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Amino Acids, Peptides and Proteins
The Amino Acids in Proteins
Polypeptides and Proteins
Protein Function
Protein Size, Composition and Properties
Four Levels of Protein Structure
Protein Primary Structure
Chromatography and Electrophoresis of
Proteins
1
Amino acids
All proteins are composed of amino acids.
•
Twenty common amino acids.
•
All are -amino acids except proline.
•
A primary amine is attached to the  carbon
- the carbon just after the acid group.
General
Structure
H
|
R-C-COOH
|
NH2
 carbon
2
Amino acids
Because an acid and base are both present,
an amino acid can form a +/- ion.
H
|
R-C-COOH
|
NH2
H
|
R-C-COO|
NH3+
How well it happens is based on pH and the
type of amino acid. Called a zwitterion.
3
-Amino acids
Except for glycine, the  carbon is attached
to four different groups - chiral center.
Carbohydrates
We use the D- form.
COO+ |
H3N - C - H
|
R
Amino Acids
We use the L- form.
4
Classification of amino acids
The -amino acid group is the same in each.
Classified by the type of side chain.
• Group I. non-polar side chains.
• Group II. polar, uncharged side chains
• Group III. acidic side chains
• Group IV. basic side chains
5
Group I. Non-polar side chains
alanine
leucine
H
|
CH3-C-COO|
+NH
3
H 3C
H
\
|
HC-CH2-C-COO/
|
+NH
H 3C
3
H 3C
valine
H
\ |
HC-C-COO/ |
H3C +NH3
H 3C H
| |
H3C-CH2-CH-C-COO|
+NH
isoleucine
3
6
Group I. Non-polar side chains
proline
phenylalanine
H 2C
|
H 2C
H
|
-CH2-C-COO|
+NH
3
CH-COO|
+NH
2
H 2C
H
|
CH3 -S-CH2-CH2-C-COO|
+NH
3
methionine
N
H
H
|
CH2-C-COO|
+NH
3
tryptophan
7
Group II. Polar side chains
glycine
HO-
tyrosine
H
|
H-C-COO|
+NH
3
serine
H
|
HO-CH2-C-COO|
+NH
3
HO H
|
|
CH3-CH-C-COO|
+NH
3
H
|
-CH2-C-COO|
+NH
3
threonine
8
Group II. Polar side chains
H
|
HS-CH2-C-COO|
+NH
3
cysteine
O
H
||
|
H2N-C-CH2-CH2-C-COO|
+NH
3
glutamine
O
H
||
|
H2N-C-CH2-C-COO|
+NH
3
asparagine
9
Group III. Acidic side chains
Based on having a pH of 7.
O
glutamic acid
H
||
|
-O-C-CH -CH -C-COO2
2
|
+NH
3
O
aspartic acid
H
||
|
-O-C-CH -C-COO2
|
+NH
3
10
Group IV. Basic side chains
Based on a pH of 7.
+NH
H
||
|
H2N-C-N-CH2-CH2-CH2-C-COOH
|
+NH
arginine
3
2
H
|
+
H3N-CH2-CH2-CH2-CH2-C-COO|
+NH
lysine
3
H
|
CH2-C-COO|
+NH
N H
3
histidine
H
H N
+
11
Polypeptides and proteins
Proteins are polymers made up of amino
acids.
Peptide bond - how the amino acids are
linked together to make
a protein.
H
|
H2NCCOOH +
|
R
H
|
H2NCCOOH
|
R’
H O
H
| ||
|
H2N - C - C - N - C - COOH
|
| |
R
H R’ + H2O
12
Polypeptides and proteins
Here is an example sequence of amino acids in a
protein.
It also shows the abbreviations commonly used.
ala
gly
pro
arg
his
ser
asn
ile
thr
asp
leu
trp
cys
lys
tyr
gln
met
glu
phe
val
13
Polypeptides and proteins
Residue - term used to refer to the amino
acid once incorporated into a polypeptide
Polypeptide - contain 10-100 residues
Protein - contain more than 100 residues.
Most peptides and proteins isolated from
cells contain between 2 - 2000 residues.
An average amino acid has a weight of 110, so
protein molecular weights are in the range of 220
- 220,000 (some are much larger).
14
Peptides
N-terminal
residue
C-terminal
residue
H O
H O
H
| ||
| ||
|
H2N - C - C - NH - C - C - N - C - COOH
|
|
| |
R
R’
H R’’
peptide
linkages
15
Protein function
Enzymes
biological catalysts.
Immunoglobulins
antibodies of immune system.
Transport
move materials around hemoglobin for O2.
Regulatory
hormones, control metabolism.
Structural
coverings and support skin, tendons, hair, nails, bone.
Movement
muscles, cilia, flagella.
