What are proteins?

Proteins are macromolecules

composed of amino acids linked
together through peptide bonds.
How are about proteins?

• the most widely distributed

biomolecules
• the most abundant biomolecules
(45% of human body)
• the most complex biomolecules
• the most diversified biological
functions
What do proteins do?

off

on

Regulation Movement

proteins B

Signaling Catalysis

Immune Transport
 Components of proteins

• major elements
C (50~55%), H (~7%), O (19~20%),
N (13~19%), S (~4%)
• trace elements
P, Fe, Cu, Zn, I, …
• The average nitrogen content in
proteins is about 16%, and
proteins are the major source of N
in biological systems.
• The protein quantity can be
estimated.
protein in 100g sample = N per gram x 6.25 x 100
 §1.1 Amino Acids
• The basic building blocks of proteins
• About 300 types of AAs in nature, but
only 20 types are used for protein
synthesis in biological systems.
• A amino group, a carboxyl group, a H
atom and a R group are connected to
a C atom.
• The C atom is an optically active
center.
 L-Amino acid

+
H3N
-
OOC

C H

R
§1.1.a Classification
• The R groups, also called side chains,
make each AA unique and distinctive.
• R groups are different in their size,
charge, hydrogen bonding capability
and chemical reactivity.
• Aas are grouped as (1) non-polar,
hydrophobic; (2) polar, neutral; (3)
basic; and (4) acidic.
 Non-polar and hydrophobic AAs

• R groups are non-polar, hydrophobic

aliphatic or aromatic groups.
• R groups are uncharged.
• AAs are insoluble in H2O.
 Polar and uncharged AAs

• R groups are polar: -OH, -SH, and

-NH2.
• R groups are highly reactive.
• AAs are soluble in H2O, that is,
hydrophilic.
 Basic AAs

• R groups have one -NH2.

• R groups are positively charged at
neutral pH (=7.0).
• AAs are highly hydrophilic.
 Acidic AAs

• R groups have –COOH.

• R groups are negatively charged at
physiological pH (=7.4).
• AAs are soluble in H2O.
Aspartic acid glutamic acid
(Asp or D) (Glu or E)
 Essential AAs
• Nutritionally essential AAs
• They cannot be synthesized in the body

• PVT TIM HALL

 Semi essential AAs
• Histidine and arginine
 Nomenclature

Starting from the carboxyl group, and

naming the rest carbon atoms
sequentially in Greek letters.

NH

     
CH3 CH COO- NH2 C NH CH2 CH2 CH2 CH COO-

NH3+ NH3+

-amino-propionic acid -amino--guanidinovaleric acid

(alanine) (arginine)
 Special amino acids - Gly

• optically inactive

+
H3N
-
OOC

C H

H
 Special amino acids - Pro

• Having a ring structure and imino

group

CH2
CHCOO-
CH2
NH2+
CH2
 Special amino acids - Cys
active thiol groups to form disulfide bond
§1.2 Peptide
§1.2.a Peptide and peptide bond
A peptide bond is a covalent bond
formed between the carboxyl group of
one AA and the amino group of its next
AA with the elimination of one H2O
molecule.
Peptides can be extended by adding
multiple AAs through multiple peptide
bonds in a sequential order.
dipeptide, tripeptide, oligopeptide, polypeptide

AAs in peptides are called as residues.

 Overview
• Proteins are composed of AAs.
• Distinctive properties of proteins are
determined by AA compositions, AA
sequences as well as the relative
positions of AAs in space.
• Proteins need well defined structures
to function properly. Their structures
are organized in a hierarchy format,
that is, primary, secondary, tertiary and
quaternary structure.
§2.1 Primary Structure

The primary structure of proteins is defined

as a linear connection of AAs along the
protein chain. It is also called amino acid
sequence.
The AA sequence must be written from the
N-terminus to the C-terminus.
Peptide bonds are responsible for
maintaining the primary structure.
 Primary structure of insulin

