the C

N-terminus C-terminus

4
N Cα C N Cα C G&G Fig. 4-2, p. 78
 20 different amino acids are found in
biological systems

Classified by their side-chains as follows:

• Non-polar (hydrophobic, “water-hating”)

• Polar (hydrophilic, “water-loving”)
– Uncharged
– Charged
• Negatively charged (also called Acidic due to pKa in pH range < 7)
• Positively charged (also called Basic due to pKa in pH range > 7)

• Know them by name!

– Full name (e.g. Glycine, Tyrosine, Arginine, Tryptophan, Asparagine)
– Three-letter abbreviation (e.g. Gly, Tyr, Arg, Trp, Asn )
– One-letter code (e.g. G, Y, R, W, N)

What is a pKa value?

• Proteins contain many ionizable groups; i.e. groups for
which it is energetically favourable to lose or gain a H+ ion
(which we often loosely call a “proton”).

• At dynamic equilibrium the fraction of molecules in the A-

or HA states remains constant, although individual
molecules are rapidly converting from one to the other.
• Adding H+ ions to the solution, which is equivalent to
decreasing the pH, will drive the equilibrium to the left
(make more HA). Similarly, increasing the pH will drive
the equilibrium to the right (make more A-).
• Remember that at low pH, the concentration of H+ ions
[H+] in water is high, whereas at high pH, [H+] is low.
• The pKa of a group is the value of the pH at which half
of the molecules are ionized.
• Note that as the pH increases past the pKa, the charge of
the group decreases by 1 unit (HA to A-).
• The value of the pKa depends on the strength of the bond
holding the H+, which in turn depends on surrounding
chemical groups. Hence the pKa of each ionizable group
will be different.
Concentration of HA and A- as a function of pH
Examples
• The pKa values of the backbone carboxylic
acid (COOH) groups of all amino acids are
in the vicinity of 2. Hence at physiological
pH (~7) these groups are always ionized
and hence negatively charged (COO-).
• However, the pKa values of the backbone
amino groups (NH3+) of all amino acids are
in the vicinity of 9.5. Hence at
physiological pH these groups are all un-
ionised and hence positively charged
(NH3+). 6
 Charge

+1 è 0
+1 è 0

0 è -1
0 è -1
0 è -1

+1 è 0

+1 è 0

The charge always decreases by 1 unit when

the pH increases past the pKa. The actual
charges are shown in red.

0 è -1
0 è -1

0 è -1

Yellow - sidechain

Hydrocarbon chains or rings

– i.e. almost all C (black) and
H (white)

The backbones of all amino

acids are identical; except for
Proline, where the side-chain
loops back and bonds with
the Nitrogen on the
backbone.

Fig. 4-3a, p. 80
 • These side-chains have
reactive groups
• Electronegative atoms
O (red), S (yellow) and
N (blue).
• -OH (Thr, Ser and Tyr),
-SH (Cys) are all
ionisable, but with pKa
values much greater
than 7.
• Hence they are neutral
at physiological pH.
• The side-chain –NH
group of His is neutral
above its pKa (6), so at
physiological pH most
molecules will be
neutral, although some
will be positive.
• Hence His is slightly
positive at
physiological pH,
which accounts for the
slightly red background
(see next slide).
• Only a small decrease
in physiological pH
will give it charge +1,
which accounts for the
involvement of His in
many pH-dependent
reactions.

Fig. 4-3b, p. 80

Why “Acidic”?
– because the pKa of the ionisable sidechain group is in the acidic region (pKa < 7)
– hence these residues are negatively charged at physiological pH

G&G Fig. 4-3c, p. 80

 Why “Basic”?
– because the pKa of the ionisable sidechain group (NH2+)
is in the basic region (pKa > 7)
– hence these residues are positively charged at physiological pH

Why is Histidine coloured redder than the other polar uncharged

residues in (b)?
– because a small fraction of molecules will be positive at pH 7.

