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Les polypeptides
Diversité et complexité
fonctionelles
Gènes
Génome +/- 30000 Gènes
Protéines
Protéome
Interaction
Intercatome
> 1000000 Protéines
?????
Complexité
In 2003, Human genome
sequence was deciphered!
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•
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Genome is the complete set of genes of a living thing.
In 2003, the human genome sequencing was completed.
The human genome contains about 3 billion base pairs.
The number of genes is estimated to be between 20,000 to
25,000.
• The difference between the genome of human and that of
chimpanzee is only 1.23%!
3 billion base pair => 6 G letters
&
1 letter => 1 byte
The whole genome can be recorded in
just 10 CD-ROMs!
Our life is maintained by
molecular network systems
Molecular network
system in a cell
(From ExPASy Biochemical Pathways; http://www.expasy.org/cgi-bin/show_thumbnails.pl?2)
Diversité structurale
Protéines simples
Protéines conjuguées
Goupe Prosthétique
Diversité fonctionelle
Protéines globulaires
Protéines fibreuses
structure
Proteins play key roles in a
living system
• Three examples of protein functions
Alcohol
dehydrogenase
oxidizes alcohols
to aldehydes or
ketones
– Catalysis:
Almost all chemical reactions in a
living cell are catalyzed by protein
enzymes.
– Transport:
Some proteins transports various
substances, such as oxygen, ions,
and so on.
– Information transfer:
For example, hormones.
Haemoglobin
carries oxygen
Insulin controls
the amount of
sugar in the
blood
Classification des protéines selon leurs fonctions biologiques
Enzymes
Ribonucléase
Hydrolyse du RNA
Cytochrome C
Transfère des électrons
Trypsine
Hydrolyse de certains peptides
Protéines de stockage
Ovalbumine
Protéine du blanc d’oeuf
Caséine
Protéine du lait
ferritine
Stockage du fer dans la rate
Protéines de transport
Hémoglobine
Transport de O2 dans le sang des
vertébrés
Myoglobine
Transport de O2 dans le muscle
Sérum-albumine
Transport des acides gras dans le sang
Transferrine
Transport du fer dans le sang
Protéines contractiles
Myosine
Filaments stationnaires dans la myofibrille
Actine
Filaments mobiles dans la myofibrille
Protéines de protection dans le sang
des vertébrés
Anticorps
Forment des complexes avec des
protéines étrangères
Fibrinogène
Précurseur de la fibrine dans la
coagulation sanguine
Thrombine
Composé intervenant dals la coagulation
sanguine
Toxines
Toxine de Clostridium botulinum
Provoque des intoxications alimentaires
Toxine diphtérique
Toxine bactérienne
Venin de serpent
Enzyme qui hydrolysent des
phosphoglycérides
ricine
Protéine toxique du ricin
Hormones
Insuline
Régularise le métabolisme glucidique
Hormone de croissance
Stimule la croissance ex: des os
Protéines de structures
Protéine de la coque virale
Gaine autour du chromosome
Glycoprotéines
Gaine des cellules et parois
Collagène
Tissu conjonctif fibreux (tendons os
cartilage)
Alpha kératine
Peau plume ongles et sabots
Protéines conjuguées
Catégories
Groupement prosthétique
Complexes nucléoprotéiques ex
ribosome
RNA
Lipoprotéines ex lipoprotéines
plasmatiques
Phospholipides, cholesterol, lipides
neutres
Glycoproteines ex gamma globulines
Hexosamine, galactose, mannose, acide
sialique
Phosphoprotéines ex Caséine
Phosphate
Hémoprotéines ex hémoglobine
Protoporphyrine
Flavoprotéines ex Succinate
déshydrogénase
Nucléotide flavinique
Métalloprotéines ex alcool
déshydrogénase
Zn
Masses moléculaires de quelques protéines
Masses moléculaires
Nombre de résidus
Nombre de chaines
Insuline
5733
51
2
Lysozyme
13930
129
1
Chymotrypsine
(Pancreas du boeuf)
21600
228
1
Hémoglobine
64500
574
4
Hexokinase (levure)
96000
800
4
Gamma globuline
(cheval)
149900
1250
4
Glycogène
phosphorylase (muscle
de lapin)
495000
4100
4
Glutamate
déshydrogénase (foie de
boeuf)
1000000
8300
40
Acide gras synthétase
(levure)
2300000
20000
21
Virus de la mosaique du
tabc
40000000
336500
2130
Stick
La structure tridimensionnelle
est directement liée à la fonction
Space
filling
Ribbon
• There are 20 “standard” amino acids that make up
all the proteins in all organisms
• There are more potential combinations of these 20
amino acids than visible stars in the sky
Acide aminé
• The a-carbon has two functional groups:
– a primary amine (-NH2)
– a primary carboxylic acid (-COOH)
• The 20 biochemical amino acids are all
distinguished by their side chain group ‘R’
Glycine
H
(-)
(+)
L- vs. D- enantiomers
Biochemical reactions
discriminate structural
isomeric forms
COONH3 -C-R
H
L
L-alanine
COOR-C- NH3+
H
D
Amino Acid Charge Distribution
• Biphasic tritration
curve
9.69
– Protonation of amino
and carboxyl groups at
different pH
• Thus, amino acids are
never neutral in
aqueous solutions
• Isoelectric point
2.34
– The pH at which a
molecule carries no net
electric charge
– Zwitterion is in perfect
equilibrium
a-amino acids: at pH 7
• At physiological pH, AA are dipolar:
– The amino group is protonated (-NH3+)
– The carboxylic acid is deprotonated (-COO-)
• Ions with a structural polarity are:
zwitterions
– Very soluble in polar solvents (cytoplasm)
