Download The O 2

Chapter 1
Protein
Contents
1. Chemical components
2. Molecular structures
3. Biological functions
4. Structure-function relationship
5. Physical and chemical properties
6. Exploration of proteins
7. Proteomics: a new frontier
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?
Section 1
Chemical Components of
Proteins
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
R
H
Molecular weight
Dalton:
A unit of mass nearly equal to that
of a hydrogen atom
Gly
Ala
Val
Leu
Ile
C2NO2H5
C3NO2H7
C5NO2H11
C6NO2H13
C6NO2H13
75
89
117
131
131
§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
(Asp or D)
glutamic acid
(Glu or E)
Nomenclature
Starting from the carboxyl group, and
naming the rest carbon atoms
sequentially in Greek letters.
NH


CH3
CH

COO-
NH3+
-amino-propionic acid
(alanine)


NH2 C NH CH2 CH2 CH2

CH COONH3+
-amino--guanidinovaleric acid
(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
CH2
NH2+
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.
§1.2.b Biologically active peptides
Glutathione (GSH)
Glutamic acid
cystein
glycine
H2O2
2GSH
GSH
peroxidase
2H2O
GSSG
NADP+
GSH
reductase
NADPH+H+
As a reductant to protect nucleic acids and
proteins from toxin by discharging free radical
or H2O2
Peptides
• Peptide hormones secreted from
peptidergic neurons or
– Somatostatin, Noacosapeptide, Octapeptide,
– Thyrotropin-release hormone, Antidiuretic
hormone
• Neuropeptides responsible for signal
transduction
– Enkephalin, Endorphin, Dynorphin, Substance P,
Neuropeptide Y
thyrotropin-release hormone
Pyroglutamic acid
histidine
prolinamide
Neuropeptide
name
amino acid sequence
oxytocin
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
└──S───S──┘
Vasopresin
Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-Gly-NH2
└──S────S──┘
Met-enkephalin
Tyr- Gly-Gly-Phe-Met
Leu-enkephalin
Tyr- Gly-Gly-Phe-Leu
Atrial natriuetic
factor
Ser-Leu-Arg-Arg-Ser-Ser-Cys-Phe-Gly-Gly-ArgMet-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-CysAsn-Ser-Phe-Arg-Tyr
Substance P
Arg-Pro-Lys-Pro-Bln-Phe-Phe-Gly-Leu-Met-NH2
Bradykinin
Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg
Section 2
Molecular Structures of
Proteins
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.
Resonant conjugation
O
H
O
O－
C
C
N
N＋
H
H
R2
0.124
5
0. 1
C
1
C
C 0.1
32
N
46
0.1
C-N:
0.149nm
C＝N: 0.127nm
H
R1
H
Rotation of peptide unit
Peptide units can rotate
freely around C-C and
C-N bonds to form two
torsion angles  and .
“Beads on a string”
N-terminus
C
Peptide unit
Backbone
C-terminus
Linus Carl Pauling
• b. 1901, d. 1994
• California Institute of
Technology, CA
• The Nobel Prize in Chemistry
(1954), “for his research into
the nature of the chemical
bond and its application to
the elucidation of the
structure of complex
substances”
• The Nobel Peace Prize (1962)
§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
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 sideby-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.
§2.2.e Motif
When several local peptides of defined
secondary structures are close enough
in space, they are able to form a
particular “super-secondary” structure.
• Zinc finger
HLH (helix-loop-helix)
HTH (helix-turn-helix)
Leucine zipper
§2.2.f Side chains effect
• Shape: Pro having a rigid ring (–
helix disrupter)
• Size: -sheet needs AAs of small side
chain. Leu, Ile, Trp, and Asn having
bulky sides (hard to form –helix)
• Charge: Too many charged AAs in a
short region of one peptide is hard to
form –helix.
