Amino Acids & Proteins

1
 Amino Acids
• Building blocks of proteins
• Carboxylic acid group
• Amino group
• Side group R gives unique characteristics

R side chain
I
H2 N—C —COOH
I
H 3
Non-protein Amino Acids
 Examples of Amino Acids
H
I
H2N—C —COOH
I
H glycine
CH3
I
H2N—C —COOH
I
H alanine 13
 Types of Amino Acids
Nonpolar R = H, CH3, alkyl groups, aromatic
O
Polar ll
R = –CH2OH, –CH2SH, –CH2C–NH2,
(polar groups with –O-, -SH, -N-)
Polar/Acidic
R = –CH2COOH, or -COOH
Polar/ Basic
R = –CH2CH2NH2
15
 Learning Check AA1
Identify each as (1) polar or (2) nonpolar

A. NH2–CH2–COOH (Glycine)

CH3
|
CH–OH
|
B. NH2–CH–COOH (Serine)
16
 Solution AA1
Identify each as (1) polar or (2) nonpolar

A.(2) NH2–CH2–COOH (Glycine)

CH3
|
CH–OH
|
B. (1) NH2–CH–COOH (Serine)
17
 Essential Amino Acids

• 10 amino acids not synthesized by the

body
• arg, his, ile, leu, lys, met, phe, thr, trp, val
• Must obtain from the diet
• All in dairy products
• 1 or more missing in grains
and vegetables
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 The Peptide Bond
Amide bond formed by the –COOH of an amino
acid and the –NH2 of the next amino acid
O CH3
+ || + |
NH3–CH2–COH + H3N–CH–COO–
O CH3
+ || |
NH3–CH2–C – N–CH–COO–
| peptide bond
H 19
 R O R O
H H
N C C-OH N C C-OH
H H
H H

Condensation A dipeptide is formed

reaction

R O H R O
H
N C C N C C-OH + H2O
H H
H

The peptide bond

© 2007 Paul Billiet ODWS
 Peptides
• Amino acids linked by amide (peptide) bonds

Gly Lys Phe Arg Ser

H3N- -COO
end Peptide bonds end

Glycyllysylphenylalanylarginylserine
23
 Learning Check AA3
What are the possible tripeptides formed
from one each of leucine, glycine, and
alanine?

24
 Solution AA3
Tripeptides possible from one each of
leucine, glycine, and alanine
Leu-Gly-Ala
Leu-Ala-Gly
Ala-Leu-Gly
Ala-Gly-Leu
Gly-Ala-Leu
Gly-Leu-Ala
25
 Learning Check AA4
Write the three-letter abbreviations for the
following tetrapeptide:

CH 3
CH 3 S
CH CH 3 SH CH 2
CH 3 O CH O CH 2 O CH 2 O
-
H 3N CH C N CH C N CH C N CH C O
H H H

26
 Solution AA4

CH3
CH3 S
CH CH3 SH CH2
CH3 O CH O CH2 O CH2 O
-
H3N CH C N CH C N CH C N CH C O
H H H
Ala-Leu-Cys-Met

27
 Take Note:

The atoms along the side chain are named with Greek letters in 
Greek alphabetical order: α, β, γ, δ, є and so on. Cα refers to 
the carbon atom closest to the carbonyl group of that amino 
acid, Cβ the second closest and so on. The Cα is usually 
considered a part of the backbone. The dihedral angles around 
the bonds between these atoms are named χ1, χ2, χ3 etc. E.g. 
the first and second carbon atom in the side chain of lysine is 
named α and β, and the dihedral angle around the α‐β bond is 
named χ1. Side chains can be in different conformations called 
gauche(‐), trans and gauche(+). Side chains generally tend to try 
to come into a staggered conformation around χ2, driven by 
the minimization of the overlap between the electron orbitals 
of the hydrogen atoms.
PROTEINS

29
Ch. 6.1, Roitt, Ivan
Immunology
3rd ed.
 Proteins classified by function
• CATALYTIC: enzymes

• STORAGE: ovalbumen (in eggs), casein (in milk),

zein (in maize)

• TRANSPORT: haemoglobin

• COMMUNICATION: hormones (eg insulin) and

neurotransmitters

• CONTRACTILE: actin, myosin, dynein (in

microtubules)
© 2007 Paul Billiet ODWS
– PROTECTIVE: Immunoglobulin,
fibrinogen, blood clotting factors