16
Protein size, composition
and properties
One important property is molecular weight.
There are two common ways to calculate it.
• Determine the number of amino acid
residues, then multiply by 110 -- the average
molecular weight of an amino acid.
• Directly measure the mass of a protein and
report it in daltons. One dalton = One
atomic mass unit.
17
Size of some important proteins
Protein
MW
Insulin
6,000
51
Cytochrome c
16,000
104
Hemoglobin
65,000
574
Gamma globulin176,000
Myosin
800,000
Residues
1320
6100
18
Protein composition
Proteins can be classified based on the
number of polypeptides used
Monomeric - only a single polypeptide
chain is present.
Oligomeric - two or more polypeptide
chains are present.
The subunit peptide chains are typically
held together with noncovalent bonds.
19
Protein composition
Proteins are also classified based on their
composition.
Simple proteins - only contain amino acid
residues.
Conjugated proteins - contain other
biomolecules - prosthetic groups.
These groups impart additional properties
to a protein.
20
Example - cytochrome C 550
Heme structure
Contains Fe2+
Used in
metabolism.
Aggregate of
proteins and
other structures.
21
Protein solubility
Two categories.
Determined by the types of amino acid
side chains involved.
Water soluble
- globular proteins
Water insoluble
- fibrous proteins.
22
Four levels of protein structure
Primary structure
The actual sequence of amino acids in a protein.
Secondary structure
The type of regular repeating structure
(-helix, -sheet)
Tertiary structure
Interaction of side chains.
Quaternary structure
Association of two or more polypeptide chains to
form a multisubunit molecule.
23
Summary of
protein structure
primary
secondary
H O
H O
H
| ||
| ||
|
H2N - C - C- NH - C - C - N - C - COOH
|
|
| |
R
R’
H R’’
tertiary
quaternary
24
Determination of
primary structure
The first step is to isolate the protein in a
pure form from its natural source.
Typically, only very small amounts can be
obtained.
Total amino acid composition can be
determined by hydrolysis of the protein.
(6M HCl at 100oC).
The amount of each amino acid can then
be measured chromatography.
25
Protein sequencing
Methods that determine the order of each
amino acid in a protein.
Edman degradation.
• Method of choice for protein sequencing.
• Relies on a sequential degradation by
removing one amino acid at a time from
the N-terminus.
• Process can be automated and works with
peptides with up to 50 residues.
26
Edman degradation
+
N C S
phenylisothiocyanate
O
O
NH2 CH C N CH C N COOR
H R'
peptide
H S H
O
O
N C N CH C N CH C N COOH R'
R
N
O
S
C
H+
N H
H
R
phenylthiohyantoin
O
NH2 CH C N COOH
R'
remaining peptide
isolate and
react with
additional
reagent.
27
Edman degradation
Problems with the method.
•
Does not provide 100% yield - resulting in
contamination.
•
Limited to about 50 cycles so proteins
must be cut to smaller sizes. Must rely on
enzymes and reagents to cleave a protein
at known locations.
•
Disulfide bonds between cysteine
residues can present problems.
28
Protein sequencing
As of 1998, over 30,000 protein sequences
were available in a computer database.
Having such information available makes it
possible to study and compare sequence
information.
Several biochemical conclusions have been
made as a result of studying this data.
29
Protein sequencing
Identification of protein families.
• Proteins with common sequence features
have similar biological function,
• This allow for the characterization of newly
discovered proteins.
Example - protein kinases
Enzymes that catalyze the phosphorylation of
amino acid residues.
All known protein kinases have the same common
sequence region (domain) of 240 residues.
30
Protein sequencing
Evolutionary development of proteins
• Comparison of protein types for many
organisms.
• Possible to establish taxonomic
relationships.
Example - cytochrome c
Protein used in aerobic respiration.
It has been determined for over 60 organisms.
27 residues are the same for all forms.
Other variations indicate evolutionary changes.
31
Protein sequencing
Search for dysfunction.
• Normally, all residues in a protein are
identical for a species.
• Some individuals may produce a protein
with one or more ‘incorrect’ residues.
Example - sickle cell anemia.
Two ‘incorrect’ amino acid residues result in
malformed hemoglobin.
This causes deformation of red blood cells.
32
Protein sequencing
Three dimensional nature of proteins.
• Sequence data can be coupled with other
methods.
• X-ray crystallography can produce 3-D
structural information.
It is a difficult method and has not kept up
with the number of proteins that have
been isolated.
• Sequencing may offer an alternative
approach.
