Classificazione dei livelli strutturali in

proteine

1

Classificazione dei livelli strutturali in

proteineStruttura primaria

(sequenza amminoacidica)

2

struttura secondaria:

la forma della catena principale

della proteina(protein backbone)

3

DIAGRAMMA DI RAMACHANDRANGopalasamudram Narayana Iyer Ramachandran

8 October 1922 - 7 April 2001

 Journal of Molecular Biology - 1963

4

DIAGRAMMA DI RAMACHANDRAN

φ

ψ

φ

ψ

180

180-1

80 0

0

5

Conformazionedelle catene laterali

Torsion Axes and Dihedral Angles of the side chain of LysineThe sample amino acid Lysine has four torsion axes within its side chain. The torsion axes are symbolized as arrows, the dihedral angles are labeled chi1 to chi4. 6

eliche α

An α-helix in ultra-high-resolution electron density contours, with O atoms in red, N atoms in blue, and hydrogen bonds as green dotted lines (PDB file 2NRL, 17-32).

in α-helix hydrogen bonds between carbonyl i and amide i+4

7

Folding mechanism of alpha helices

8

φ

ψ

180

180-1

80 0

0

eliche α, ma non

solo

9

10

11

12

13

14

15

16

17

18

anse

19

a:

b: ripiegamento a forcina

a: motivo

d: barile

e: barile

(e)

Strutture supersecondarie o motivi(elementi di struttura secondaria uguali o diversi si combinano)

20

21

22

23

24

25

26

27

28

29

30

Domini strutturali(subunità avente stabilità strutturale ed una propria funzione)

gliceraldeide-3-fosfato deidrogenasi

dominio che lega il NAD+

dominio che lega ilgliceraldeide-3-fosfato

31

32

Piruvato chinasiEnzima bifunzionale

PRA-isomerasi

IGP-sintetasi33

Struttura quaternaria

Eteromultimeri

Omomultimeri

34

Set of keywords used:Intrinsically disordered proteins

Intrinsically Unstructured Proteins (IUPs)

35

The natively unstructured state is a simple and elegant solution adopted by evolution to avoid large protein, genome and cell sizes

Implications…

Schematic representation of dimers (a) unstable (disordered) and (b) stable (ordered) monomers. Although in both cases the interface area between the monomers is the same, the size of the ordered monomer is much larger compared with the disordered example

TiBS, 2003, 28, 81

Cell size constraints?

36

Some Structural FeaturesCombination of low overall hydrophobicity and relative high net charge under physiological conditions

Blue squares=275folded Red circle=91 IUPsGreen circles=130 Predicted IUPsCyan circle=242 Homologues of IUPs

Proteins, 2000, 41,415

A combination of low mean hydrophobicity and high net charge preclude the formation of a hydrophobic cluster and promote an extended conformation

37

IUPs are enriched in S,P,E,K (disorder promoting) and depleted in W, Y,F,C,I, L, N (order promoting)

IUPs have a distinctive aa composition

Table 2 Aminoacid Frequencies of Ordered and Disordered Proteins

TiBS, 2002, 527

Some Structural Features…

38

Linnaeus[5]

(1735)2 kingdoms

Haeckel[6]

(1866)3 kingdoms

Chatton[7]

(1925)2 groups

Copeland[8]

(1938)4 kingdoms

Whittaker[2] (1969)5 kingdoms

Woese [9][10]

(1977,1990)3 domains

Animalia Animalia

Eukaryote

Animalia Animalia

EukaryaVegetabilia Plantae

Plantae Plantae

ProtoctistaFungi

(not treated) Protista

Protista

Procaryote Monera MoneraArchaea

Bacteria

Taxonomy is the practice and science of classification

39

Phylogenetic Tree of All Life

40

41

42

estimated number of protein folds: ~ 2000 (?)

This three-dimensional map of the protein universe shows the distribution in space of the 500 most common protein folds as represented by spheres. The spheres, which are colored according to classification, reveal four distinct classes.

From http://www.lbl.gov/Science-Articles/Archive/PBD-Universe-map-Kim.html

It is conceivable that, of the primordial peptides, those containing fragments with high helix and/or strand propensity found their way to fold into small alpha, beta, and alpha plus beta structures," Kim says. "The alpha slash beta fold structures do not appear until proteins of sufficient size rose through evolution and the formation of supersecondary structural units became possible.

