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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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

A new approach towards deciphering the protein code: The protein assembly model

Claire Lesieur

[email protected]


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


A new approach towards deciphering the protein code the protein assembly model

?

?

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


A new approach towards deciphering the protein code the protein assembly model

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


Biologically active amino acids

Biologically active amino acids


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


Protein assembly

Protein assembly


Aerolysine

Aerolysine

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


A new approach towards deciphering the protein code the protein assembly model

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


Experimental approach

Experimental approach


A new approach towards deciphering the protein code the protein assembly model

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


A new approach towards deciphering the protein code the protein assembly model

CtxB5


A new approach towards deciphering the protein code the protein assembly model

LTB

CtxB

Cholera toxin B

Heat labile enterotoxin B


A new approach towards deciphering the protein code the protein assembly model

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


A new approach towards deciphering the protein code the protein assembly model

LTB5


A new approach towards deciphering the protein code the protein assembly model

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?


A new approach towards deciphering the protein code the protein assembly model

INTERFACES:

Zone de contact entre monomeres voisins


Analyses des interfaces

Analyses des interfaces

InterfaceTrimer pentamerheptamer

Brin 1

Brin 2

0101 0101 Ch111Ch

n.a. Ch111Ch 1111/1


Trimeric domain

Trimeric Domain


A new approach towards deciphering the protein code the protein assembly model

Fibritin like domain


Oligomeric proteins

Oligomeric proteins

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

Nombre de cas5722 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


A new approach towards deciphering the protein code the protein assembly model

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


Putative lipoprotein from e carotovora

PUTATIVE LIPOPROTEIN from E. CAROTOVORA

3BDU 20-29, 38-53


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-


A new approach towards deciphering the protein code the protein assembly model

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


A new approach towards deciphering the protein code the protein assembly model

1N9R

yeast

Methanobacterium Thermautriophicum: extremophile

1JBM

P. aerophilum: bacterium

1LNX


A new approach towards deciphering the protein code the protein assembly model

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


A new approach towards deciphering the protein code the protein assembly model

2CBY


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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