33
Protein sequencing
Ala
Ala
Lys
Phe
Glu
Arg
Glu
His
Met
Asp
Ser
Ser
Thr
Ser
Ala
Ala
Ala
Ser
Thr
Thr
Asp
Glu
Glu
Gly
Lys
NH2
Ser
Cys
Cys
Asp
Tyr
Glu
Ser
Tyr
Ser
Thr
Met
Ala
Lys
HOOC - Val
Val
Ser
Asp
Ala
Glu
Val
Val
Val
Asp
Ile
Ala
Ile
Leu
Lys
Ser
Asp
Ala
Glu
His
Val
Cys
Ala
Glu
Phe
Cys
Thr
Thr
Ser
Glu
Thr
Asp
Glu
Gly
Ser
Ala
Lys
Ile
Ser
Thr
Asp
Asp
Try
Cys
Cys
Arg
Asp
Glu
Glu
Thr
Met
Gly
Met
Ser
Lys
Asp
Asp
Pro
Lys
Tyr
Ala
Val
Pro
Lys
Tyr
Cys
Cys
Val
Asp
Arg
Pro
Val
Pro
Asp
His
Try
Lys
Example - ribonuclease
Phe
Lys
Thr
Ser
Ser
Leu
Asp
Arg
34
Protein sequencing
Example - ribonuclease
35
Chromatography and
electrophoresis of proteins
For a protein to be assayed by X-ray
crystallography or protein sequencing, a
pure sample must be produced.
After preparation of a cell extract, an
appropriate separation method must be
employed. Two such methods are:
Chromatography
Electrophoresis
36
Chromatography
Several chromatographic methods have
been attempted to isolate pure protein
fractions.
ion exchange
thin layer chromatography
column liquid chromatography
size exclusion chromatography
affinity chromatography
Affinity chromatography is becoming
increasingly more important.
37
Affinity Chromatography
The method dates back to 1910.
Modern method was first published in 1967,
by Axen, et al. -- ‘Cyanogen bromide
Method for the Immobilization of Ligands
on Agarose.’
Ohlson (1978) was the first to demonstrate
the use of a rigid, microparticulate
support - beginnings of instrumental
method.
38
Affinity Chromatography
The method involves the interaction of a ligand
with the solute of interest. It can be viewed
as being comparable to ion-exchange.
Two general types of ligands
Specific
Binds only to one species.
Antibody/antigen
General
Group specific
Binds to specific groups
on target species.
39
Affinity chromatography
Support
The material that the ligand is bound to.
Ideally, it should be rigid, stable and have
a high surface area.
Agarose is the most popular although
cellulose, dextran and polyacrylamide
have been evaluated.
40
Affinity chromatography
Agarose gel
A polymer of D-galactose and 3,6anhydro-L-galactose.
It can be used at pressures up to 1 psi and
over a pH range of 4-9.
Cross-linking can be used to extend the
pressure range.
41
Affinity chromatography
The separation is conducted in four basic
steps.
Sample introduction
Adsorption of components of interest
Removal of impurities
Elution of components.
42
Affinity chromatography
Sample introduction
You must make sure
that your column
has adequate
capacity.
ligand
spacer
matrix
43
Affinity chromatography
Absorption
Using a slow flow, your
sample is then allowed
to pass through the
column.
The flow helps drive
your sample
components towards
‘fresh’ sites.
44
Affinity chromatography
Washing
Next, you can
remove impurities
by passing several
volumes of fresh
solvent through the
column.
45
Affinity chromatography
Elution
The component of
interest must then
be removed and
collected.
This also acts to
regenerate the
column.
46
Affinity chromatography
Elution methods
Biospecific
An inhibitor is added to the mobile phase
(free ligand).
The free ligand will compete for the
solute.
This approach is most often used when a
low molecular weight inhibitor is
available.
47
Affinity chromatography
Elution methods
Nonspecific
A reagent is added that denatures the
solute (pH, KSCN, urea, ionic strength...)
Once denatured, the solute is released
from the ligand.
If the solute is to be further used, it must
not be irreversibly altered.
48
Affinity chromatography
An example.
Column: 50 mm x 30 mm
containing 60 ml of
Protein A Sepharose
Sample: 5 liter cell culture
supernatant with mouse
IgGa2 and 0.5% fetal calf
serum.
Starting buffer:
0.1 M Na2HPO4, pH 7
60
70
minutes
80
Elution buffer:
0.1 M citric acid, pH 4
Flow rate: 66.6 ml/min
49
Electrophoresis
A separation method that relies on both the
size and the charge of a species.
• Samples are placed in an electrical field.
• They tend to migrate to specific positions
in the field.
• With gel electrophoresis, a cross-linked
polymer acts like a molecular sieve smaller proteins move faster than larger
ones.
50
Electrophoresis
51
Electrophoresis
Bands can then be
compared to
standards as a
means of identifying
the molecular
weight. Band
patterns can be
used to indicate a
protein’s origin.
MW
200,000
100,000
50,000
25,000
12,500
6,250
standard
sample
52