Two peptides of 21 and 30 AAs

Two inter-chain -S-S- bonds
One intra-chain -S-S- bond
§2.2 Secondary Structure

The secondary structure of a protein is

defined as a local spatial structure of a
certain peptide segment, that is, the
relative positions of backbone atoms of this
peptide segment.
Repeating units of N(-H), C, and C(=O)
constitute the backbone.
H-bonds are responsible for stabilizing the
secondary structure.
The side chains are not considered.
-helix
-pleated sheet
-turn (-bend)
random coil
 Peptide unit

• Six atoms, C-C(=O)-N(-H)-C,

constitute a planer peptide unit.
• The peptide unit is rigid due to the
partial double bond property.
• C=O and N-H groups are in trans
conformation and cannot rotate
around the peptide bond.
 Peptide unit

• Six atoms, C-C(=O)-N(-H)-C,

constitute a planer peptide unit.
• The peptide unit is rigid due to the
partial double bond property.
• C=O and N-H groups are in trans
conformation and cannot rotate
around the peptide bond.
 Rotation of peptide unit

Peptide units can rotate

freely around C-C and
C-N bonds to form two
torsion angles  and .
§2.2.a -helix

• A helical conformation is right-handed.

• 3.6 AAs per turn and a 0.15 nm vertical
distance, creating a pitch of 0.54 nm.
• Side chains of AA residues protrude
outward from the helical backbone.
• The hydrogen-bonds are parallel to the
helical axis.
Left-hand versus right-hand
This image cannot currently be display ed.
 0.54 nm
3.6 个残基
C原子

O原子

N原子

第n＋3个肽键的H原子 H原子

第n个肽键的O原子

肽链走向

0.5 nm

（a ） （b）
The -CO group of residue n is H-bonded to the -
NH group of residue (n+4).
§2.2.b -pleated sheet
• An extended zigzag conformation of
protein backbones
• Protein backbones are arranged side-
by-side through H-bonds.
• H-bonds are perpendicular to the
backbone direction.
• The side chains of adjacent AAs
protrude in opposite directions.
• The adjacent protein backbones can
be either parallel or anti-parallel.
§2.2.c -turn

• One -turn involves four AAs. The -CO

and -NH groups of the first AA are
hydrogen bonded to the -NH and -CO
groups of the fourth AA, respectively.
• The -turn reverses abruptly the
direction of a protein backbone.
• H-bonds are perpendicular to the
protein backbone.
§2.2.d Random coil

• There is no consistent relationship

between planes.
§2.3 Tertiary Structure

The tertiary structure is defined as the spatial

positions of all atoms of a protein, i.e., the
three-dimensional (3D) arrangement of all
atoms.
Four types of interactions stabilize the
protein tertiary structure.
• hydrophobic interaction
• ionic interaction
• hydrogen bond
• van der Waals interaction
§2.3.a Hydrophobic interaction
Nonpolar molecules tend to cluster together in
water, that is, aqueous environment tends to
squeeze nonpolar molecules together.
§2.3.b Ionic interaction
• A charged group is able to attract another
group of opposite charges.
• The force is determined by Coulomb’s law.
§2.3.c Hydrogen bond
A hydrogen atom is shared by two other atoms.
H-donor: the atom to which H atom is more tightly
attached, and the other is H-acceptor.
§2.3.d van der Waals force
• An asymmetric
electronic charge
around an atom causes
a similar asymmetry
around its neighboring
atoms.
• The attraction between
a pair of atoms
increases as they
come closer, until they
are repelled by van der
Waals contact distance.
Interactions stabilizing proteins
Myoglobin (Mb)

Located in muscle to
supply O2
1st protein in high
resolution
153 AAs
75% of structure is -
helix in 8 regions.
the interior almost
entirely nonpolar
residues
§2.4 Quaternary Structure