– TOXINS: snake venom

– STRUCTURAL: cell membrane proteins,

keratin (hair), collagen
 BioMedical Importance of
Proteins
9 An internal protein network, the cytoskeleton, maintains
cellular shape and physical integrity

9 Actin and myosin filaments form the contractile

machinery of muscle

9 Hemoglobin transports oxygen

9 Antibodies search for foreign invaders

9 Enzymes catalyze reactions that generate energy,

synthesize and degrade biomolecules, replicates and
transcribe genes, process mRNAs, etc….
 Proteins

9 Receptors enables cells to sense and respond

to environmental cues

9 An important goal of molecular medicine is the

identification of CHONS whose presence,
absence, or deficiency is associated with
specific physiologic states or diseases
 Proteins
9 are organic compounds of high molecular weight

9 Made up of a-amino acids joined by means of

peptide linkage

9 Mulder (1839) gave the name proteins (Gr. Proteios,

meaning preeminence)

9 They are the most complex and most diverse in

chemical composition .

9 Most functionally diverse of all biological

compounds.
 Proteins

9 Made up of 20 amino acids

9 Can be arranged in almost infinite number of

sequences to make almost an infinite number of
different proteins.

9 They supply the body not only with heat and

energy but also materials for building and repair.
 Proteins
9The first group of food substances
whose absence in the diet was definitely
established to be fatal.

9Temporarily stored in the body.

9Provide materials for growth.

 Proteins
9Molecular weight is ranging from 5,000
to 8,000,000 daltons (possess the
properties and characteristics of a
colloid).
 Proteins
9 The protein molecule is composed of hundreds or
even thousands of amino acids linked by peptide
linkage (carboxyl of one amino acid is linked with the
amine group of the other).

H
I
9 R – C – COOH
I
NH2
 General Chracteristics of CHONs

A. Specificity – while carbohydrates, lipids, etc.

may be found in all animals and plants,
proteins are specific:
1) Differing species have different proteins
2) Organs/tissues have proteins that are unique
3) Individuals have proteins specific for
that individual
 General Chracteristics of CHONs

B. All proteins contain carbon, hydrogen, oxygen,

sulfur and nitrogen

C. Nitrogen sets proteins apart from carbohydrates

and fats

D. Depending on their specific function, proteins

may also contain phosphorous, iodine, iron,
copper, etc.
 Properties of Proteins

• Most are soluble in water, in alcohol, in dilute

base or in various concentrations of salt
solutions.

• Proteins are heat labile exhibiting various

degrees of lability depending upon type of
protein, solution and temperature profile.
 Properties of Proteins
• Proteins can be reversible or
irreversible, denatured by heating, by
salt concentration, by freezing, by
ultrasonic stress or by aging.
Levels of Protein Structure
 Configuration vs Conformation

• CONFIGURATION
- refers to the geometric relationship between
a given set of atoms

• CONFORMATION
- refers to the spatial relationship of every
atom in a molecule
 Configuration vs Conformation
• Several Neurologic Disorders Result from Altered
Protein Conformation

• Prions: CHONS which lack nucleic acid, seen in

Creutzfeldt-Jakob disease, scrapie in sheep,
bovine spongiform encephalopathy (mad cow
disease)

• Alzheimer’s Disease: refolding or misfolding of

another another protein endogenous to human
brain tissue, B-amyloid is prominent in
Alzheimer’s disease.
 Levels of Protein Structure

1° structure: the sequence of amino acids in a

polypeptide chain, read from the N-terminal end to
the C-terminal end
• 2° structure: the ordered 3-dimensional
arrangements (conformations) in localized regions of
a polypeptide chain; refers only to interactions of the
peptide backbone
• e. g., α-helix and β-pleated sheet
• 3˚ structure: 3-D arrangement of all atoms
• 4˚ structure: arrangement of monomer subunits with
respect to each other
 1˚ Structure
• The 1˚ sequence of proteins determines its 3-D
conformation

• Changes in just one amino acid in sequence

can alter biological function, e.g. hemoglobin
associated with sickle-cell anemia

