Transcript Protein - UniMAP Portal

Chapter 5: Outline
Amino Acids
Amino acid classes
Bioactive AA
Modified AA
Peptides
Proteins (We are here)
Protein structure
Fibrous proteins
Globular proteins
Stereoisomers
Titration of AA
AA reactions
5P2-1
Protein Function
1. Catalysis
2. Structure
3. Movement
4. Defense
5. Regulation
6. Transport
7. Storage
8. Stress Response
5P2-2
PEPTIDES – re-caps
Peptide: a polymer of about 2-100
AAs linked by the peptide (amide)
bond. As the amino group and the
carboxyl group link, water is lost.
5P2-3
A peptide is written with the Nterminal end to the left and the Cterminal end to the right.
H2N-Tyr-Ala-Cys-Gly-COOH
Name =
Tyrosylalanylcysteinylglycine
The peptide bond is rigid and planar
due to the resonance contribution
shown right.
5P2-4
Amino acids consists of carboxyl
groups, amino groups, and various R
groups can undergo numerous
chemical reactions.
Polypeptides are linear polymers.
They are composed of amino acids that
linked together by peptide bonds.
Peptides bonds are amide linkages.
The linkages formed when the unshared
electron pair of the -amino nitrogen
atom of one amino acid attacks the carboxyl carbon.
5P2-5
The linked amino acids are referred to as
amino acid residues.
The amino acid residue with the free
amino group is called the N-terminal
residue and written to the left of
polypeptide.
H2N-Tyr-Ala-Cys-Gly-COOH
Name = Tyrosylalanylcysteinylglycine
The amino acid residue with free
carboxyl group is called the C-terminal
residue and written to the right of a
polypeptide.
5P2-6
The C-terminal residues can be
identified by a group of enzymes
called the carboxypeptidases.
The N-terminal residues is
determined using Sanger’s method
where the polypeptide chain is
reacted with 1-fluoro-2,4dinitrobenzene.
5P2-7
DEFRAGMENTATION OF POLYPEPTIDE
# The cleavage or broken up of polypeptides
done by using several reagents, mostly the
enzymes.
# The pancreatic enzyme, trypsin is most
commonly used.
# Trypsin cleaves peptide bonds on the
carboxyl side of either lysine or arginine
residues.
# Chymotrypsin, another pancreatic enzyme,
breaks peptide bonds on the carboxyl side
of phenylalanine, tyrosine or trptophan.
5P2-8
# Cyanogen bromide is a reagent
specifically cleaves peptide bonds on the
carboxyl side of methionine residues.
# 1-fluoro-2,4-dinitrobenzene (DNFB) has
been used to determine the N-terminal
amino acids.
# The N-terminal is identified as the
dinitrophenyl (DNP) is attached to the
terminal after reaction.
# Carboxypeptidases hydrolizes peptides
one residue at a time from the C-terminal
end.
5P2-9
Class discussion
page 155
Problem 1 and 2
5P2-10
Proteins by Shape-1
Fibrous proteins exist as long stranded molecules:
Eg. Silk, collagen, wool. A collagen segment in
space-filling model illustrates this point.
Red spheres represent oxygen,
grey carbon, and blue nitrogen
5P2-11
Proteins by Shape-2
Globular proteins
have somewhat
spherical shapes.
Most enzymes are
globular. Eg.
myoglobin,
hemoglobin.
Myoglobin in
space-filling model
is the chosen
example.
5P2-12
Proteins by Composition
Simple
Contain only amino acids
Conjugated
simple protein (apoprotein)
prosthetic group (nonprotein)
Hologlycoproteins
protein
lipoproteins
metaloproteins
etc.
5P2-13
Four Levels of Protein Structure
Primary, 1o
the amino acid sequence
Secondary, 2o
3-D arrangement of backbone atoms in
space
Tertiary, 3o
3-D arrangement of all the atoms in
space
Quaternary, 4o
3-D arrangement of subunit chains
5P2-14
Determining Primary Structure
1. Hydrolyze protein with hot 6M HCl.
Identify AA and % of each.
Usually done by chromatography
2. Identify the N-term and C-term AAs
C-term via carboxypeptidase
N-term via Sanger’s Reagent, DNFB
2,4-dinitrofluorobenzene
Often step 2 can be skipped today.
5P2-15
Det. Primary Structure: 2
3. Selectively fragment large proteins
into smaller ones.
