Download Diapositive 1

Claire Lesieur
[email protected]
Membrane (Lipids)
Proteins
Nucleus
(chromosome)
Nucleus
DNA
Protein
Lipids
CHON
Chromosome
Cutting
- Recognition
- Enzyme
- Signaling
- Carrier
- Shape generator
- Road networks
-
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Function
Shape
How the shape provides a particular function
How the shape is acquired
?
GKKHDGATTYQW
?
ADRTGGILLKMHGGARECVVP
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How it folds: Mechanisms of protein folding
How the information is encrypted in the
sequences: CODING problem
All the information necessary for the protein folding is
within the protein primary sequence
C.B. Anfinsen, Haber, E., Sela, M. & White, F. H. , Proc.
Nati. Acad. Sci. USA 47 (1961) 1309-1314.
Levinthal’s paradox(1968): not random search but directed
Levinthal, C. (1968) J. Chim. Phys. 65, 44-45.
COOH
H2N
Structure primaire
ms
s-hours
Short range interaction
Structure Secondaire
short-range
interactions
Structure Tertiaire
long-range
interactions
X-ray crystallography + NMR: PDB
3D modeling: PDB
~ 70 % Sequence similarity: 3D modeling
70 % similarity: different shape
Low sequence similarity: similar shape
Amino acids on the surface of proteins: changeable
b-strands transmembrane domain: 1010101
a-helice transmembrane domain: 11111111111111111
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Geometrical constrain
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Chemical constrain
?Sequence Pattern
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Domains
Shape and role
?
Sequence Pattern
Trends in Microbiology (2000). Vol 8 (4):169-172
Cholera toxin
CtxA
CtxB5
•
AB5 toxin
– A catalytic subunit
– B receptor binding subunit
•
•
GM1: cell receptor
Endocytosed and traffic to the
ER
ADP ribosylation of Ga subunit
Increase of cAMP leading to
water loss
•
•
ER
Assembly in vitro
pH 1
pH 7
15 min
Pentamere
Monomere
Mean residue Molecular Ellipticity
2 10
5
0
-2 10
-4 10
-6 10
-8 10
-1 10
5
5
5
5
6
6
-1,2 10
200
pH 1
pH 7
Native
210
220
230
240
Wavelength (nm)
250
Trp-fluorescence
300
200
100
0
320
unfolded
340
360
380
Wavelength (nm)
Fluorescence Intensity (a.u.)
Fluorescence Intensity
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350
300
250
lex= 295 nm
lem=352 nm
200
150
100
50
0
0
20
40
Time (min)
60
100
Function
CtxB
80
60
His
40
HISTIDINE
20
0
4,5 5
5,5 6
pH
6,5 7
7,5 8
…
…
CtxB5
LTB
Heat labile enterotoxin B
CtxB
Cholera toxin B
Function
100
80
LTB
CtxB
60
N-terminal
40
20
N-terminal
0
4,5 5
5,5 6
pH
6,5 7
7,5 8
LTB5
Kinetics differences
On pathway intermediates
differences
It is particular amino acids that are
responsible for each individual step
of assembly and folding
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Alzheimer, Parkinson, Prion diseases
Protein X: FOLD state: healthy
Information for interfaces
(Protein X)n: Assembly state: Lethal
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Protein Interface formation
Rules?
Mechanism?
Preferential geometries related to preferential
sequences of amino acids?
INTERFACES:
Zone de contact entre
monomeres voisins
Interface
Trimer
pentamer
Brin 1
0101
0101
Brin 2
n.a.
Ch111Ch
heptamer
Ch111Ch
1111/1
Fibritin like domain
Nombre de monomer
Nombre de cas
2
3
4
5
6
7
8
9 10 11 12
5722 1035 2340 168 721 46 512 45 87 8
205
Monomer M
513 -524
LMITTECMVTDL
aaa-bbbbbbb-
Monomer M+ 1
35-49
GRNVVLDKSFGAPTI
--bbbb-------bb
Distances
2HY6 (30)
1
1N9R (68)
19
1WNR (94)
1
2F86 (129)
344
1JBM (78)
10
1G31 (107)
5
1LNX (74)
8
1Q57 (483)
64
2RAQ (94)
3
1GRL (518)
6
1IOK (524)
2
1PZN (240)
96
1J2P (229)
4
1Y7O(194)
2F6I (189)
beta
alpha
a+b
rc
86
94
471
88
111
80
549
97
523
526
336
233
1
194
177
1TG6 (193)
1
2CBY (179)
15
1OEL (525)
2
1LEP (92)
1
3BDU (51)
2
1HX5 (92)
30
5
367
193
194
525
92
53
97
3BDU 20-29, 38-53
3BDU
1G31
1JBM
1LNX
1N9R
1J2P
1HX5
1LEP
Con2
1--111011-110110--10
0--1-1001-100100--00
11001000101100101101
1--0100010110000---1
0--0100011110010--11
----1000101100101--1
------0011110010--11
0---10001000--00--11
----1-001-1100-0-
1LEP: 1-8, 88-94, 40-57
1WNR: 1-8, 88-94, 44-57, 62-77
1G31: 8-15, 104-111, 68-85
1HX5: 5-11, 94-97, 51-62, 68-80, 27-30
yeast
1N9R
Methanobacterium
Thermautriophicum:
extremophile
1JBM
P. aerophilum: bacterium
1LNX
1
1N9R: 66-82
yeast
1+1
Methanobacterium
Thermautriophicum:
extremophile
1JBM: 12-18, 42-50, 64-83
1 +1 +1
P. Aerophilum
Hyperthermophilic bacterium
1LNX: 10-15, 25-32, 40-48, 63-77
2CBY
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Geometry and function related
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Family of protein interfaces
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Assembly keys
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Classification of protein interfaces: Database
Systematic analysis of protein interfacessubjective classification
Mathematical approach: Laurent Vuillon (LAMA)
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Functional analysis of protein interfaces
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Protein Assembly mechanism from block: Giovanni
Feverati
Stoechiometry/Symmetry: Paul Sorba
Experimental tests: Claire Lesieur
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Alicia Ng Ling
Mun Keat Chong
Boon Leng Chua
Danyang Kong
Giovanni Feverati
Paul Sorba