G&G Fig. 4-3, p. 81

Charge of amino acids at pH 7?

backbone

• Because the pH of biological environments is always close to 7, that is the pH that is

of most interest to us.
• So we ask, based on the previous table, which amino acids are neutral at pH 7,
which negative and which positive?
• pH 7 is well above pH 2-3, so in a sample of identical molecules, all of the
backbone carboxyl groups will be ionized (or “deprotonated”). Hence they are all
COO- rather than COOH.
• pH 7 is also well below pH 9-10, so in a sample of identical molecules, all the
backbone amino groups will be un-ionized (or “protonated”). Hence they are all
NH3+ rather than NH2.
• Hence the backbones of all amino acids are neutral (charge = 0) at pH 7.

12
 Charge of amino acids at pH 7?
side-chain

Now, looking at the sidechain ionizable groups:

• e.g. Arginine, pH 7 is below the pKa of 9.0, hence in a sample of identical molecules, all of
these groups are completely un-ionized (protonated). Hence the charge on the left (the higher
charge) applies and Arg has charge +1 at pH 7.
• e.g. Aspartic acid, pH 7 is above the pKa of 3.9, hence in a sample of identical molecules, all
of these groups are completely ionized (deprotonated). Hence the charge on the right (the
lower charge) applies and Asp has charge -1 at pH 7.
• e.g. Serine, pH 7 is well below the pKa of 13, hence in a sample of identical molecules, all of
these groups are completely un-ionized (protonated). Hence the charge on the left (the higher
charge) applies, and Serine is neutral at pH 7.
• Finally, for Histidine, pH 7 is only 1 pH unit above the pKa of 6.0. Hence in a sample of
identical molecules, almost all of these groups are ionized, and therefore have the lower
charge, 0. However a small fraction will have the higher charge, +1. Hence the average charge
over the whole population will be very slightly positive.

13

α- and β-amino acids

Cα
• Amino acids can be thought of chains of C atoms (-C-C-C-)
decorated with other groups.
• In each case the chain starts with the carboxyl carbon (CO), followed
by carbons numbered α, β, γ, δ, ε...
• The amino group does not have to be attached to the Cα carbon; if it Cβ
is, it’s called an α-amino acid.
• If it’s attached to the β-carbon, it’s called a β-amino acid. Cγ
• The only β-amino acid found in nature is β-alanine.
Cδ

Cα Cβ
Cε

Lysine

14
 Effect of pH on amino acids (1)

Question
• What is the net charge on a sample of Glycine molecules when the pH is 7?
Answer
• From the table, Glycine has a backbone carboxyl group with a pKa of 2.3 and a
backbone amino group with a pKa of 9.6.
• pH 7 is far above the pKa of the carboxyl group, so all the molecules will be ionized
(–COO-) and the average charge will be (very close to) -1.
• pH 7 is far below the pKa of the amino group, so all the molecules will be un-
ionized (–NH3+) and the average charge will be (very close to) +1.
• Hence the net average charge of the molecule is (-1) + (+1) = 0.
• Conclusion
– Ignoring the side-chain ionizable groups, at physiological pH (close to 7), the backbones of
all 20 amino acids have a group (–COO-) with charge -1, and a group (–NH3+) with charge
+1, and so the backbones always have net 0 charge.
15

Effect of pH on amino acids (2)

Question
• What is the net charge on a sample of Isoleucine molecules when the pH is 9?
Answer
• From the table, Isoleucine has a backbone carboxyl group with a pKa of 2.4 and a backbone
amino group with a pKa of 9.7. It has no additional ionizable groups.
• pH 9 is very far above the pKa of the carboxyl group, so this group will be almost completely
ionized (i.e. all molecules have lost the H in –COOH, to become –COO-. Hence the average
charge (sum of the charges on all molecules, divide by the number of molecules) is -1.
• But pH 9 is only just less than the pKa of the backbone amino group, so some of the molecules
will have become ionized (–NH2, charge 0), but most will still be un-ionised (–NH3+, charge
+1). Hence the average net charge on all the molecules will be the weighted average of +1 and
0.
• Let’s assume, by way of example, that 80 % of the molecules are un-ionized (charge +1) and 20
% are ionized (charge 0). The weighted average will therefore be 0.80 * (+1) + 0.20 * (0) =
0.8.
• Adding the charge of -1 on the carboxyl group, we conclude that the net average charge on the
molecule is between -1 and 0. 16
 Example of weighted averages