Eg. Tripsin: cleave to leave Arg or Lys
as C-term AA
Eg. Chymotrypsin: cleave to leave Tyr
or Trp or Phe as C-term AA
Eg. Cyanogen bromide cleaves at
internal Met leaving Met as C-term
homoserine lactone
5P2-16
Det. Primary Structure: 3
4. Determine AA sequence of peptides
with AA sequencer using Edman’s
reagent:
phenyl isothiocyanate which reacts
with the N-term AA
See the next slide
5P2-17
Det. Primary Structure: 3b
+
H3N
1
2
-
COO
3
protein
Edman’s reagent
N C S
S
NH C NH
1
2
3
Thiazoline derivative
N
CH R1
NH C
S
C
O
C
C
S
NH
+ H3N
+
2
3
-
COO
O
aqueous acid
RAR
CH R1
N
-
COO
Phenylthiohydantoin (PTH)
derivative of N-term AA
5P2-18
Det. Primary Structure: 4
5. Reassemble peptide fragments from
step 3 to give protein.
An example follows on the next slide.
5P2-19
Det. Primary Structure: 4b
A twelve AA peptide was hydrolyzed.
Trypsin hydrolysis:
Leu-Ser-Tyr-Gly-Ile-Arg
One is
Thr-Ala-Met-Phe-Val-Lys
C-term
Chymotrypsin hydrolysis
Val-Lys-Leu-Ser-Tyr
Lys is internal!
Gly-Ile-Arg
Thr-Ala-Met-Phe
Deduce the AA sequence
5P2-20
Det. Primary Structure: 4c
Keeping in mind the N-term AA and overlaping the
sequences properly gives:
Tr
Leu-Ser-Tyr-Gly-Ile-Arg
Ct
Gly-Ile-Arg
Ct
Val-Lys-Leu-Ser-Tyr
Tr Thr-Ala-Met-Phe-Val-Lys
Ct Thr-Ala-Met-Phe
The complete sequence is:
Thr-Ala-Met-Phe-Val-Lys-Leu-Ser-Tyr-Gly-Ile-Arg
5P2-21
Secondary Structure
The two very important secondary
structures of proteins are:
-helix
b-pleated sheet
Both depend on hydrogen bonding
between the amide H and the
carbonyl O further down the chain
or on a parallel chain.
5P2-22
 Helix: Peptide w Hbonds
First six C=O to N hydrogen bonds shown
5P2-23
b Sheet: stick form Protein G
H bonds in dotted red-blue
H bonds shown in dotted red-blue
Chain
segment 1
Seg 2
Seg 3
Chain 1
Seg 4
5P2-24
B Sheet: Lewis Structure
N-term
C-term
CH
C O
H N
HC
O C
N H
CH
C O
H N
HC
H N
C O
CH
N H
O C
HC
H N
C O
C-term
N-term
Antiparallel sheet
N-term
CH
C O
H N
HC
O C
N H
CH
C O
H N
C-term
N-term
CH
C O
H N
HC
O C
N H
CH
C O
H N
C-term
Parallel sheet
5P2-25
Supersecondary Structure
Reverse turns in a protein chain allow
helices and sheets to align side-by-side
Common AA found at turns are:
glycine: small size allows a turn
proline: geometry favors a turn
5P2-26
Supersecondary Structure: 2
Combinations of  helix and b sheet.
bb

b meander
5P2-27
Tertiary Structure
The configuration of all the atoms in the
protein chain:
side chains
prosthetic groups
helical and pleated sheet regions
5P2-28
Tertiary Structure: 2
Protein folding attractions:
1. Noncovalent forces
a. Inter and intrachain H bonding
b. Hydrophobic interactions
c. Electrostatic attractions
+ to - ionic attraction
d. Complexation with metal ions
e. Ion-dipole
2. Covalent disulfide bridges
5P2-29
Tertiary interactions: diag.
disulfide
+
NH3 O
O C
Polypeptide Chain
CH2 S
S CH2
CH3
CH3
HO CH2
CH2 OH
metal coord’n
Mg2+
H3C CH CH3
O O
H3C CH CH3
O H C
ionic
Ion-dipole
hydrophobic
+
NH3 O
O C
H bonds
or dipole
5P2-30
Domains
Domains are common structural units
within the protein that bind an ion or
small molecule.
5P2-31
Quaternary Structure-1
Quaternary structure is the result of
noncovalent interactions between two
or more protein chains.
Oligomers are multisubunit proteins with
all or some identical subunits.
The subunits are called protomers.
two subunits are called dimers
four subunits are called tetramers
5P2-32
Quaternary Structure-2
If a change in structure on one chain
causes changes in structure at another
site, the protein is said to be allosteric.
Many enzymes exhibit allosteric control
features.
Hemoglobin is a classic example of an
allosteric protein.
5P2-33
Denaturation
-loss of protein structure,
not 1o.