43

Statistics of new folds in PDB

44

Top Folds in Yeast Transcriptome

45

TIM barrel nelle strutture depositate nel PDB

46

ENERGIA TOTALE CONFORMAZIONALE

E=Ea+Er+Ees+El+Et+E+EH+EH

Ea attrazioneEr repulsioneEes potenziale elettrostaticoEl variazione di lunghezze di legameEt variazione di angoli di legameEpotenziale torsionaleEH legame ad idrogenoEhinterazione idrofobica

47

48

Protein structure dynamics

ENERGIA TOTALE CONFORMAZIONALE

E=Ea+Er+Ees+El+Et+E+EH+EH

49

50

51

Molecular Dynamics simulation

http://www.youtube.com/watch?v=AeYAHOCaRbk

52

Molecular Dynamics simulation

53

Nel paradosso di Levinthal una proteina di 100 residui raggiungerebbe la struttura nativa attraverso una ricerca casuale nello spazio conformazionale in 1087 s.

Ogni aa con tre possibilità di e : 3200 conformazioni diverse. Tempo minimo di interconversione 10-13 s

3200/ 10-13 circa 1087 s

54

55

Ipotetico meccanismo di ripiegamento di una proteina con due domini: formazione di elementi di struttura secondaria locali con formazione dei domini e loro assemblaggio finale. La struttura terziaria è raggiunta in pochi secondi.

Illustration of the main driving force behind protein structure formation. In the compact fold (to the right), the hydrophobic amino acids (shown as black spheres) are in general shielded from the solvent.

56

This is the E. coli major cold shock protein, CspA. This folding pathway is completely fictitious. The structure of CspA was solved by Hermann Schindelin and coworkers (PNAS, 91:5119-5123, 1994). The coordinates for the cold shock protein (ID code "1mjc") may be retrieved from the Brookhaven Protein Data Bank. Below is a step by step presentation. 57

58

59

60

thiol-disulfide oxidoreductase

Chaperone-assisted protein foldingThe unfolded polypeptide enters the central cavity of chaperonin, where it folds. The hydrolysis of several ATP molecules is required for chaperonin function. The three dimensional proteins structure is shown in Figure. The lines on the chaperonin cylinder are to represent the 7 identical GroEL subunits that make up each ring. Not shown is the end cap composed of GroES subunits.

61

E. coli chaperonin (GroE)The core structure of chaperonin consists of two identical rings composed of seven GroEL subunits. Unfolded prteins bind to the central cavity. Bound ATP molecules can be identified by their red oxygen atoms (spacefill). The quaternary structure is shown from (a) the side, and (b) the top. [PDB 1DER] (c) During folding, the size of the central cavity of one of the rings increases and the end is capped by a protein containing seven GroES subunits. [PDB 1AON]. Highlighted in green is one of the GroEL subunits. 62

63

• The enclosed cavity is the site of protein folding

• Misfolded proteins in the cavity are given about 20 seconds to refold. After 20 seconds, they are expelled either refolded successfully, or given another chance to enter the same or a different chaperon complex to try again

• A misfolded protein will be kept in “solitary confinement” until it has reformed correctly.

Christian P. Schultz Illuminating folding intermediates Nature Structural Biology 7, 7 - 10 (2000)

Schematic of the folding energy landscape of a protein molecule where the energy of the protein is displayed as a function of the topological arrangements of the atoms.The multiple states of the unfolded protein located at the top fall into a folding funnel consisting of an almost infinite number of local minima, each of which describes possible folding arrangements in the protein. Most of these states represent transient folding intermediates in the process of attaining the correct native fold. Some of these intermediates retain a more stable structure such as the molten globule, whereas other local minima act as folding traps irreversibly capturing the protein in a misfolded state.

64

65

The ability of proteins to change conformation is the essence of the amyloidoses — in these diseases, the proteins have converted into the ‘primordial’ structure rather than remaining in their evolved states. 66

amiloidosi

67

68

69

Proposed three-dimensional structure (a) PrPC and (b) PrPSc

The structure of the normal prion protein, PrPC, is characterised by four -helices. Conversion of PrPC to the disease-associated form of PrPSc results in the loss of two of the helical structures (shown shaded in brown), which are converted to linear structures known as -sheets. It is this conversion that is associated with the aquisition of prion infectivity.

70

71

72

73

mutazione E/V in posizione 6 nella emoglobina

Anemia falciforme

74