• If 20 % of a group of people has mass 120 kg, 50 % has mass 50 kg and 30 %

has mass 80 kg, then the weighted average mass of the group is:
• (20/100) * 120 kg + (50/100) * 50 kg + (30/100) * 80 kg = 73 kg

17

Effect of pH on amino acids (3)

Question
• What is the net charge on a sample of Glutamic acid molecules when the pH is 5?
Answer
• From the table, Glutamic acid has a backbone carboxyl group with a pKa of 2.2 and a
backbone amino group with a pKa of 9.7. It also has a backbone carboxyl group with a
pKa of 4.3.
• pH 5 is far above the pKa of the !-carboxyl group, so all the molecules will be ionized
(–COO-) and the average charge will be (very close to) -1.
• pH 5 is far below the pKa of the !-amino group, so all the molecules will be un-ionized
(–NH3+) and the average charge will be (very close to) +1.
• But pH 5 is only just higher than the pKa of the "-carboxyl group (backbone), so most
of the molecules will have become ionized (–COO-, charge -1), but some will still be
un-ionised (–COOH, charge 0). Hence the average net charge on all the molecules will
be the weighted average of -1 and 0, which is between -1 and 0.
• Adding the charge of -1 on the backbone groups (-1 and +1, which cancel each other
out), we conclude that the net average charge on the molecule is between -1 and 0.
18
 Reactions involving amino acids

19

Proteases
• Proteases (also called proteinases) are proteins that cleave
peptide bonds within other proteins.
• They do so by providing a binding cleft into which the
substrate protein can bind, positioning the scissile bond close
to reactive (electronegative) groups on the side chains of the
protease.
• In consequence, proteases have been named for the amino acid
most responsible for cleavage of the peptide bond:
– Serine proteases (-OH hydroxyl; pKa 8.3)
– Cysteine proteases (-SH sulfhydryl; pKa ~13)
– Aspartyl proteases (-COOH carboxyl, pKa 3.9)
– Threonine proteases (-OH; pKa ~ 13)
• Metalloproteases use a different strategy:
– they bind metal ions (e.g. Mg2+) which are positively charged and
highly reactive.
 Disulphide bridge
• This is a covalent bond between side-
chain thiol (-SH, also called
sulfhydral) groups of cysteines in two
proteins.
• For example, antibody molecules are
made up of 4 separate amino acid
chains, stabilised by intra- and inter-
molecular disulphide bonds.
• Disulphide bonds are more common
outside of eukaryotic cells than inside,
because they cannot survive in intra
reducing environment found inside
cells.
inter
• Reducing environments can be created
in the laboratory by adding reducing
agents such as dithiothreitol (DTT) or
β-mercaptoethanol. Antibody molecules contain many
intra- and inter-molecular S-S bonds

Reducing agents
• When working with proteins in vitro we often add a reducing agent to abolish disulphide bonds.
• Breaking of disulphide bonds corresponds to reduction, i.e. the gain of electrons, which makes the disulphide
bond unnecessary (since covalent bonds are just another way of gaining electrons to fill shells). 2 electrons
are required per bond. Those electrons are supplied by the reducing agent, which becomes oxidized in the
process (forming its own disulphide in the process).
• The reduction potential (Eh) of an atom or group is the potential for attracting electrons away from other
groups; i.e. to become reduced. It is similar to electronegativity. The greater the reduction potential, the
more willingness a group has to being reduced (accept e-s).
• DTT has Eh = -330 mV
• -RSSR- has Eh = -250 mV
• Hence a disuphide bond will become reduced in the presence of DTT.

reduced

reduced

oxidised
oxidised

Reduction of disulphide bond by

Reduction of disulphide bond by DTT β-mercaptoethanol
 Phosphorylation

• Transfer of γ-phosphate group

(PO43-) from ATP to hydroxyl
group of Ser, Thr, Tyr.
• Catalysed by enzyme that binds
both the protein and ATP; called a
phosphoryl transferase or, more
commonly, a kinase.
• Addition of 2 negative charges can
radically alter the conformation
and/or function of proteins.
• Phosphorylation is a major
signaling process, driving many
processes within the cell.
• Phosphorylation is reversed by
phosphatases.

