Erez Podoly Introduction to Molecular Neurobiology

From Primary to Quanternary Structure

1º 3º 4º There are three major 2º components: helices, β-sheets and what’s in-between them (turns and loops).

 helices and  sheets are stabilized by hydrogen bonds between backbone oxygen and hydrogen atoms.

The Protein Folding Problem

In the 1960’s, C.B. Anfinsen performed a series of

in vitro

experiments that lead him to the “Thermodynamic Hypothesis”: As he stated ten years later, in his 1972 Nobel acceptance speech, “The native conformation is determined by the totality of interatomic interactions and hence by the amino acid sequence, in a given environment”.

Anfinsen’s some proteins exceptions: have conformations proteins get hypothesis and multiple folding from chaperones.

has Some some help

Chou and Fasman

Amino Acid  -Helix β-Sheet Turn Ala 1.29

0.90

0.78

Cys Leu Met Glu Gln His Lys Val Ile Phe Tyr Trp Thr Gly Ser Asp Asn Pro 1.11

1.30

1.47

1.44

1.27

1.22

1.23

0.91

0.97

1.07

0.72

0.99

0.82

0.56

0.82

1.04

0.90

0.52

0.74

1.02

0.97

0.75

0.80

1.08

0.77

1.49

1.45

1.32

1.25

1.14

1.21

0.92

0.95

0.72

0.76

0.64

0.80

0.59

0.39

1.00

0.97

0.69

0.96

0.47

0.51

0.58

1.05

0.75

1.03

1.64

1.33

1.41

1.23

1.91

Arg 0.96

0.99

0.88

Favors  -Helix Favors β -strand Favors turn

Leventhal Paradox

How much time does it take to a given protein (100aa) to fold into a single stable native conformation (assuming three conformations/peptide bond)?

Leventhal Paradox

• 3 100 = 5.15 × 10 47 conformations.

• Fastest motions 10 -15 sec.

• Sampling all conformations would take 5.15 × 10 32 • 60 × 60 × 24 × 365 = 3.15 × 10 7 seconds in a year.

• Sampling all conformations will take 1.6 × 10 25 • The age of the universe is ~ 11-20 × 10 9 years.

sec.

years.

The Leventhal Paradox: proteins are able to quickly fold into their conformations despite such an overwhelming number of possibilities.

Quick Overview of Energy

Bond

H-bonds Ionic bonds Hydrophobic interactions Van der vaals interactions Disulfide bridge

Strength (kcal/mole)

3-7 10 1-2 1 51

The Protein Folding Problem

Proteins could fold more quickly if they retain native-like intermediates along the way.

The Protein Folding Problem

Much of conformation space is already restricted by allowed phi/psi angles (Ramachandran plot). Gopalasamudram Narayana Iyer Ramachandran (1922 – 2001) Y F

The Dihedral Angles –

Φ

,

Ψ Each unit can rotate around two such bonds: C α -N (phi) & C α -C (psi) .

Most combinations of Φ & Ψ angles are not allowed due to steric collisions.

C α -N bond (phi) C α -C bond (psi)

Ramachandran Plot

The angle pairs Φ & Ψ are usually plotted against each other in a diagram called a Ramachandran plot.

Most of Ramachandran plot area includes values that are not allowed.

Colored areas show sterically allowed regions.

Y F

Secondary Structures

The major allowed regions in conformational components of proteins.

Ramachandran plot define

Structural Classification of Proteins

CLASS:

 , β,  /β,  +β (but also: multi-domain, membrane and cell surface, small proteins, coiled coil proteins).

FOLD:

secondary structures in same arrangement.

SUPERFAMILY:

function/structure similarity.

FAMILY:

>30% sequence similarity, and similar known structure/function.

Protein Class Protein Fold Protein Superfamily Protein Family

Structural Classification of Proteins

• • •

SCOP

Manual classification http://scop.berkeley.edu/

CATH

Semi-manual classification http://www.cathdb.info/latest/index.html

FSSP

Automatic classification http://ekhidna.biocenter.helsinki.fi/dali/start

Class

All  All β  /β  +β Multi-domain Membrane & cell surface Small proteins Total

Folds

218 144 136 279 46 47 75 945

Superfamilies

376 290 222 409 46 88 108 1539

Families

608 560 629 717 61 99 171 2845

Proteins’ Classes

All



All β



/β

Proteins’ Folds

Proteins are defined as having a common fold if they have the same major secondary structures in the same arrangement and with the same topological connections.

A structural domain is an element of overall structure that is self stabilizing and often folds independently of the rest of the protein chain; Most domains can be classified into "folds".

Because they are self-stabilizing, domains can be "swapped" by genetic engineering between one protein and another to make chimera proteins.

http://pawsonlab.mshri.on.ca/index.php?option=com_content&task=view&id=30&Itemid=63

The Structure/function paradigm

In parallel with the growth in structural knowledge, there has been an increasing conviction that the biological function of proteins is encoded in their 3D structure. Most molecular biologists believe that determining protein functions depends on the protein structure.

The Structure/function paradigm: the amino acid sequence determines protein 3D structure and the structure determines the function.

Intrinsically Disordered Proteins

A significant proportion of proteins contain regions, sometimes quite large, that apparently don't fold into specific structure, but rather remain as flexible ensembles.

These regions are termed "intrinsically disordered”, but also “intrinsically unstructured” or “Naturally unfolded”.

Disordered regions are sequences within proteins that fail to fold into one fixed structure. It doesn’t mean they don’t have a structure, on the contrary: we may consider them as “multiple folded”.

Expansion of the Structure/Function Paradigm

“Creating a new theory is not like destroying an old barn and erecting a skyscraper in its place. It is rather like climbing a mountain, gaining new and wider views, discovering unexpected connections between our starting points and its rich environment.

But the point from which we started out still exists and can be seen, although it appears smaller and forms a tiny part of our broad view gained by the mastery of the obstacles on our adventurous way up”.

-- Albert Einstein,

The Evolution of Physics

.

Function can arise from the two protein forms (the ordered state, but also the random coil state) and transitions between them. Thus, proteins that lack a 3D structure may carry out function.

The History of Structural Biology

1895

: W. C. Roentgen discovers X rays.

1912

: Max von Laue discovers X-ray diffraction by crystals.

1913

: W. L. Bragg reports the crystal structure of NaCl .

1935

: J. M. Robertson solves the structure of pthalocyanin.

1948

: Bijvoet solves strychnine (cryst. decides bet. alternatives).

1958

: Kendrew reports the crystal structure of Myoglobin

.

1962

: M. F. Perutz and Sir J. C. Kendrew win the Nobel Prize for their studies on the structures of globlular proteins.

1965

: Lysozyme.

1968

: Haemoglobin .

1971

: Insulin.

1971

: PDB is established at Brookhaven National Lab., NY.

Methods for Structure Determination

Methods for Structure Determination

Rate limiting Step Physical principle Hydrogens # structures in solution Size limitation Wavelength Range Quality measure The result Thermodynamics

X-Ray

Crystallization Electron diffraction Invisible 1 None (MDa) 0.1 – 100 Å Resolution and R factor Snapshot Not possible

NMR

Resonance assignment Spin of nuclei in magnetic field The only detectable atom 15-20 64KDa 0.6 – 10 m RMSD Protein movements Kinetic measurements

Methods for Structure Determination

NMR X-Ray