a new approach towards deciphering the protein code the protein assembly model
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A new approach towards deciphering the protein code: The protein assembly model. Claire Lesieur [email protected] Membrane (Lipids). Proteins. Nucleus (chromosome). Elements of the living world. Protein. Nucleus. Lipids. DNA. CHON. Chromosome. Protein Biological activities.

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elements of the living world
Membrane (Lipids)

Proteins

Nucleus

(chromosome)

Elements of the living world

Protein

Nucleus

Lipids

DNA

CHON

Chromosome

protein biological activities
Protein Biological activities
  • Cutting
  • Recognition
  • Enzyme
  • Signaling
  • Carrier
  • Shape generator
  • Road networks
structure function relationship in proteins
Structure-function relationshipin proteins
  • Function
  • Shape
  • How the shape provides a particular function
  • How the shape is acquired
slide7
?

?

GKKHDGATTYQW

the protein folding problem
The protein folding problem
  • How it folds: Mechanisms of protein folding
  • How the information is encrypted in the sequences: CODING problem

ADRTGGILLKMHGGARECVVP

slide9
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.

mechanism
COOH

H2N

s-hours

ms

Structure Tertiaire

Structure primaire

Structure Secondaire

Mechanism

Short range interaction

long-range

interactions

short-range

interactions

code still unknown
Code: still unknown

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

transmembrane domains of membrane proteins
Transmembrane domains of Membrane proteins

b-strands transmembrane domain: 1010101

a-helicetransmembrane domain: 11111111111111111

sequence shape predictions
Sequence-Shape predictions
  • Geometrical constrain
  • Chemical constrain
to read sequences you need to determined comparable sequences
To read sequences you need to determined comparable sequences
  • Domains
  • Shape and role

?

Sequence Pattern

?

Sequence Pattern

aerolysine
Aerolysine

Trends in Microbiology (2000). Vol 8 (4):169-172

slide18
ER

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
slide20
Assembly in vitro

pH 7

pH 1

15 min

Pentamere

Monomere

2d structural level short range interaction
2D structural level: short range interaction

5

2 10

0

5

-2 10

5

-4 10

pH 1

5

-6 10

Mean residue Molecular Ellipticity

pH 7

5

-8 10

Native

6

-1 10

6

-1,2 10

200

210

220

230

240

250

Wavelength (nm)

3d structural level long range interaction
3D structural level: long range interaction
  • Trp-fluorescence

300

lex= 295 nm

lem=352 nm

Fluorescence Intensity (a.u.)

200

Fluorescence Intensity

100

unfolded

0

Time (min)

320

340

360

380

Wavelength (nm)

functional test
Functional test

His

CtxB

100

80

Function

60

HISTIDINE

40

20

4,5

5

5,5

6

6,5

7

7,5

8

0

pH

slide24

CtxB5

slide26
LTB

CtxB

Cholera toxin B

Heat labile enterotoxin B

slide27
N-terminal

100

LTB

CtxB

80

Function

60

N-terminal

40

20

0

4,5

5

5,5

6

6,5

7

7,5

8

pH

slide29
Kinetics differences

On pathway intermediates differences

It is particular amino acids that are responsible for each individual step of assembly and folding

fundamental question
Fundamental question
  • Alzheimer, Parkinson, Prion diseases

Protein X: FOLD state: healthy

Information for interfaces

(Protein X)n: Assembly state: Lethal

theoritical approach
Theoritical approach
  • Protein Interface formation
  • Rules?
  • Mechanism?
  • Preferential geometries related to preferential sequences of amino acids?
slide32
INTERFACES:

Zone de contact entre monomeres voisins

analyses des interfaces
Analyses des interfaces

Interface Trimer pentamer heptamer

Brin 1

Brin 2

0101 0101 Ch111Ch

n.a. Ch111Ch 1111/1

oligomeric proteins
Oligomeric proteins

Nombre de monomer 2 3 4 5 6 7 8 9 10 11 12

Nombre de cas 5722 1035 2340 168 721 46 512 45 87 8 205

programme detection protein interfaces
Programme detection Protein Interfaces

Monomer M

513 -524

LMITTECMVTDL

aaa-bbbbbbb-

Monomer M+ 1

35-49

GRNVVLDKSFGAPTI

--bbbb-------bb

Distances

slide39
2HY6 (30)

1 30

beta

1N9R (68)

19 86

alpha

1WNR (94)

1 94

a+b

2F86 (129)

344 471

1JBM (78)

10 88

rc

1G31 (107)

5 111

1LNX (74)

8 80

1Q57 (483)

64 549

2RAQ (94)

3 97

1GRL (518)

6 523

1IOK (524)

2 526

1PZN (240)

96 336

1J2P (229)

4 233

1Y7O(194)

1 194

2F6I (189)

177 367

1TG6 (193)

1 193

2CBY (179)

15 194

1OEL (525)

2 525

1LEP (92)

1 92

3BDU (51)

2 53

1HX5 (92)

5 97

common protein interfaces of unrelated proteins
Common protein interfaces of unrelated proteins

3BDU 1--111011-110110--10

1G31 0--1-1001-100100--00

1JBM 11001000101100101101

1LNX 1--0100010110000---1

1N9R 0--0100011110010--11

1J2P ----1000101100101--1

1HX5 ------0011110010--11

1LEP 0---10001000--00--11

Con2 ----1-001-1100-0-

slide42
1LEP: 1-8, 88-94, 40-57

1WNR: 1-8, 88-94, 44-57, 62-77

1HX5: 5-11, 94-97, 51-62, 68-80,27-30

1G31: 8-15, 104-111, 68-85

slide44
1N9R

yeast

Methanobacterium Thermautriophicum: extremophile

1JBM

P. aerophilum: bacterium

1LNX

slide45
1

yeast

1 + 1

Methanobacterium Thermautriophicum: extremophile

1JBM: 12-18, 42-50, 64-83

1 +1 +1

1N9R: 66-82

P. Aerophilum

Hyperthermophilic bacterium

1LNX: 10-15, 25-32, 40-48, 63-77

conclusion
Conclusion
  • Geometry and function related
  • Family of protein interfaces
  • Assembly keys
future
Future
  • Classification of protein interfaces: Database
  • Systematic analysis of protein interfaces-subjective classification
      • Mathematical approach: Laurent Vuillon (LAMA)
  • Functional analysis of protein interfaces
    • Protein Assembly mechanism from block: Giovanni Feverati
    • Stoechiometry/Symmetry: Paul Sorba
    • Experimental tests: Claire Lesieur
acknowledgment
Acknowledgment
  • Alicia Ng Ling
  • Mun Keat Chong
  • Boon Leng Chua
  • Danyang Kong
  • Giovanni Feverati
  • Paul Sorba
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