The quaternary structure is defined as

the spatial arrangement of multiple
subunits of a protein.
• Proteins need to have two or more
polypeptide chains to function properly.
• Each individual peptide is called
subunit.
• These subunits are associated through
H-bonds, ionic interactions, and
hydrophobic interactions.
• Polypeptide chains can be in dimer,
trimer .., as well as homo- or hetero-
form.
 Hemoglobin(Hb)
• O2 transporter in erythrocyte
• 2  subunits, 141 AAs
2  subunits, 146 AAs
• 4 subunits are maintained together
by 8 pairs of ionic interactions.
• Each subunit contains one heme
group.
• The conserved hydrophobic core
stabilizes the 3D structure.
Structure of hemoglobin
From primary to quaternary structure
§2.5 Protein classification
• Constituents
simple protein
conjugated protein = protein + prosthetic
groups

Prosthetic group is non-protein part,

binding to protein by covalent bond. This
group can be carbohydrates, lipids,
nucleic acids, phosphates, pigments, or
metal ions.
• Classification based on the overall
shape
• Globular protein:
long/short < 10，soluble in water;
including enzymes, transportors,
receptors, regulators, …
• Fibrous protein:
highly elongated; insoluble in water;
including collage, elastin, α-
keratin, …
Structural similarity of Mb and Hb
Structure of hemoglobin
§3.2 Collagen

insoluble fibers that have high

tensile strength
25% of total protein weight of
human body
consisting of three chains of
same size (285kd)
Collagen in different organisms

Tissue Content
Bone 88.0
Calcaneal tendon 86.0
Skin 71.9
Cornea 68.1
Cartilage 46-63
Ligament 17.0
Aorta 12-24
Liver 3.9
 Unusual components

AA components
Gly (1/3), proline (1/4), 4-hydroxyproline (1/10),
5-hydroxylysine (1%)

AA sequences
(Gly-Pro-Y)n or (Gly-X-Hyp)n
X and Y can be any AAs.
n can be as high as a few hundreds.
 Intermolecular cross-link

Lys at N- and C-termini and Hly in helical regions

are responsible for the cross-link.
The linkage varies with the physiological function
and the tissue age.
30 genes encode for collagens, and 8 post-
translational modifications are needed collagen
maturation.
§5.1 Amphoteric
Isoelectric point
AAs in solution at certain pH are predominantly in
dipolar form, fully ionized but without net
charge due to -COO- and -NH3+ groups.
This characteristic pH is called isoelectric point,
designated as pI.
pI is determined by pK, the ionization constant of
the ionizable groups.
 R CH COOH
NH2

- -
R CH COO- R CH COO-
+OH +OH
R CH COOH
+H+ +H+
NH3+ NH3 + NH2

pH<pI pH=pI pH>pI

cation amphoteric anion
Amino acid pI M.W. Amino acid pI M.W.

Glycine 5.97 75 cystein 5.07 121

Alanine 6.00 89 methionine 5.74 149

Valine 5.96 117 asparagine 5.41 132

Leucine 5.98 131 glutamine 5.65 146

cystein 5.60 119

Isoleucine 6.02 131
aspartic 2.97 133
Phenylalani 5.48 165 acid
ne
glutamic 3.22 147
Proline 6.30 115 acid
tryptophan 5.89 204 Lysine 9.74 146

serine 5.68 105 Arginine 10.76 174

tyrosine 5.66 181 Histidine 7.59 155

• Side-chains of a protein have many
ionizable groups, making the protein
either positively or negatively
charged in response to the pH of the
solution.
• The pH at which the protein has zero
net-charge is referred to as
isoelectric point (pI).
 COOH COO- COO-
+ OH- + OH-
P P P
+ H+ +
+ H+
NH3+ NH3 NH2

cation amphoteric anion

pH < pI pH = pI pH > pI

• pI of most protein is ~ 5.0, and negatively charges in

body fluid (pH7.4)
• pI > 7.4: basic proteins: protamine, histone
• pI < 7.4: acidic proteins: pepsin
§5.5 Coloring reactions

• Biuret reaction: peptide bonds and Cu2+

under the heating condition to form red or
purple chelates.
• Used for determine the hydrolysis of
proteins since free amino acids do not
react.