Classement des aas en fonction de leur
réaction avec l’eau
Indole
20 Amino acids
Glycine (G)
Alanine (A)
Valine (V)
Isoleucine (I)
Leucine (L)
Proline (P)
Methionine (M)
Phenylalanine (F)
Tryptophan (W)
Asparagine (N)
Glutamine (Q)
Serine (S)
Threonine (T)
Tyrosine (Y)
Cysteine (C)
Lysine (K)
Arginine (R)
Histidine (H)
Asparatic acid (D) Glutamic acid (E)
White: Hydrophobic, Green: Hydrophilic, Red: Acidic, Blue: Basic
Polaires Chargés
pK1 α-COOH
pK2 α-NH2
pK3
pI
D
1.88
9.60
3.65
2.77
E
2.19
9.67
4.25
3.22
K
2.18
8.95
10.53
9.74
R
2.17
9.04
12.48
10.76
H
1.82
9.17
6.00
7.59
Y
2.20
9.11
10.07
5.66
C
1.96
10.28
8.18
5.07
N
2.02
8.80
5.41
Q
2.17
9.13
5.65
S
2.21
9.15
T
2.09
9.10
5.60
G
2.34
9.60
5.97
A
2.34
9.69
6.00
V
2.32
9.62
5.96
L
2.36
9.60
5.98
I
2.36
9.60
6.02
M
2.28
9.21
5.74
P
1.99
10.60
6.30
F
1.83
9.13
5.48
W
2.83
9.39
5.89
Polaires neutres
pH >15
5.68
Apolaires
Modifications Post- traductionelles
Les peptides
Protein Structure
• Primary Structure
– The sequence of amino acid residues
• Secondary Structure
– The spatial arrangement of the backbone
atoms of the peptide bonds
• Tertiary Structure
– 3D spatial arrangement of all atoms in the
backbone and side-chain groups
N aas >>>> N-1 lien peptidiques
Peptide Resonance
• Resonance interactions give the peptide bond
~40% of the structural character of a double-bond
• Configuration Trans <> Cis
• Lien peptidique polaire
Structure primaire
Amino acid oligomers:
polypeptides
Serine
aC
NH3+
COO-
COO-
aC
Glycine
NH3+
Glutamine
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Rotational flexibility
only possible
between different
planar units
Ca – N:
– f bond angle
Ca – C:
– y bond angle
These angle are
constrained by
– Amide hydrogens
– Carbonyl oxygen
– Side chain groups
Steric Clashes
Reduce the
Number of
Allowed
Combinations
of Dihedral
Angles
Ø = 180°
Ψ= 0° CO
Ø = 0°
Ψ= 180° NH
Experimental data
Solution Proteique
• Conservation -800C > -200C > -40C
• lyophilisation
• Eviter congélation/décongélation (perte d’activité
et précipitation)
• Stable hautement concentrée (1-10mg/ml)
• Manipulation -40C > température ambiante
• Solution tamponnée (50-100mM)
• Antibactérien (NaN3 ou stérile)
• Antiproteases
• Agent stabilisant (antioxidant…)
Gel Filtration Chromatography
Filtration moléculaire
•
Partition coefficient, Kav = Ve – Vo/Vt – Vo
Ve = the volume required to elute a component,
Vt = the total liquid volume of the column
and Vo = the volume of liquid outside the
beads. For an example, see the attached
elution profile for a Superdex column.
•
Kav is strictly determined by the Stoke's
radius (rotational volume) of the molecule,
which is proportional to its molecular weight
and shape.
Edman reaction
Edman Degradation chemistry is a stepwise process that has been automated
(instruments perform the reactions without human intervention). PITC labeling of the
unprotonated, terminal amine is followed by cleavage in anhydrous trifluoroacetic acid
in the gas phase. The free, derivitized aa is moved away from the rest of the peptide,
where it is dissolved in aqueous acid. The structure rearranges to a form that absorbs
UV light at 254 nm. The derivitization cyclizes the aa, so that each PTH-aa is more
hydrophobic (less polar) than the corresponding parent aa. In order to differentiate
between the 20 hydrophobic PTH-aa, a reversed-phase HPLC column is used. The most
common application is to determine the NH2-terminal sequence of proteins that have
been separated by SDS-PAGE or 2D electrophoresis.
Protein identification strategies
Edman Degradation
Electro-Transfert
-
Blocked N-terminus
Slow
Loss of material
Edman
Degradation
Identify by HPLC
Repeat cycle to
identify the next
amino acid
Couplage
This reaction can be understood if we look for some
analogies that will help us apply the patterns we
used in the past. The -N=C=S group resembles a
CO2 (O=C=O) molecule in that the carbon atom is
connected to two electronegative atoms by a double
(sigma and pi) bond. We know from reacting
Grignard reagents with CO2 that the nucleophile
attacks the carbon in CO2, so we can expect the
same type of pattern in the Edman degradation.
The nucleophile is the free NH2 group at the N
terminus of the peptide, formed by loss of a proton
from the NH3+to some unspecified base. As we
have seen with other reactions of NH2 groups, this
step is followed by a proton shift.
The product of the addition of N and H to the C=N
double bond has a nucleophilic sulfur atom located just
in reach of the carbonyl carbon at the other end of the
N terminal amino acid. Attack of this sulfur at that
carbonyl group is followed by departure of the NH
group of the next amino acid. This cleaves the peptide
bond between the N terminal amino acid and the next
amino acid. Further reshuffling of protons yields an
isomer of the phenylthiohydantoin. This isomer is
converted to the phenylthiohydantoin during the
treatment with HCl and the phenylthiohydantoin is
identified. Since the phenylthiohydantoin includes the
R group of the N terminal amino acid, identification of
the phenylthiohydantoin also identifies the N terminal
amino acid.
Clivage