Transcript CHMI 2227E Biochemistry I

CHMI 2227E
Biochemistry I
Peptides
-General
structure and properties
CHMI 2227 - E.R. Gauthier, Ph.D.
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Peptides

Peptides are polymers of amino acids;

Amino acids building blocks (residues) are
linked to each other through a covalent bond:
the peptide bond.
1
1
2
2
CHMI 2227 - E.R. Gauthier, Ph.D.
A dipeptide
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Peptides

Polypeptides and proteins are simply chains of amino acids linked together through peptides
bonds:




If less than 20 residues: oligopeptide;
If more than 20 residues but molecular mass (Mr) < 10,000 Da: polypeptides.
If Mr > 10 kDa: protein.
Particular terminology:

Dipeptide (2 residues) / Tripeptide (3 residues) / Tetrapeptide (4 residues) / Pentapeptide (5
residues) / Ect, ect, ect.

Note: 1 Da (dalton) = 1 g /mol.

Little trick: Mr of a polypeptide/protein
number
amino acids x 110 Da.
CHMI 2227 - ~
E.R.
Gauthier,of
Ph.D.
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Peptides - polarity


Each peptide has a polarity:

One extremity with the NH2
(the one bonded to the Ca)
which is not part of a peptide
bond: N-terminal end;

One extremity with the COOH
(the one bonded to the Ca)
which is not part of a peptide
bond: C-terminal end;
By convention: the N-terminal
is always placed on the left,
and the C-terminal on the
right.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Peptides - nomenclature

Different ways to write this peptide (hyphen = peptide bond) :





Tyrosyl-glycyl-glycyl-phenylalanyl-leucine
Tyr-Gly-Gly-Phe-Leu
Y-G-G-F-L
YGGFL
Note that the peptide is always written with the N-ter to the left and the C-ter to the
right (NH2COOH).
CHMI 2227 - E.R. Gauthier, Ph.D.
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Peptide: hydrolysis

The composition (NOT the sequence) of a peptide in its amino acid
constituents is determined by first hydrolysing the peptide bond, and
then identifying the amino acids:

Tyr-Gly-Gly-Phe-Leu  Gly2, Leu, Phe, Tyr
6 M HCl

The amino acids are then purified by High Pressure Liquid
Chromatography (HPLC). Detection is done by UV absorbance.

To detect those amino acids that cannot absorb UV (you know
which ones…), the amino acid are derivatized, meaning they are
chemically coupled with a compound that absorbs UV.

Quantification and identification of the amino acids is often done
with the help of standards (analyzed with the same system but in a
separate experiment);
CHMI 2227 - E.R. Gauthier, Ph.D.
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Analysis of amino acids
Detection of amino acids: ninhydrin reagent
O
COOH
OH
OH
C
NH3+
R-HC=O
CO2
O
O
R
N
O
Ninhydrin
2
Amino acid
O
O
Purple!!

While Trp, Phe and Tyr can be detected by their A260-280nm, the other amino acids
cannot;

Ninhydrin reacts with the amine group of amino acids, generating a purple product
(yellow in the case of Pro).

The ninhydrin reaction allows one to detect and quantify (A570nm) the amino acids
contained in the fractions of CHMI
the IEX
2227column.
- E.R. Gauthier, Ph.D.
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High Pressure Liquid Chromatography
(HPLC)
PITC = phenylisothiocyanate
PTC = phenylthiocarbamyl
http://www.protein.iastate.edu/aaa.html
CHMI 2227 - E.R. Gauthier, Ph.D.
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High Pressure Liquid Chromatography
(HPLC)

http://www.protein.iastate.edu/aaa_figure3.html
CHMI 2227 - E.R. Gauthier, Ph.D.
The relative
amount of each
amino acid is
given by
calculating the
area under
each curve.
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Peptide - ionization

Each peptide will exist in different protonated forms, depending on the pH
and its amino acid composition:

Terminal amino and carboxyl groups can be protonated/ionized as in the free
amino acid;

The side chain can also be ionized, if an appropriate group is present;

The NH2 and COOH groups that are part of the peptide bond are NOT ionized.

Therefore, there will be a pH where a given peptide/protein will carry no net
charges: this pH value will be the isoelectric point of the peptide/protein in
question.

Example: Ionization of the peptide GAVFD at pH 2, 6 and 12.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Example of peptides
1. Aspartame: artificial sweetener
COOCH2
O
H3N+-CH-C-NH-CH-C-OCH3
CH2
O
Asp-Phe-methyl ester
2. Oxytocin: stimulates uterine contractions
Disulfide bond
S
S
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly-NH2
Glycinamide residue: 2HN-CH2-CONH2
CHMI 2227 - E.R. Gauthier, Ph.D.
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Example of peptides
3.Insulin
Intrachain disulfide bond
Interchain disulfide bonds
CHMI 2227 - E.R. Gauthier, Ph.D.
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Example of peptides
4.Cystic Fibrosis Transductance Regulator
CHMI 2227 - E.R. Gauthier, Ph.D.