§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
Ribonuclease
• A pancreatic
enzyme that
hydrolyzes RNA
• 124 AAs
• Mainly -sheet
• Highly compact
and nonpolar
interior
• 4 disulfide bonds
Rhodopsin
• Photoreceptor
protein
• 7 transmembrane
helices
• 11-cis-retinal
chromophore
in the pocket
• Residues are
modified.
§2.3.e Domain
Large polypeptides may be organized
into structurally close but functionally
independent units.
Fiberousis protein
Methyl-accepting chemotaxin
• Highly conservative
cytosolic domain
• Divergent
periplasmic domain
serving as a
chemosensor
• Transducing the
external singles into
the cell
§2.3.f Chaperon
Chaperones are large, multisubunit
proteins that promote protein foldings
by providing a protective environment
where polypeptides fold correctly into
native conformations or quaternary
structures.
How does chaperon work?
• Reversibly bind to the hydrophobic
portions to advance the formation of
correct peptide conformations
• Bind to misfolded peptides to induce
them to the proper conformations
• Assist the formation of correct
disulfide bonds
§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 heteroform.
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
Ionic forces among Hb subunits
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, αkeratin, …
Section 3
Biological Functions of
Proteins
§3.1 Hemoglobin
• Hb can bind O2 reversibly, just like Mb.
• Both  and  chains are strikingly
similar to that of Mb.
• Although only 24 of 146 AAs of their
sequences are identical, 9 critical
residues are conserved in sixty species.
• Residues on the surface are highly
variable, but the nonpolar core is
conserved.
Structural similarity of Mb and Hb
Fe-porphyrin complex
Fe lies at
the center
of picketfence
porphyrin to
form 4
coordinate
bonds with
4 N atoms.
Heme group
The 5th coordinate
of Fe is formed
with histidine F8,
and the 6th one is
for either histidine
E7 or O2.
Heme group
Oxygen-disassociation curve
• The saturation Y
is defined as the
fractional
occupancy of all
O2-binding sites.
• Y varies with the
concentration of
O2 . The
equilibrium
constants for Hb
subunits are
different.
Binding behavior of Hb
• Hb has a lower affinity for O2 than Mb
(lower P50).
• The O2–binding to the 1st subunit
enhances the O2–binding to the 2nd and
3rd subunits. Such process further
enhances the O2–binding to the 4th
subunit significantly.
• Hb binds O2 in a positive cooperative
manner, which enhances the O2
transport.
Local structural change
Upon oxygenation, the Fe atom is moved into
the porphyrin plane, leading to the formation
of a strong bond with O2.
CO and O2 binding
Hb forces CO to bind at an angle due to steric
hindrance of His E7, which weakens the
binding of CO with the heme.
Conformational changes
The quaternary structure of Hb changes
markedly upon oxygenation ( subunit
shifts by 0.6nm and rotates by 15°).
Global structural change
The quaternary
structure of Hb
changes markedly
for the tense (T)
form to the relaxed
(R) form upon
oxygenation.
Allosteric effect
• The behavior that the lignad-binding
to one subunit causes structural
changes and stimulate the further
binding to other subunits is termed
as allosteric effect.
• The protein is allosteric protein, and
the substrate is allosteric effector.
• Allosteric effect can be influenced by
activators as well as inhibitors.
Concerted versus sequential



1

non-oxygenized Hb
（T conformation）






oxygenized Hb
（R conformation）











§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.
Unusual triplex
• Each helix is Lhanded and 3 AAs
per turn.
• Three helixes wind
together through Hbonds in the righthanded form.
• Unusual helical
conformation (0.312
nm versus 0.15nm)
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 posttranslational modifications are needed
collagen maturation.
Type of collagens
Diseases and collagen
pK curve
Structure of hemoglobin
Concerted versus sequential
Model comparison
Concerted Model
Sequential Model
The symmetry is essential Subunits can interact
for the subunit interaction. even if they are in
different forms.