1. Strong acid or base
2. Organic solvents
3. Detergents
4. Reducing agents
5. Salt concentrations
6. Heavy metal ions
7. Temperature changes
8. Mechanical stress
o
2 
o
4 ,
but
5P2-34
Denaturation-2
Denaturing destroys the physiological
function of the protein.
Function may be restored if the correct
conditions for the protein function are
restored.
But! Cooling a hardboiled egg does not
restore protein function!!
5P2-35
Fibrous Proteins
Fibrous proteins have a high
concentration of -helix or b-sheet.
Most are structural proteins.
Examples include:
a-keratin
collagen
silk fibroin
5P2-36
Globular Proteins
Usually bind substrates within a
hydrophobic cleft in the structure.
Myoglobin and hemoglobin are typical
examples of globular proteins.
Both are hemoproteins and each is
involved in oxygen metabolism.
5P2-37
Roles of Globular proteins
- Globular proteins are compact, spherical
molecules which are usually water-soluble.
- In their functions, usually requires them to
bind precisely to other molecules.
- Therefore the identity and arrangement of
surface amino acids are important.
- The acid aminos interact to form a specific
binding cavities which are complementary
to the structure of a specific molecule,
called ligand.
- Others is devoted to the binding of
regulatory molecules to ensure the
maintenance of the polypeptide’s three
dimensional structure.
5P2-38
Myoglobin:
o
2
and
o
3
aspects
Globular myoglobin has 153 AA arranged in
eight -helical regions labeled A-H.
The prosthetic heme group is necessary for
its function, oxygen storage in mammalian
muscle tissue.
His E7 and F8 are important for locating the
heme group within the protein and for binding
oxygen.
A representation of myoglobin follows with
the helical regions shown as ribbons.
5P2-39
Myoglobin: 2o and 3o aspects
Some helical regions
Heme group with iron (orange)
at the center
5P2-40
The Heme Group
-
-
CH2CH2COO
OOC CH2CH2
N of His
F8 binds
CH3
H3C
to
N
N
fifth site on
the iron.
Fe(II)
Pyrrole ring
H2C
N
N
CH
CH3
His E7 acts
as a ”gate”
CH3
CH CH2
for oxygen.
5P2-41
Binding Site for Heme
Lower His bonds covalently
to iron(II)
Oxygen coordinates to
sixth site on iron and the
upper His acts as a “gate”
for the oxygen.
N
N
H
O
N
O
N
Fe
N
N
N
N
5P2-42
Hemoglobin
A tetrameric protein
two -chains (141 AA)
two b-chains (146 AA)
four heme units, one in each chain
Oxygen binds to heme in hemoglobin
cooperatively: as one O2 is bound, it
becomes easier for the next to bind.
Lengthy segments of the  and b chains
homologous to myoglobin.
5P2-43
Hemoglobin: ribbons + hemes
Each chain
is in ribbon
form and
color coded.
The heme
groups are
in space
filling form
5P2-44
Oxygen Binding Curves
Oxygen bonds differently to hemoglobin
and myoglobin.
Myoglobin shows normal behavior while
hemoglobinn shows cooperative
behavior. Each oxygen added to a
heme makes additon of the next one
easier.
The myoglobin curve is hyperbolic.
The hemoglobin curve is sigmoidal.
5P2-45
Differences in structures of Myoglobin and
Hemoglobin
Myoglobin
Hemoglobin
1. The protein component
of myoglobin, called
globin, composed of
single polypeptide chain
that contains eight
sections of -helix.
2. The oxygen
dissociation curve has a
hyperbolic shape in a
simple pattern.
1. The protein component
of haemoglobin, a
tetramer, composed of
two -chains and two b–
chains.
2. The oxygen
dissociation curve has
a complicated sigmoidal
pattern by the
noncovalent interactions
among its four subunits.
5P2-46
Continue….
Myoglobin
Hemoglobin
3. Binds the molecule
tightly and release it only
when the cell’s oxygen
concentration is very low.
4. Has a greater affinity
for oxygen, binds more
oxygen from blood to
muscle.
3. Each b-dimer remain
largely unchanged when
hemoglobin interconverts
between its oxygenated
and deoxygenated
forms.
4. Has smaller affinity for
oxygen, easy to release
oxygen to muscle.
5P2-47
Oxygen Binding Curves-2
5P2-48
The Bohr Effect (H+ and Hb)
Lungs:
pH higher than in actively metabolizing
tissue. (Low H+). Hb binds oxygen and
+
releases H .
Muscle at Work:
+
pH lower (H product of metabolism). Hb
releases oxygen and binds H+.
HbO2 +
H+
+ CO2
metabolism
in lungs
O2 + H+-Hb-CO2
5P2-49
The End
Amino Acids, Peptides
And Proteins
Part 2
5P2-50