Aside: Phosphorylation-activated signalling

• Epidermal Growth Factor (EGF) is a small protein
secreted by specialised cells into the inter-cellular
space whose job is to signal to cells that they should
divide (undergo mitosis).
• The EGF receptor (EGFR) is a large protein which
spans the membrane. How does binding of EGF to the
extra-cellular portion of EGFR cause proteins to be
transcribed inside the nucleus of the cell?
• Binding of EGF induces two receptors to dimerize.
• The intracellular portion of EGFR contains a kinase domain; dimerisation allows one receptor to
phosphorylate key tyrosine residues on the other receptor and vice versa.
• When phosphorylated these tyrosines have high affinity for domains called SH2 domains
• Many signalling proteins contain SH2 domains and therefore become instantly recruited to the
dimerised receptors, causing them to become activated.
• One of these, known as RAF, is a serine/threoning kinase (EGFR is a tyrosine kinase) which
phosphorylates, and activates, another kinase called MEK.
• Phosphorylation of MEK causes it to phosphorylate and activate another kinase called MAPK.
Once activated, MAPK enters the nucleus where it activates transcription factors which directly
cause expression of proteins causing the cell to divide.
• The above is known as the MAPK pathway. It is only one of a number of similar pathways
transferring the growth signal carried by growth factors outside the cell to expression of proteins
and ultimately cell division inside the nucleus of the cell.
 Lysine acetylation

Acetyl group

• Acetyl group has chemical formula Cε

COCH3.
• ε-amino group of lysines (-NH3+) can be
modified to acetyl groups.
• Acetylation is carried out by Lysine
Acetyl Transferases (KATs), using
Acetyl-CoA as the acetyl donor.
• De-acetylation is carried out by Lysine
Deacetylases (KDACs).
• Since histones contain many acetylated
lysines, KATs and KDACs are also often
referred to as Histone ATs (HATs) and
Histone deacetylases (HDACs).

Lysine acetylation plays an important

role in gene regulation

• When not being actively transcribed,

genomic DNA is packaged around
drum-like structures called
nucleosomes.
• Four different proteins, called histones
H2A, H2B, H3 and H4, associate to
form each nucleosome.
• The N-terminal tails of the histones are
rich in lysine residues, which are
positively charged. The positive charges
lead to electrostatic attraction between
the negatively charged DNA and the
histones.
• Acetylation neutralises the positive
charge on the histones, thereby opening
up the packaging of DNA.
• This promotes access to polymerases
DNA wrapped around histones
and thereby promotes gene transcription.
 Isopeptide bonds
• An isopeptide bond is any peptide bond Peptide bond
found other than on the backbone of a
protein.
• For example, Glutathione is a molecule
made up of the amino acids Glu-Cys-Gly.
• However it isn’t a tri-peptide, because the
bond between Glu and Cys is between the
sidechain carboxyl of Glu and the
backbone amine of Cys.
• This is an example of an isopeptide bond. Glu Cys Gly
• Isopeptide bonds can form between the
sidechain carboxyl groups of aspartic or
Isopeptide bond
glutamic acids and backbone or side-chain
amide groups (as shown). Glutathione
• They can also form between side-chain
amino group and the backbone carboxyl
group, as in ubiquitination (next slide).

Ubiquitination
• Isopeptide bonds form
between the sidechain ε-
amino group of lysine
residues and the backbone
Ubiquitin
carboxyl groups of
ubiquitin molecules.
• Ubiquitin (Ub) is an
evolutionarily-conserved,
highly stable 76 aa protein
• Additional ubiquitin
molecules can be attached
to lysines on the first
ubiquitin (of which there
are 7), to form “poly-
ubiquitin” chains.

Formation of poly-ubiquitin chains

 Ubiquitin-mediated protein
degradation
• Ubiquitination is a newly-recognised
form of cell-signalling (like
phosphorylation) that serves a
variety of purposes.
• For example, poly-ubiquitinated
proteins are recognised by a large
molecular complex called the
proteasome and degraded.
• This is an important mechanism
allowing cells to switch off the
effects of important signalling
proteins such as p53, which initiates
the process of apoptosis (“cell
suicide”), which is one of the body’s
defences against cancer.