T and R forms are in
T to R is induced by the
equilibrium in the absence binding of substrate.
of substrate
Homotropic interaction is
positive.
Homotropic interaction
can be positive and
negative.
Section 4
Structure-Function
Relationship of Proteins
§4.1 Primary Structure and Function
• Primary structure is the
fundamental to the spatial
structures and biological functions
of proteins.
• For a protein of particular sequence,
many conformers are possible, but
only the correct one has the
biological functions.
1. Proteins having similar amino acid
sequences demonstrate the
functional similarity.
2. Proteins of incorrect structures have
no proper biological functions, even
their amino sequences are remained
in a right order.
3. The alternation of key AAs in a protein
will cause the lose of its biological
functions.
Sequences of Cytochrome C
________10 ________20 ________30 ________40 ________50 ________60 ________70 ________80 ________90 _______100 ____
Human
GDVEKGKKIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGYSYTA ANKNKGIIWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE
Chimpanzee
GDVEKGKKIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGYSYTA ANKNKGIIWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE
Monkey
GDVEKGKRIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQASGFTYTE ANKNKGIIWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE
Macaque
GDVEKGKKIF IMKCSQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGYSYTA ANKNKGITWG EDTLMEYLEN PKKYIPGTKM IFVGIKKKEE RADLIAYLKK ATNE
Cow
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKKGE RADLIAYLKK ATNE
Dog
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKKGE RADLIAYLKK ATKE
Grey whale
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAVGFSYTD ANKNKGITWG EETLMEYLEN PKKYIPGTKM IFAGIKKKGE RADLIAYLKK ATNE
Horse
GDVEKGKKIF VQKCAQCHTV EKGGKHKTGP NLHGLFGRKT GQAPGFSYTD ANKNKGITWK EETLMEYLEN PKKYIPGTKM IFAGIKKKTE RADLIAYLKK ATNE
• Cytochrome C is a protein which can
be found in all aerobic organisms.
Structures of Cytochrome C
tuna-heart
mitochondria
photosynthetic
bacterium
denitrifying
bacterium
1. Proteins having similar amino acid
sequences demonstrate the functional
similarity.
2. Proteins of incorrect structures
have no proper biological functions,
even their amino acid sequences
are remained in a right order.
3. The alternation of key AAs in a protein
will cause the lose of its biological
functions.
Bovine nuclease
• 124 AAs, 4
disulfide
bonds (105
possibilities)
• The denatured protein remains its primary
structure, but no biological function.
• Only the correct form has the enzymatic
activity.
The renatured protein will restore its
functions partially or fully depending
upon the correctness of the refolded
structure.
1. Proteins having similar amino acid
sequences demonstrate the functional
similarity.
2. Proteins of incorrect structures have
no proper biological functions, even
their amino sequences are remained
in a right order.
3. The alternation of key AAs in a
protein will cause the lose of its
biological functions.
Sickle-cell of anemia
Patient’s symptoms:
Cough, fever and headache, a
tinge of yellow in whites of eyes,
visible pale mucous membrane,
enlarged heart, well developed
physically, anemic, much less
RD cells
clinical test:
The shape of the red cells was
very irregular, large number of
thin, elongated, sickle-shaped
and crescent-shaped forms.
Identifying the cause
• pI of sickle-cell Hb was higher than
normal one by 0.23, which is equivalent to
2 to 4 net positive charges per Hb
molecule. (1949, Pauling)
• 2-D electrophoresis showed only one
peptide of 28 digested Hb peptides is
different (1954, Ingram).
Identifying the cause
Difference in primary structure of Hb
• Sequence analysis showed the
difference in AA sequence.
Hb A : Val-His-Leu-Thr-Pro-Glu-Glu-LysHb S : Val-His-Leu-Thr-Pro-Val -Glu-Lys-
• This is the first case of molecular
disease identified in history. Further
studies showed that the AA variation is
due to the gene mutation.