Single polypeptide chain
of 1480 amino acids;

Responsible for the
transport of chloride ions
across the cell
membrane;

Mutation of F508 yields a
non-functional protein and
cystic fibrosis.

What is the approximate
Mr of CFTR?
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General properties of proteins
1. Proteins differ in their Mr.
Mr
(kDa)
Insulin
5.7
Cytochrome c
13
Ribonuclease A
13.7
Lysozyme
13.9
Myoglobin
16.9
Chymotrypsin
21.6
Chymotrypsinogen
22
Hemoglobin
64.5
Serum albumin
68.5
Hexokinase
102
Immunoglobulin G
145
RNA polymerase
450
Apolipoprotein B
513
Glutamate
1,000
dehydrogenase
Protein
#
# chains
residues
51
2
104
1
124
1
129
1
153
1
241
3
245
1
574
4
550
1
800
2
1,320
4
4,100
5
4,536
1
8,300
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Source: Biochemistry. Lehninger.
CHMI 2227 - E.R. Gauthier, Ph.D.
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Multimeric proteins

Proteins with more than one polypeptide
chains are called multimeric proteins;

Different types of multimeric proteins exist:



Homo/oligomeric: 2 or more copies of the
same polypeptide chain;
Heteromeric: different polypeptide chains
make up the proteins.
The different polypeptides of a multimeric
protein (i.e. the protein’s subunits) can be
held together in different ways:




Disulfide bonds
Hydrogen bonds
Hydrophobic interactions
Electrostatic interactions
Monomer/subunit
1
2
Heterodimer
1
1
Homodimer
Hydrogen bonds:
N-H ----- O-H
N-H ----- N
O-H ----- O=C
N-H ----- O=C
Electrostatic interactions:
COO- ----- H3+N

Multimeric proteins most often require all
their parts in order to be functional.

Very often proteins can change partners,
providing them leading to their in/activation
or giving them a different function.
Hydrophobic interactions:
-CH3 CH3CH3
CHMI 2227 - E.R. Gauthier, Ph.D.
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Importance of multimeric
proteins – planar cell polarity
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Importance of multimeric
proteins – planar cell polarity
Nature Genetics 38, 21 - 23 (2006)
CHMI 2227 - E.R. Gauthier, Ph.D.
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General properties of proteins
2. Proteins differ in their pI.
Protein
Pepsin
pI
1
Egg albumin
4.6
Serum albumin
4.9
Urease
5
b-lactoglobulin
5.2
Hemoglobin
6.8
Myoglobin
7
Chymotrypsinogen
9.5
Cytochrome c
10.7
Lysozyme
11
Source: Biochemistry. Lehninger.
CHMI 2227 - E.R. Gauthier, Ph.D.
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General properties of proteins
3. Frequency of amino acid composition
Number of residues per
molecule of protein
Amino
Acid
Human
cytochrome c
Bovine
chymotrypsinogen
Ala
6
22
Arg
2
4
Asn
5
15
Asp
3
8
Cys
2
10
Gln
2
10
Glu
8
5
Gly
13
23
His
3
2
Ile
8
10
Number of residues per
molecule of protein
Amino
Acid
Human
cytochrome c
Bovine
chymotrypsinogen
Leu
6
19
Lys
18
14
Met
3
2
Phe
3
6
Pro
4
9
Ser
2
28
Thr
7
23
Trp
1
8
Tyr
5
4
Val
3
23
Total
104
245
CHMI 2227 - E.R. Gauthier, Ph.D.
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General properties of proteins
3. Frequency of amino acid composition
CHMI 2227 - E.R. Gauthier, Ph.D.
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General properties of proteins
4. Proteins can include other chemical
groups in addition to amino acids
Class
Prosthetic
group
Example
Lipoprotein
Lipids
b1-lipoprotein (blood)
Glycoprotein
Carbohydrates
(sugars)
Immunoglobulin G
(blood)
Phosphoprotein
Phosphate
groups
Casein (milk)
Hemoprotein
Heme (iron
porphyrin)
Hemoglobin
Flavoprotein
Flavin
nucleotides
Succinate
dehydrogenase
Metalloprotein
Fe
Zn
Ca
Cu
Ferritin
Alcohol dehydrogenase
Calmodulin
Plastocyanin
CHMI 2227 - E.R. Gauthier, Ph.D.
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General properties of proteins
5. Proteins have a specific shape

Each polypeptide spontaneously
adopts a shape or conformation.

A protein in its correct
conformation is said to be native;

This conformation is unique to
each protein;

Disruptions in the conformation
(e.g. by heating) denatures the
protein and usually leads to its
inactivation.
Globular proteins
Fibrillar (rod-like) proteins
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