Chirality

30
 • Does it matter in which positions the 4 groups are
added?
• If their positions were randomly swopped around,
surely we could we always get back to the same
configuration just by rotating the molecule?
A • For example, what about structure B? Can it be
rotated to give A?
Ø Yes.
Ø A rotation of 180 °about the axis shown will turn B
into A.

B • And C?
Ø No, no rotation will make C the same as A.
Ø Note that C can be generated from A by swopping the
positions of the amino and carboxyl groups. But that
corresponds to a physical change to the molecule.
• And D?
Ø Again, no rotation of D will give the same
C configuration as A.
Ø Note that D can be generated from A by swopping the
positions of the H and amino groups.

31
D

• So, if C and D are both different from A, are they

the same as each other?
Ø Yes
Ø A rotation about the axis shown will generate C
from D.
• Any odd number of swops shifts the
configuration from the one “group” to the other.
• Any even number of swops keeps it in the same
group.
• It turns out that the mirror image of one group is
always in the other group, so these groups are
often described as mirror images of each other.
• But most of us have very little idea how a mirror
changes an image, so it’s not always easy to see
how swopping 2 ligands creates the mirror image.
• So let’s rather stick to swopping ligands.

32
D
• Each amino acid can be made in two different ways,
using exactly the same components. The Cα is a chiral
• The two different ways are called stereoisomers.
• They cannot be transformed into each other using
physical operations such as rotations. They can be
centre
transformed into each other using mirror images, but a
mirror image is not a physical operation.
• We say that the Cα is a chiral centre. The fact that there
R
are 2 groups is due to the fact that there are 4 different
groups attached to the Cα. If two or more were the same,
it would no longer be a chiral centre.
• Only one of the stereoisomers of amino acids is
produced in biological systems, so we need to be able to
distinguish between them.
• How do we decide which stereoisomer a given amino N CO
acid belongs to, and what should we call them?
• CORN Law:
– Rotate the molecule so the CO, R and N groups are in the
plane of the page and the H is oriented away from the
viewer.
– If they spell CORN in a clockwise direction the amino acid
is called “D” for dextro (right); if in an anti-clockwise
direction it is “L” for levo (left).
• Almost all amino acids produced in living systems on
Earth are L-isomers.
• Glycine is neither L or D, since two of the groups
attached to the α-carbon are identical. Hence the Cα of
glycine is not a chiral centre. We say it is achiral.
• Isoleucine and threonine each have two chiral centres,
one on the backbone and the other on their side chains.

D or L?
and which amino acid?
• The filled wedge
means it is
pointing out of the
Cα page. All simple
lines lie in the
plane of the page.
• Hence the H must
be pointing into
the page.
CO
R