• Amino acids can react with ninhydrin to

form a chemicals having maximal
adsorption at 570 nm.
• Used for quantifying the free amino acids.
§6.1 Isolation and purification

• Homogenization and centrifugation

• Dialysis
• Chromatography
• Electrophoresis
§6.1.a Homogenization

• Rupture the plasma membrane to

release the intracellular components
into the buffered solution

• Sonication, French pressure,

mechanical grinding,
• Chemical reagents, lysozymes
 Centrifugation
• Because of the differences in size and
shape, proteins will sediment gradually
under the centrifugal force until the
sedimentation force and buoyant force
reach the balance.
• The sedimentation behavior is described
in sedimentation coefficient (S) which is
proportional to the molecular weight.
Differential centrifugation
 Differential centrifugation
homogenate

600 g，3 min

Pellet supernatant
(nuclei)
6,000 g，8 min

Pellet supernatant
(mitochondria, chloroplasts,
lysosomes, peroxisomes) 40,000 g，30 min

Pellet
supernatant
(plasma membrane,
fragments of Golgi and ER)
100,000 g，90 min

Pellet supernatant
(ribosomal subunits) (cytosol)
Rate-zone centrifugation
§6.1.b Dialysis

• Proteins, as macromolecules, cannot

pass through the semipermeable
membrane containing pores of smaller
than protein dimension, thus large
proteins and small molecules can be
separated.
• Dialysis can be used for protein
purification, desalting, and condensation.
§6.1.d Chromatography

When a protein solution (called as mobile

phase) passes through a stationary phase,
proteins can interact with the stationary
phase due to the differences in size,
charge, and affinity, making the different
proteins flow through the stationary phase
at different speeds.
 Elution
buffer
Protein mixture

Solid
phase
capable
of
reacting
with
proteins
to be
separated

Protein 1 Protein 2
OD280nm

Elution volume
 Gel filtration
Proteins are separated based on their
sizes and shapes. The stationary phase is
of semi-uniform pores. When the protein
solution flows through porous beads,
smaller proteins can enter the pores and
stay there for a longer period, but larger
proteins flow directly through the column,
resulting in the separation of proteins. It is
also called molecular sieve or size
exclusion.
 Gel filtration
Small proteins can
enter the porous
beads, and have a
longer stationary
time

Porous beads
Large proteins that
allow the small
are unable to enter
proteins enter
the porous beads will
pass by and flow out
directly
 Type of chromatography

• Ion exchange: based on the ionic

interactions
• Affinity: based on the binding strengths
• Filtration: based on the protein sizes
• Hydrophobicity: based on the
hydrophobic forces
Ion-exchange chromatography
More negatively charged
proteins bind to the solid
phase tightly, and stronger
elution buffer is needed to
elute them out the column

＝ －
＝ －
－ ＋ ＋
＝
＋ ＋ ＝ ＋ ＋
＝ － ＝
＋ ＋ ＝ ＋ ＋
＝ －
＋ ＋ ＋ ＋
＝ ＝
＋ ＋
＝
－ ＝
－
－
－
－ Ionic exchange
column with
Less negatively charged positive charge
proteins bind to the solid
phase loosely, and weak
elution buffer can be used to
elute them out the column
Affinity chromatography
Exchange column
with the ligands
for binding
special proteins

Proteins having Proteins having

weak binding strong binding
affinity with the affinity with the
ligads ligads
§6.2 Electrophoresis Analysis

Used mainly for determination of

proteins

SDS-PAGE = Sodium dodecyl sulfate

polyacrylamide gel electrophoresis
IEF = isoelectric focusing electrophoresis
2D = two dimensional electrophoresis