§4.2 Spatial Structure and Function
• Proteins will experience multiple
processed to become correctly folded,
that is, having a correct structure.
• The incorrect protein structure may lead
to function alternation or diseases.
• A particular spatial structure of a protein is
strongly correlated with its specific
biological functions.
Mad cow disease and prion proteins
• A transmissible, inheritable neural
disease, destroying brain tissues by
converting them to a spongy appearance
• the conformational changes of prion
protein (PrP)
– PrPc: -helix, water soluble
– PrPsc: -sheet, water insoluble
Structural changes of prion protein
PrPc
PrPsc
Section 5
Physical and Chemical
Properties of Proteins
§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
+OH-
R CH COOH
+H+
NH3+
+OH
R CH COOR CH COO-
NH3+
+H+
NH2
pH<pI
pH=pI
pH>pI
cation
amphoteric
anion
Amino acid
pI
M.W.
Glycine
5.97
75
Alanine
6.00
Valine
Amino acid
pI
M.W.
cystein
5.07
121
89
methionine
5.74
149
5.96
117
asparagine
5.41
132
Leucine
5.98
131
glutamine
5.65
146
Isoleucine
6.02
131
cystein
5.60
119
Phenylalani
ne
5.48
165
aspartic
acid
2.97
133
3.22
147
Proline
6.30
115
glutamic
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).
COO-
COOH
+ OH-
P
COO+ OH-
P
NH3+
+ H+
P
NH3+
cation
amphoteric
pH < pI
pH = pI
+ H+
NH2
anion
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
§5.2 Colloid property
• Diameter: 1~100nm,
in the range of colloid;
• Hydrophilic groups
on the surface form a
hydration shell;
• Hydration shell and
electric repulsion
make proteins stable
in solution.
- - + +
+ - +
- + - +
+
-+
+ - +
+
- +- -
Precipitation of protein colloid
+ +
+
+
+
+ +
positively charged
(hydrophilic)
dehydration
+ + +
+
+
+
+ +
positively charged
(hydrophobic)
acid
base
base
acid
isoelectric point
(hydrophilic)
dehydration
base
Instable protein
(deposition)
－
－
－
－
－
－－ －
negatively charged
(hydrophilic)
dehydration
acid
－ －
－
－
－
－
－ －
negatively charged
(hydrophobic)
§5.3 Protein denaturation
The process in which a protein loses
its native conformation under the
treatment of denaturants is referred to
as protein denaturation.
• The denatured proteins tend to
- decrease in solubility;
- increase the viscosity;
- lose the biological activity;
- lose crystalizability;
- be susceptible to enzymatic digestion.
• Cause of denaturation
the disruption of hydration shell and
electric repulsion
• Denaturants
physical: heat, ultraviolet light, violent
shaking, …
chemical: strong acids, bases, organic
solvents, detergents, …
• Applications
sterilization, lyophilization
Renaturation
• Once the denaturants are removed, the
denatured proteins tend to fold back to
their native conformations partially or
fully.
• The renatured proteins can restore their
biological functions.
Renaturation
Protein precipitation
The denatured proteins expose their side
chains or the inner part to the aqueous
environment, which causes the proteins
aggregated and separated out from the
aqueous solution.
Protein coagulation
• When the denatured proteins become
insoluble fluffy materials, heating
denatured proteins will turn them into a
hard solid which are not soluble even
strong acids and bases are applied.
• Coagulation is an irreversible process.
§5.4 UV absorption
• Trp, Tyr, and Phe have aromatic groups
of resonance double bonds.
• AAs have a strong absorption at 280nm.
• Both free and incorporated AAs show
this absorption.
§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.