L-Serine
N

1. Which is the Cα? The one attached to the COOH (and the NH2)
2. Which is the sidechain?
3. Which are the CO, R, N and H? H is not shown but its presence is inferred (4th valence)
4. What is the sidechain? -CH2-OH
5. Which amino acid? Serine
6. Is the H pointing into the page? Can’t tell
7. Is it D or L? Can’t tell
34
There’s no 3D information!
 Is the Cα the only chiral centre?
• The double bonded O counts as 2 bonded O
atoms, so the backbone carbonyl carbon of
every amino acid is achiral.
• The long hydrocarbon chain of lysine is
made up of repeated –CH2- groups; because
of the 2 H-atoms, these carbons are all
achiral.
• But the C! carbon of threonine is attached to
4 different groups (-CH3, -OH, -C" and –H)
and so is chiral; the wedge in the diagram
tells us the -OH is pointing out of the page,
the -H (not drawn) is pointing into the page.
The 2 chiral carbons give 4 possible
stereoisomers; the one shown is the only one
found in nature.
• Similarly, the C! carbon of isoleucine is also
chiral. The isomer shown is the only one
found in nature.
• There are no other chiral groups in amino
acids found in biological systems.
D/L system
• As we will see in the next lecture, the two chiral isomers
rotate the plane of polarization of light passing through it in
different directions;
• e.g. a solution of L-serine rotated the polarization to the
left and R-serine rotated it to the right.
• This effect was used to distinguish between the two
isomers.
• But that method only works for a molecule with a single
chiral centre; where there are two chiral centres, the optical
rotation is an average of the two, and it is not possible to
separate the two rotations.
• Furthermore, chirality was assigned using the CORN law,
which only works with amino acids (because CO, R and N
are only found in amino acids).
• The CORN law is really just a way of ordering the 4
groups attached to the chiral centre.
L-alanine
= CH3
S-alanine
carbonyl
CH2 carbon
lysine
threonine
isoleucine
R/S system
• A new way of assigning chirality was developed in 1966,
which works for all molecules.
• Instead of labeling the entire molecule, each chiral centre has
its own label (R/S).
• As a generalization of the CORN Law, the Cahn-Ingold-
Prelog rules set up an ordering of all chemical groups found in
biological systems. Restricting this ordering to the groups
found in proteins, we have:
SH > OH > NH3+ > CH2SH > COOH > CH2OH > CH3 > H
• Then, with the lowest priority group facing into the page, the
chirality is R (“rectus” - “right”) if the remaining 3 groups
decrease in priority in a clockwise direction, otherwise S
(“sinistrus” - “left”)
• Hence for alanine the L-enantiomer (shown on left) is also the
S-enantiomer since NH3+, COOH and R = CH3 follow each
other in an anti-clockwise direction.
• In fact all L-amino acids, apart from cysteine, are also the S
isomer. But due to the thiol group (R = CH2-SH), L-Cys
corresponds to R-Cys.
• Molecules containing 2 chiral centres will have two S/R labels.
For example, the form of threonine found in biology is (2S,
3R), meaning that the 2-carbon (the C!) is S, but the 3-carbon
(C") is R.
• However the form of Isoleucine found in biology is (2S, 3S).
• Since we are most interested in amino acids in the course, we
will not consider the R/S system further.
36
 L or D?
R

CO

Cα N

L-Glutamic acid L-Cysteine

1. Which is the Cα?

2. Which is the sidechain?
3. What is the sidechain? CH2-CH2-COOH
4. Which are the CO, R, N and H?
5. Is the H pointing into the page? Yes
6. Which amino acid Glutamic acid
7. Is it D or L? L
37

Spectroscopy of amino acids

Spectroscopy: The study of how radiation interacts with matter,

particularly as a function of the wavelength of the radiation.

38
 Optical activity
Polarisation of light

• Light oscillates transversely (side-to-side) in a plane

perpendicular to the direction of motion. This is called
the plane of polarization. Two examples, red and blue,
are shown opposite.
• Each wave can have a different plane of polarization.
The polarization can be expressed as the sum of a
horizonal component and a vertical component.
• A polarizer is a material that only allows one
component of the polarization to pass through. Polaroid
is the most famous trade name for such a material.
• The principles behind Polaroid sunglasses are (1) that
all light reflected off horizonal surfaces (such as a road,
or the ocean) is horizontally polarized. And (2), that
light reflected off horizontal surfaces is the most
problematic.
• A second polarizer can be added and rotated relative to
the first so it doesn’t allow through the component
allowed through by the first. The light will then be
totally extinguished. This defines the plane of
polarization of the light.

• In 1815 Jean-Baptise Biot used the setup

shown on the right to discover that
Optical activity
solutions of carbon-based molecules Polarisation and chirality
rotate the plane of polarized light; we say
these molecules are optically active.
• In 1848 Louis Pasteur demonstrated that
optical activity is a consequence of the
chirality of carbon atoms.
• From this he deduced that 3-dimensional
shape is important in biology: living
processes are based on physical
interactions of molecules in 3-
dimensional space (the “lock and key”
model).
– A more recently-discovered example of this is is
that different stereoisomers can smell different to
us – the “olfactory receptors” in our noses bind
only to molecules with specific shapes. 1. Use a polariser to select a single plane of
• Pasteur further discovered that living polarisation. With only buffer in the tube, rotate
processes are able to distinguish between the second polarizer (called the analyser) until no
light is transmitted.
chiral isomers. 2. Replace the buffer with a biological solution, in
• This is in contrast to the reactions used to the same buffer. If some light is now transmitted,
synthesize the same molecules in rotate the analyser until, once again, the light is
extinguished.
chemistry laboratories, which usually 3. The angle through which the analyser was turned
cannot distinguish between chiral isomers corresponds to the optical activity.
and produce equal amounts of both.
 Absorbance spectroscopy
Absorbance spectrum

• Solutions of proteins and amino acids

are transparent to visible light (400-
700 nm).
• However they absorb strongly in the
ultra-violet (UV) region (250-290
nm), due to ring structures present on
side-chains of Phe, Tyr and Trp.