• Determination of 1˚ sequence is routine

biochemistry lab work (See Ch. 5).
 Protein Structure Levels

• PRIMARY: the linear sequence of amino acids

linked together by peptide
bonds

• Contributes to the native confirmation of proteins

 PRIMARY STRUCTURE

The sequence of amino acids

MIL1 sequence:
>gi|7662506|ref|NP_056182.1| MIL1 protein [Homo
sapiens]
MEDCLAHLGEKVSQELKEPLHKALQMLLSQPVTYQAFRECTLETTVHASGWN
KILVPLVLLRQMLLELTRLGQEPLSALLQFGVTYLEDYSAEYIIQQGGWGTV
FSLESEEEEYPGITAEDSNDIYILPSDNSGQVSPPESPTVTTSWQSESLPVS
LSASQSWHTESLPVSLGPESWQQIAMDPEEVKSLDSNGAGEKSENNSSNSDI
VHVEKEEVPEGMEEAAVASVVLPARELQEALPEAPAPLLPHITATSLLGTRE
PDTEVITVEKSSPATSLFVELDEEEVKAATTEPTEVEEVVPALEPTETLLSE
KEINAREESLVEELSPASEKKPVPPSEGKSRLSPAGEMKPMPLSEGKSILLF
GGAAAVAILAVAIGVALALRKK

length: 386amino acids

 PRIMARY STRUCTURE

• The numbers of amino acids vary

(e.g. insulin 51, lysozyme 129, haemoglobin 574,
gamma globulin 1250)

• The primary structure determines the folding of

the polypeptide to give a functional protein

• Polar amino acids (acidic, basic and neutral) are

hydrophilic and tend to be placed on the outside
of the protein.

• Non-polar (hydrophobic) amino acids tend to be

placed on the inside of the protein
© 2007 Paul Billiet ODWS
 Infinite variety

• The number of possible sequences is infinite

An average protein has 300 amino acids,
At each position there could be one of 20
different amino acids
= 10390 possible combinations

• Most are useless

Natural selection picks out the best

© 2007 Paul Billiet ODWS

 SECONDARY STRUCTURE

• This produces the alpha helix, beta pleating, beta

bends, non-repetitive structures, supersecondary
structures

• The length of the helix or pleat is determined by

certain amino acids that will not participate in
these structures (e.g. proline)

• Contributes to the native confirmation of proteins

 SECONDARY STRUCTURE

The folding of the

N-C-C backbone of
the polypeptide
chain using weak
hydrogen bonds

© Text 2007 Paul Billiet ODWS

 β-Pleated Sheet
• Polypeptide chains lie adjacent to one
another; may be parallel or antiparallel
• R groups alternate, first above and then below
plane
• Each peptide bond is s-trans and planar
• C=O and N-H groups of each peptide bond are
perpendicular to axis of the sheet
• C=O---H-N hydrogen bonds are between
adjacent sheets and perpendicular to the
direction of the sheet
β-Pleated Sheet (Cont’d)
 β-Pleated Sheet (Cont’d)
β-bulge- a common nonrepetive irregular 2˚
motif in anti-parallel structure
An alpha helix:
The backbone is
formed as a helix.
An ideal alpha helix
consists
of 3.6 residues per
complete turn.

An antiparallel beta
sheet.
Beta sheets are
created,
when atoms of beta
strands are hydrogen
bound.
Beta sheets may
consist of parallel
strands,
antiparallel strands or
out of a mixture
of parallel and
antiparallel strands
 Structures of Reverse Turns
• Glycine found in reverse turns
• Spatial (steric) reasons
• Polypeptide changes direction
• Proline also encountered in reverse turns.
 α-Helices and β-Sheets
• Supersecondary structures: the combination
of α- and β-sections, as for example
– βαβ unit: two parallel strands of β-sheet connected
by a stretch of α-helix
– αα unit: two antiparallel α-helices
– β-meander: an antiparallel sheet formed by a
series of tight reverse turns connecting stretches of
a polypeptide chain
– Greek key: a repetitive supersecondary structure
formed when an antiparallel sheet doubles back on
itself
– β-barrel: created when β-sheets are extensive
enough to fold back on themselves
Schematic Diagrams of Supersecondary
Structures
 Collagen Triple Helix
• Consists of three polypeptide chains wrapped around
each other in a ropelike twist to form a triple helix
called tropocollagen; MW approx. 300,000