Section 6
Exploration of Protein
§6.1 Isolation and purification
• Homogenization and centrifugation
• Dialysis
• Precipitation
• 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
(nuclei)
supernatant
6,000 g，8 min
Pellet
(mitochondria, chloroplasts,
lysosomes, peroxisomes)
supernatant
40,000 g，30 min
Pellet
supernatant
(plasma membrane,
fragments of Golgi and ER)
100,000 g，90 min
Pellet
(ribosomal subunits)
supernatant
(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.c Precipitation
• Adding a large quantity of salts, such as
Ammonia sulfate, into the protein solution
will neutralize the surface charges and the
destruct the hydration shell of proteins,
causing them to precipitate.
• Acetone has the similar function.
• An efficient way to concentrate proteins.
§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.
Protein mixture
Elution
buffer
Solid
phase
capable
of
reacting
with
proteins
to be
separated
Protein 2
OD280nm
Protein 1
Elution volume
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
－
＝
＋
－
＋
＝
＝
＋
＋
＋
＋
＝
＝
＝
＋
－
＝
＝
－
＋
＋
＋
＝
＋
－
－
＋
＋
＝
＝
－
－
－
Less negatively charged
proteins bind to the solid
phase loosely, and weak
elution buffer can be used to
elute them out the column
＋
＋
－
＝
＋
Ionic exchange
column with
positive charge
Affinity chromatography
Exchange column
with the ligands
for binding
special proteins
Proteins having
weak binding
affinity with the
ligads
Proteins having
strong binding
affinity with the
ligads
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
Large proteins that
are unable to enter
the porous beads will
pass by and flow out
directly
Porous beads
allow the small
proteins enter
§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
§6.2.a SDS-PAGE
• Sodium dodecyl sulfate is a kind of
detergent to denature the proteins
• Anionic SDS bind to protein in the ratio of
1 SDS per 2 AAs.
• The protein-SDS complex is roughly
charged proportional to the mass.
• The smaller the protein, the faster the
moving speed in the electric field.
• The gel polymer material for protein
discrimination is composed of
methylenebisacrylamide and acrylamide.
• The pore size can be controlled by
changing the concentration of crosslinking reagent.
• The gel with different concentration of
cross-linking reagent can be used for
different size proteins.
§ 6.2.b Isoelectric focusing
• Depend upon the electric properties of
proteins
• The charged proteins, either positively or
negatively, will migrate in the electric field.
• The proteins having net zero charges
stop moving in the electric field.
Principle of IFE
§6.2.c 2D electrophoresis
• 1st dimension: isoelectric focusing
electrophoresis
• 2nd dimension: SDS-PAGE
• A high throughput approach to identify
proteins
§6.3 Composition and Sequence
• Composition
• Determination of terminal residues
• Edman degradation
• Sequencing strategy
• Mass spectroscopy
• Deduction form DNA sequences
§6.3.a Composition analysis
• Hydrolyzing the purified protein samples
in an evacuated and sealed tube by
heating it in 6 M HCl at 100°C
• Analyzing the AA components using
chromatography
• (Alaa, Argb, Asnc, Aspd, … Valz)
Chromatography of AA
§6.3.b Terminal residues
• The amino-terminal residue reacts with
fluorodinitrobenzene or dabsyl chloride
to form a stable product which can be
analyzed using chromatography.
• The carboxyl-terminal residue can react
with fluorodinitrobenzene or dabsyl
chloride to form a stable product.
N-terminal reaction
§6.3.c Edman degradation
1. Labeling the N-terminal residue with a
fluorophore.
2. Cleaving the labeled residue without
breaking the peptide bonds of the rest
part of the peptide.
3. Determining the N-terminal residue with
chromatography.
4. Repeating the same procedure to the rest
peptide until the whole sequence is
determined.
First cycle of Edman degradation
N
C
S
H2N
H
O
H
O
C
C
N
C
C
H
R2
H
O
N
C
C
H
R2
R1
phenyl
isothiocyanate
labeling
H
S
H
H
O
N
C
N
C
C
R1
releasing
S
N
C
C
N
O
C
H
R1
H2N
H
H
O
C
C
R2
H
O
N
C
C
H
R3
Edman degradation
§6.3.d Overlapping approach
1. Cleaving a protein into small peptides by
chemical or enzymatic methods, and
purify these peptides.