Light is absorbed exponentially inside a

protein solution

• Experiments using UV light showed that

if the intensity of light is attenuated
(reduced in intensity) by a factor α (<1)
when it penetrates a certain distance into
a solution of protein, then it will be
attenuated by a factor α2 when it
penetrates twice the distance. P = αP0
• It is not difficult to show that this means
that the intensity falls exponentially with
the distance b:

P = P0 10 − k b

• where k is an unknown positive

constant.
P = α 2P 0
 Absorption depends on the concentration
as well as the distance
• The graph on the right shows how the
intensity of the transmitted light falls as it
penetrates further into the solution.
• The larger k, the faster the intensity falls.
P = P0 10 − k b
• What could determine the value of the
constant k? k=1
– Probably the concentration of the dissolved
molecules.
k=2
– But also the make-up of the molecules themselves.
k=5
• It turns out that k = ! C, where
– C is the concentration of the solution
– ! is a constant that is characteristic of the molecules
themselves
• Hence
P = P0 10 − εC b
• Transmitted intensity depends exponentially
on concentration C.

Beer-Lambert Law
Examples
• Exponential behaviour is not easy to work
with. So instead we define the absorbance A, • if P = P0 (i.e. no absorption)
as follows: then A = 0 (log10(1) = 0)
• if P = 0.1 P0 (i.e. 90 % absorption)
A = log10 (P0 P) then A = 1 (log10(10) = 1)
then • if A = 0.3,
then P0/P = 10 0.3 = 2
A = log10 (P0 P) i.e. half the light has been
= log10 (P0 P0 10 − εC b ) absorbed
= log10 (10 εC b )
= εC b
Conclusion
• This is the Beer Lambert Law: A = # Cb • The B-L law can only be applied when
• C is the concentration of the solution; units M less than half of the incident light is
• b is the optical path length; units cm absorbed
• ε is the extinction coefficient; units M-1cm-1
• The Law only holds when A is between 0 and 0.3 • What if the sample is more concentrated?
(corresponding to max. 50 % absorption) Ø Dilute by known amount until A
• Absorbance depends linearly on C. falls in the range 0 – 0.3
• When C = 0 there is no absorbance, as we Ø Calculate C, then multiply by
would hope. dilution factor
 Examples
1. A sample absorbs 95 % of the light incident on it at 280 nm. What is the
absorbance, A280?
• If P0 = 100, then P = 5
• Hence A280 = log10(100/5) = log10(20) = 1.3

2. A sample has an absorbance of 0.5. What percentage of the light is absorbed

by the sample?
• Firstly, if y = log10(x), then x = 10y. That is the definition of log10(x).
• So A280 = 0.5 = log10(P0/P)
• Hence P0/P = 100.5 = 3.16
• Hence P/P0 = 1/3.16 ~ 0.316 = 32 %
• Hence P = 32% of P0 (this is the transmitted light, not the absorbed light)
• Hence 68 % of the incident light is absorbed

45

Example
10% of incident light has been absorbed by the time it has travelled 1 cm into the
sample. How much has been absorbed absorbed by the time it has travelled 3.5 cm
into the sample.

α = P/P0 = 0.90 (Careful, it’s not 10 %. ! is the fraction of light transmitted)

After the light has travelled 1 cm, P/P0 = 0.90
After the light has travelled 2 cm, P/P0 = (0.90)2 = 0.81
After the light has travelled 3 cm, P/P0 = (0.90)3 = 0.73

What about 3.5 cm? Yes, you guessed it…!