• 30% of amino acids in each chain are Pro and Hyp

(hydroxyproline); hydroxylysine also occurs

• Every third position is Gly and repeating sequences

are X-Pro-Gly and X-Hyp-Gly

• Each polypeptide chain is a helix but not an α-helix

• The three strands are held together by hydrogen

bonding involving hydroxyproline and hydroxylysine

• With age, collagen helices become cross linked by

covalent bonds formed between Lys and His residues
 Fibrous Proteins
• Fibrous proteins:
- contain polypeptide chains organized
approximately parallel along a
single axis
– consist of long fibers or large sheets
– tend to be mechanically strong
– are insoluble in water and dilute salt solutions
– play important structural roles in nature
• Examples are
– keratin of hair and wool
– collagen of connective tissue of animals
including cartilage, bones, teeth, skin, and
blood vessels
 Fibrous proteins

• Involved in structure: tendons ligaments blood clots

(e.g. collagen and keratin)

• Contractile proteins in movement: muscle,

microtubules
(cytoskelton, mitotic spindle, cilia, flagella)

© 2007 Paul Billiet ODWS

 Globular Proteins
• Globular proteins:
- proteins which are folded to a more or
less spherical shape
– they tend to be soluble in water and salt
solutions
– most of their polar side chains are on the
outside and interact with the aqueous
environment by hydrogen bonding and ion-
dipole interactions
– most of their nonpolar side chains are buried
inside
– nearly all have substantial sections of α-helix
and β-sheet
 Globular proteins

• most proteins which move around (e.g.

albumin, casein in milk)

• Proteins with binding sites:

enzymes, haemoglobin, immunoglobulins,
membrane receptor sites

© 2007 Paul Billiet ODWS

Comparison of Shapes of Fibrous and
Globular Proteins
 TERTIARY STRUCTURE

The folding of
the polypeptide
into domains
whose chemical
properties are
determined by
the amino acids
in the chain

MIL1 protein
© 2007 Paul Billiet ODWS
© Anne-Marie Ternes
 TERTIARY STRUCTURE
• This folding is sometimes held together by
strong covalent bonds
(e.g. cysteine-cysteine disulphide bridge)

• Bending of the chain takes place at certain

amino acids
(e.g. proline)

• Hydrophobic amino acids tend to arrange

themselves inside the molecule

• Hydrophilic amino acids arrange themselves

on the outside
© 2007 Paul Billiet ODWS
 3˚ Structure

• The 3-dimensional arrangement of atoms in the

molecule.

• In fibrous protein, backbone of protein does not fall

back on itself, it is important aspect of 3˚ not specified
by 2˚ structure.

• In globular protein, more information needed. 3k

structure allows for the determination of the way
helical and pleated-sheet sections fold back on each
other.

• Interactions between side chains also plays a role.

 Forces in 3˚ Structure
• Noncovalent interactions, including
– hydrogen bonding between polar side chains, e.g.,
Ser and Thr
– hydrophobic interaction between nonpolar side
chains, e.g., Val and Ile
– electrostatic attraction between side chains of
opposite charge, e.g., Lys and Glu
– electrostatic repulsion between side chains of like
charge, e.g., Lys and Arg, Glu and Asp
• Covalent interactions: Disulfide (-S-S-) bonds
between side chains of cysteines
Forces That Stabilize Protein Structure
 Helices are visualized
as ribbons and
extended strands of
betasheets by broad
arrows.

Chain B of Protein Kinase C © Max Planck Institute for Molecular Genetics

 Myoglobin
• A single polypeptide chain of 153 amino acids

• A single heme group in a hydrophobic pocket

• 8 regions of α-helix; no regions of β-sheet

• Most polar side chains are on the surface

• Nonpolar side chains are folded to the interior

• Two His side chains are in the interior, involved with

interaction with the heme group

• Fe(II) of heme has 6 coordinates sites; 4 interact with

N atoms of heme, 1 with N of a His side chain, and 1
with either an O2 molecule or an N of the second His
side chain
The Structure of Myoglobin
Oxygen Binding Site of Myoglobin
 Quaternary Structure
• Quaternary (4°) structure: the association of
polypepetide monomers into multisubunit
proteins
- dimers
- trimers
- tetramers

Noncovalent interactions
- electrostatics,
- hydrogen bonds,
- hydrophobic
 QUATERNARY STRUCTURE

Some proteins are

made of several
polypeptide
subunits
(e.g. haemoglobin
has four)

Protein Kinase C

© Max Planck Institute for Molecular Genetics

© Text 2007 Paul Billiet ODWS

Oxygen Binding of Hemoglobin (Hb)