2. Sequencing each peptide using Edman
degradation approach.
3. Overlapping peptide fragments to
arrange them in a right order, and
accomplishing the AA sequencing.
Cleavage of peptides
Cleavage reagent
Cleavage site
Cyanogen bromide
Met (C)
O-Iodosobenzoate
Try (C)
Hydroxylamine
Asp-Gly
Trypsin
Lys and Arg (C)
Clostripain
Arg (C)
Staphylococcal protease
Asp and Glu (C)
Thrombin
Arg (C)
Overlapping approach
1. Tryptic cleavage generates two peptides
Gly-Phe-Val-Glu-Arg, Val-Phe-Asp-Lys
2. Chymotryptic cleavage generates three
peptides
Val-Phe, Val-Glu-Arg, Asp-Lys-Gly-Phe
3. Overlapping the sequenced peptides
Tryptic peptide
Tryptic peptide
Val - Phe - Asp - Lys - Gly - Phe - Val - Glu - Arg
Chymotryptic peptide
§6.3.e Mass spectroscopy
• Newly developed approach applied to
biology and medicine areas
• Revolutionized bioanalytical technique
• Offering a fast, high accuracy, and high
throughput determination for analyzing
peptides and proteins.
Matrix-aided ionization
1. Deposit samples
on a plate.
2. Introduce a beam
of laser.
3. Ionize samples.
4. Analyze ionized
molecules.
5. Determine the AA
sequence.
Fragmentation of peptide
xn+3
yn+3
xn+2
yn+2
xn+1
O
O
O
O
yn+1
O
—N—C—C —N—C—C—N—C—C —N—C—C —N—C—C
H Ri
H Ri+1
ai+1
H
Ri+2
H
Ri+3
bi+1
ai+2
bi+2
ai+3
bi+3
H
Ri+4
From MS to AA sequence
§6.3.f Deduction from DNA sequence
Isolate the genes encoding the protein
DNA sequencing
mRNA sequencing
Determine the AA sequence according to the
3-letter genetic codons
§5.4 Structure Determination
• Circular dichroism spectroscopy
• X-ray crystallography
• Nuclear magnetic resonance
spectroscopy
• Prediction based on the protein
sequence homology
• Computer simulation
Section 7
Proteomics:
A New Frontier
Proteomics
a comprehensive knowledge
about all the proteins of a cell at
a specific given time.
Objectives
•
Biological process
–
•
Molecular function
–
•
The overall process toward which this
protein contributes
The biological activity the protein
accomplishes
Cellular component
–
The location of protein activity
Proteomics approaches
•
•
•
•
•
•
Separation
Sequence determination
3D-structure
Functionality
Expression regulation
Post-translation modification
Protein chips
Incorrect conformation and diseases
• Proteins synthesis, post-translation
modification and maturation is a very
complex process.
• Only the correct folding process leads to
the correct conformation and the proper
functions of proteins.
• Incorrect folding process may lead to
diseases.
Structures of Cytochrome C
Rice
Yeast
Bacterium
Phylogenetic tree of cytochrome C
Human,
Chimpanzee
Candida
krusei
Horse,
Zebra
Debaryomyces
kloeckeri
Macaque
Pig, cow, sheep
Dog
Gray whale
Shark
Baker’s
yeast
Hornworm
moth
Penguin
Turkey, chicken
Duck
Pigeon
Turtle
Tuna
Alligator
Kangaroo
Carp
Silk moth
Neurospora
crassa
Bullfrog
Monkey
Rabbit
Bacteria
Bonito
Fruit fly
Screwworm
fly
Pacific
lamprey
Mungbean
Pumpkin
Wheat
Tomato
Sunflower
Homology comparison