After the light has travelled 3.5 cm, P/P0 = (0.90)3.5 = 0.69 = 69 %

On your calculator: 0.9 xy 3.5

46
 Types of spectroscopy

• Circular dichroism (CD) spectroscopy (an extension of optical activity)

• Absorbance spectroscopy
• Fluorescence spectroscopy
• Infra-red spectroscopy
• Raman spectroscopy
• Nuclear Magnetic Resonance Spectroscopy
• X-ray diffraction BTN322
• Electron microscopy

Fluorescence spectroscopy
• Fluorescence happens when a
molecule re-emits light it has
previously absorbed.
• The re-emitted light always has
longer wavelength (lower energy)
than the incident light (Stokes’
shift), allowing the fluorescence to
be distinguished from the much
stronger incident light.
• The quantum yield is the efficiency
with which the absorbed light is re-
emitted; a yield of 1 corresponds to
100% re-emission.
• Most of the fluorescence in a
protein comes from Trp and Tyr,
which absorb at 280 nm and emit at
350 nm and 320 nm respectively
 Investigating ligand binding and
folding using fluorescence
• The fluorescence efficiency can change
depending on biophysical conditions. For
example:
– Unfolding of a protein can expose Trp, Phe or Tyr residues
to the solvent, reducing the fluorescence (“quenching”).
– Binding of a ligand molecule to a protein can change its
fluorescence, for example by shielding a fluorophore from
the solvent, as in the case shown on the right.
• Fluorescence is a simple indicator of the
fraction of protein molecules that are bound to
ligand or folded.
• The figure shows that the fraction of protein
molecules bound to ligand changes from 0 to
100% in the region 10-8 - 10-6 M (10 - 1000 nM).
• The KD of a ligand is the concentration of ligand
Acta Cryst (2011). D67, 119-12Acta Cryst (2011). D67, 119-12
required to 50 % saturate the protein.
• We deduce that the KD of this ligand is around Change in fluorescence of a protein when it binds
100 nM (10-7 M). a ligand (small molecule).
• Measurements such as these are crucial to
determining whether pharmaceuticals (enzyme
inhibitors etc) will be effective.

Green Fluorescent Protein

• Green Fluorescent Protein (GFP)

exhibits strong green fluorescence
when excited with blue light.
• GFP is produced naturally by the
the jellyfish Aequorea victoria.
• The fluorescence is stronger than
usual because the fluorescent Aequorea victoria The β-barrel structure of GFP
residues are protected from the
solvent by the barrel structure of
the protein.
• The gene was cloned from the
jellyfish and introduced into a
multitude of other organism and
cells.
• By fusing GFP to proteins
forming part of the cytoskeleton
of cells, the structure of the
cytoskeleton is revealed in minute
detail.
GFP expressed as a fusion with β- Live mice expressing GFP
tubulin, a major cytoskeletal protein
 These and other images can be found on “The GFP Site”
http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm

Different types of neurons in the mouse brain,

labelled with different flavours of fluorescent
protein.

Fluorescence and immunofluorescence

microscopy (IF)
• In fluorescence microscopy (previous slide), microscopes
are equipped with light sources of different wavelengths
used to excite fluorescence from proteins fused to GFP or
other naturally fluorescent proteins.
• Genes coding for the fusion proteins are exogenously
introduced into cells or whole organisms using a range of
techniques, including transfection, viral infections and
genome editing (e.g. CRISPR-Cas9).
• In immunofluorescence microscopy, antibodies targeting
the protein of interest are used, conjugated to a small
fluorescent molecule. The proteins are therefore not
unnaturally modified by being fused to GFP.
• Cells are fixed onto slides, permeabilized and the antibody
allowed to diffuse into the cell and bind to the protein of
interest. The cells are then imaged using a fluorescent Fluorescence microscope
microscope.
• Alternatively, scanning confocal microscopy is a new form
of microscopy in which a laser is used to scan the object
pixel by pixel and the fluorescence detected. The complete
image is then reconstructed by a computer.
• This allows very thin cross-sections of cells to be visualized
at a time, just as if the tissue or cell was physically cut into
very thin slices, producing very high-contrast images, as
shown in the next slide.
http://www.olympusmicro.com/