• A tetramer of two α-chains (141 amino acids

each) and two β-chains (153 amino acids
each); α2β2

• Each chain has 1 heme group; hemoglobin can

bind up to 4 molecules of O2
• Binding of O2 exhibited by positive
cooperativity; when one O2 is bound, it
becomes easier for the next O2 to bind
Structure of Hemoglobin
 Conformation Changes That Accompany
Hb Function
• Structural changes occur during binding of
small molecules
• Characteristic of allosteric behavior
• Hb exhibits different 4˚ structure in the bound
and unbound oxygenated forms
• Other ligands are involved in cooperative effect
of Hb can affect protein’s affinity for O2 by
altering structure
Oxy- and Deoxyhemoglobin
Immunoglobulin Structure

Immunoglobulins consist
of two types of
polypeptides, H chains
for "heavy" molecular
weight and L chains for
“light” molecular weight.
 Variable Region -
Antigen Binding Site
Immunoglobulin Structure
(cont)
Structure of Immunoglobulin
Fold Motif
Space-Filling IgG Structure
Other binding
proteins having
the
immunoglobulin
fold motif
 Denaturation
• Denaturation: the loss of the structural order (2°, 3°,
4°, or a combination of these) that gives a protein its
biological activity; that is, the loss of biological activity

• Denaturation can be brought about by

– heat
– large changes in pH, which alter charges on side
chains, e.g., -COO- to -COOH or -NH3+ to -NH2
– detergents such as sodium dodecyl sulfate (SDS)
which disrupt hydrophobic interactions
– urea or guanidine, which disrupt hydrogen bonding
– mercaptoethanol, which reduces disulfide bonds
Denaturation of a Protein
Denaturation and Refolding in
Ribonuclease

Several ways to denature

proteins
• Heat
• pH
• Detergents
• Urea
• Guanadine hydrochloride
Biochemical Methods to Analyze
Proteins

• Electrophoresis
• Chromatography: Gel filtration, ion
exchange, affinity
• Mass Spectrometry, X-ray
Crystallography, NMR
 Determination of 3° Structure
• X-ray crystallography
– uses a perfect crystal; that is, one in which all
individual protein molecules have the same 3D
structure and orientation
– exposure to a beam of x-rays gives a series
diffraction patterns
– information on molecular coordinates is extracted
by a mathematical analysis called a Fourier series
• 2-D Nuclear magnetic resonance
– can be done on protein samples in aqueous
solution
X-Ray and NMR Data

High resolution method to determine 3˚

structure of proteins (from crystal)
Diffraction pattern produced by electrons
scattering X-rays
Series of patterns taken at different
angles gives structural information
Determines solution structure
Structural info. Gained from
determining distances between
nuclei that aid in structure
determination
A representation of the 3D structure of myoglobin showing
coloured alpha helices. This protein was the first to have its
structure solved by X-ray crystallography.
Presence of SDS, a detergent,
denatures and linearizes a protein (Na and sulfate
bind to charged amino acids, the hydrocarbon
chain interacts with hydrophobic residues). An
applied electric field leads to separation of
proteins based on size through a defined gel pore
matrix.

For electrophoresis in the absence

of SDS, separation is based on size,
charge and shape of the protein
(proteins are not denatured and can
potentially retain function or activity)
Separation of proteins
based on their size is
linear in relation to the
distance migrated in the
gel. Using protein standards of
known mass and staining of the
separated proteins with dye, the
mass of the proteins in the
sample can be determined. This
is useful for purification and
diagnostic purposes.
 Separation is based on protein size.
Dextran or polyacrylamide beads of
uniform diameter are manufactured
with different pore sizes. Depending
on the sizes of the proteins to be
separated, they will enter the pore if
small enough, or be excluded if they
are too large.

Hydrophobic Chromatography
Proteins are separated based on their
net content of hydrophobic amino
acids. A hydrocarbon chain of 4-16
carbons is the usual type of resin.
Separation of proteins based on
the net charge of their constituent
amino acids. Different salt
concentrations can be used to elute
the bound proteins into tubes in a
fraction collector. As shown below,
resins for binding (+) or (-) charged
proteins can be used
• Based on the target proteins ability to bind a specific
ligand, only proteins that bind to this ligand will be
retained on the column bead. This is especially
useful for immunoaffinity purification of proteins
using specific antibodies for them.
• Example: