HGP(Human Genome Project)

HPP(Human Proteome Proyect)

D:\SPLASH.EXE

..\..\LINKS\Ho Nature(2002).pdf

Ho et al. (2002) Nature 415, 180

Protein network in Saccharomyces cerevisiae

Determination of Protein Structures

National Institutes of Health, USA

The two most common methods used to investigate molecular structures are:

1. X-ray crystallography (also called X-ray diffraction)

2. Nuclear magnetic resonance (NMR) spectroscopy

X-ray crystallography

National Institutes of Health, USA

X-Ray Beam

Scattered X-RaysCrystal Detector

The First X-Ray Structure: MyoglobinKendrew (1959)

Why X-Rays?

National Institutes of Health, USA

Radio waves UltravioletMicrowaves Infrared X-Rays

Visib

leProtein

Watermolecule

CellTennisballHouse

Soccerfield

Period

Wavelength (meters)

ESRF - The European Synchrotron Radiation FacilityGrenoble, Francia

National Institutes of Health, USA

One of the first three-dimensional NMR solution structures determined by Wüthrich, in 1985.

a) A schematic view of the topology of the polypeptide backbone of BUSI IIA (bull seminal plasma proteinase inhibitor IIA). The structure represents an average of several computed structures that fulfil the structural constraints. b) A set of five backbone structures of BUSI IIA, calculated with distance geometry using the NOE distance constraints.

The Nobel Prize in Chemistry 2002

For the development of methods for identification and structure analyses of biological macromolecules

for his development of nuclear magnetic resonance spectroscopy for determining the 3D structure of biological macromolecules in solution"

for their development of soft desorption ionisation methods for mass spectrometric analyses of biological macromolecules

J.B. Fenn K. Tanaka K. Wüthrich

Electrospray ionisation (John B. Fenn)The biomolecule starts out as an entity or complex, usually charged and dissolved in a water-rich environment. At the end of the process the same biomolecule is represented and harvested through the orifice of a mass analyser as a series of ‘naked’ multicharged ions. In a vacuum, the biomolecular ions then are selectively analysed according to their mass/charge ratio.

Soft laser desorption (SLD) (Koishi Tanaka)Gaseous macromolecular ions can be formed using a low-energy (nitrogen) laser. The figure shows the signals from singly- and doubly charged molecular ions and a protein cluster-ion with a single charge.

NMR structure determination (Kurt Wüthrich)The most important parameter for structure determination based on NMR is the nuclear Overhauser enhancement (NOE) effect. This provides information about inter-atomic distances between nuclei close in space.

Different types of two-dimensional NMR spectra: a COSY spectrum, which gives crosspeaks between resonances from protons bound to adjacent carbons or nitrogens, and a NOESY spectrum, which gives crosspeaks between resonances from protons close in space. Then paste together the upper half of a NOESY spectrum with the lower half of a COSY spectrum, so that they coincide in the diagonal, providing a connectivity diagram.

If one knows all the measurements of a house, one can draw a three-dimensional picture of the house. In the same way, by measuring a vast number of short distances in a protein it is possible to create a three-dimensional picture of its structure.

NMR Spectroscopy

National Institutes of Health, USA

Most NMR spectroscopists use magnets that are 500 megahertz to 800 megahertz.

This magnet is 900 megahertz—the strongest one available.

L-am inoácido

( ~ 100 Da)M

C

H N3+ C O O -

HR

Los Elementos y Moléculas de la VidaLosada, Vargas, Florencio y De la Rosa (1998-9)Editorial Rueda, Madrid

C C

H

H

N

N

3

3

+

+

C O O -

C O O -H

H

H H

H

H H

R H

R

R

R R

R

R R

A M I N O Á C I D O S Y P É P T I D O S

Los Elementos y Moléculas de la VidaLosada, Vargas, Florencio y De la Rosa (1998-9)Editorial Rueda, Madrid

-

+

ESTRUCTURA PLANA DE LA UNIDAD PEPTÍDICA

Los Elementos y Moléculas de la VidaLosada, Vargas, Florencio y De la Rosa (1998-9)Editorial Rueda, Madrid

-

+

CO NFO RM A CIÓ N EN -HÉ LICE(estructura secundaria)

Los Elementos y Moléculas de la VidaLosada, Vargas, Florencio y De la Rosa (1998-9)Editorial Rueda, Madrid

CONFORMACIÓN EN HOJA PLEGADA ANTIPARALELA

O O

O O

OH

H H

H HH H H

H H

H H

HO

O O

O OO O O

N

C

C

N

Los Elementos y Moléculas de la VidaLosada, Vargas, Florencio y De la Rosa (1998-9)Editorial Rueda, Madrid

..\..\..\Mis documentos\webdpto\biomoleculas\biomodel\model1\INICIO.HTM

The CATH Hierarchy

C: Class

A: Architecture

T: Topology

H: Homologous Superfamily

CATH database of structural domains

Protein Structure Classification

http://scop.mrc-lmb.cam.ac.uk/scop/

SCOP: Structural Classification of Proteins

Proteínas:Evolución a nivel molecular

A strict principle of economy:

The same economy that reuses a few motifs to subserve different functions (divergent evolution) increased the chance of different biological systems coming up with different solutions to the same problem (convergent evolution).

Evolutionary tree showing how the globin protein family arose, starting from the most primitive oxygen-binding proteins, leghemoglobins, in plants.

Divergent Evolution

Gomis et al. (2001) Nature 409, 637-641

Lateral view

View along the 6-fold axis

Divergent Evolution

The bacterial conjugation protein TrwB resembles F1-ATPase

Left, Ribbon diagram of the structure of AIF. Right, Superposition of AIF and BphA4, a bacterial oxygenase-coupled NADH-dependent ferredoxin reductase (BphA4 in light blue).

H. Ye et al. (2002) Nature Struct Biol 9, 680

The Apoptosis Inducing Factor (AIF)

Divergent Evolution

H. Ye et al. (2002) Nature Struct Biol 9, 680

Apoptosis (or programmed cell death, PCD) is a highly organized multi-step process, with the induction of mitochondrial membrane permeabilization as a decisive event in the commitment to cell death.

The execution of apoptosis comprises both caspase-dependent and caspase-independent processes.

The Apoptosis Inducing Factor (AIF), a resident protein of the inter-mitochondrial space, has been implicated as a crucial early effector of caspase-independent apoptosis, acting before or in parallel with the onset of caspase-dependent processes.

The ectopic presence of AIF in the extra-mitochondrial compartment suffices to kill cells.

Apoptosis

S. Hunot and R.A. Flavel (2002) Science 292, 865

Apoptosis

The caspase-dependent (right) and AIF-dependent (left) apoptotic pathways

H. Ye et al. (2002) Nature Struct Biol 9, 680

The Apoptosis Inducing Factor (AIF)

Human AIF is synthesized as a precursor protein of 67 kDa and converted to mature AIF of 57 kDa upon mitochondrial import and removal of the N-terminal mitochondrial localization signal.

Mature AIF is a flavoprotein with significant structural similarity to bacterial nicotinamide adenine dinucleotide (NAD)-dependent ferredoxin oxidoreductases (FNR).

This suggests that AIF is a bifunctional protein with a mitochondrial resident function and an apoptogenic function.

Because the flavin adenine dinucleotide (FAD) cofactor is dispensable for the apoptogenic function but required for the oxidoreductase activity of AIF, the structural bases for the mitochondrial and ectopic functions of AIF are probably entirely different.

H. Ye et al. (2002) Nature Struct Biol 9, 680

The Apoptosis Inducing Factor (AIF)

When released from the mitochondria or added to purified nuclei, AIF enters the nucleus and induces chromatin condensation and large-scale DNA fragmentation to ca. 50 kilobases (kb) in a caspase-independent fashion.

AIF induces chromatin condensation and initial DNA cleavage via an unknown molecular mechanism.

DNA binding is required for the apoptogenic action of AIF, which interacts with DNA in a sequence-independent manner.

The structure reveals the presence of a strong positive electrostatic potential at the AIF surface.

The structure reveals the presence of a strong positive electrostatic potential at the AIF surface.

H. Ye et al. (2002) Nature Struct Biol 9, 680

The Apoptosis Inducing Factor (AIF)

Cyt c6Pc

PS I

b6f

PSI-driven Electron Transfer

Fd

light

Convergent Evolution

Convergent Evolution

A strict principle of economy: The same economy that reuses a few motifs to subserve different functions (divergent evolution) increased the chance of different biological systems coming up with different solutions to the same problem (convergent evolution).

Dragonfly Bats

Pterosauria (pterosaurs)

Chiroptera (bats)Aves (birds)

The Three Solutions to Vertebrate Flight

Functional and mechanistic convergence in biological systems: The pterosaur,the seagull and the bat all take to the air using an analogous mechanism.

Protein evolution: convergence or divergence?

Structure and Mechanism in Protein ScienceA. Fersht (1999)WH Freeman and Company, New York, USA

Six criteria for testing whether two proteins have evolved from a common precursor:

1. The DNA sequences of their genes are similar

2. Their amino acid sequences are similar

3. Their three-dimensional structures are similar

4. Their enzyme-substrate interactions are similar

5. Their catalytic mechanisms are similar

6. The segments of polypeptide chain essential for catalysis are in the same sequence (i.e., not transposed).

Protein building blocks preserved by recombination

Voigt et al. (2002) Nature Struct Biol 9, 553

Recombination of beta-lactamases TEM-1 (gray line) and PSE-4 (black line)

Protein building blocks preserved by recombination

Voigt et al. (2002) Nature Struct Biol 9, 553

Structures of the designed hybrids of -lactamase TEM-1 (red) and PSE-4 (blue), shown in order of increasing disruption

There exists a threshold in the amount of schema disruption that the hybrid protein can tolerate. To the extent that introns function to promote recombination within proteins, natural selection would serve to bias their locations to schema boundaries.

Dinámica Molecularde las Estructuras Proteicas

La biología es inconcebible sin movimiento

http://www.life.uiuc.edu/crofts/bioph354/lect10.html

ATP synthaseAnimation of the complete mechanism

Lecture 10, ATP synthase

Schnitzer (2001) Nature 410, 878 - 881

Molecular machines

They use ATP binding at one catalytic site to trigger a large conformational change and the release of ADP from another catalytic site.

Microtubule filament (left) with the bound motor domain of Neurospora crassa conventional kinesin

Song, Y.-H. et al. (2001) EMBO J. 20, 6213-6225

The 'conventional' kinesin from the fungus Neurospora crassa is incredibly quick, moving along filamentous tracks called microtubules at speeds of 2.5 m per second — some five times faster than other conventional kinesins

Microtubule-motor protein interactions

A. Hoenger, EMBL 2000 Research Reports

Myosin, a cellular motor proteinIt takes 37-nm steps by placing one “foot” after the other

Cover - Science 27 June 2003

The Actomyosin Cross Bridge Cycle

ATP binding to either a resting length myosin head (c) or to a head bearing a load (b) results a change in conformation in the myosin head, causing a rapid, almost irreversible dissociation of the myosin head from actin (d). Following detachment from actin, the ATP is hydrolysed to ADP and Pi, both of which remain very tightly bound to the myosin head (e).

The Actomyosin Cross Bridge Cycle

http://www.mrc-lmb.cam.ac.uk/myosin/motility/XBcycle.html

XBcycle

http://molmovdb.mbb.yale.edu/MolMovDB/cgi-bin/morph.cgi?ID=12221-32592

Myosin

A major question is: Are the modes of mobility observed in enzymes just incidental (...) or are they essential for catalysis?

Flexibility could be useful in aiding the access of ligands to active sites.

Protein mobility and enzyme mechanism

Structure and Mechanism in Protein ScienceA. Fersht (1999)WH Freeman and Company, New York, USA

Untangling Protein Folding

National Institutes of Health, USA

Least Flexible

MostFlexible

Unfolded Partially folded Completely folded

E. Myshkin & G. Bullerjahn Bowling Green, Ohio,USA

Dynamics of Plastocyanin

The compact globular regions of proteins have structural fluctuations

“Breathing” of proteins

Mioglobina

Hemoglobina

Domain movements: segment flexibility

Structure and Mechanism in Protein ScienceA. Fersht (1999)WH Freeman and Company, New York, USA

All domains movements may be constructucted from a combination of hinge and shear motions:

Hinge motions, in which two elements of structure open and close as if connected by a hinge.

Shear motions, in which one element of structure slides relative to the other.

Movimiento de bisagra

Movimiento de deslizamiento

Tipos de movimientos de las estructuras proteicas

Movimientos internos de las proteínas

Empaquetamiento Mantenido No mantenido de las interfases

Empaquetamiento Restringido Libre en la bisagra de la cadena principal

Torsiones de la cadena Cambios pequeños Cambios grandesprincipal y numerosos pero pocos

Movimiento global Movimientos locales Igual al de la bisagrapequeños y encadenados

Movimiento en la Paralelo al plano Perpendicular ainterfase de la interfase la interfase

Empaquetamiento Cierto empaquetamiento Nuevos contactos en de las cadenas laterales en las dos mitades la base de la bisagra

Torsiones de las Cambios pequeños Cambios grandescadenas laterales y numerosos pero pocos

Características Mecanismo de Mecanismo deestructurales deslizamiento bisagra

TransferrinIron Transport Protein

Database of Macromolecular Movements

Database of Macromolecular Movements

Database of Macromolecular Movements

Ca - ATPase

L-Alanina (Ala) :

L-Glicina (Gly) :

L-Valina (Val) :

L-Leucina (Leu) :

L-Metionina (Met) :

L-Isoleucina (Ileu) :

C

C

C

C

C

C

C

C

C

C

C C

C

C

C

S

H

H

H

H

H

H

H

H

H

H

H

H H

H

H

H

-

-

-

-

- -

-

-

- - --

3

3

3

2

2

3

3

2 2

3

3

3

I. Grupos R no polares (a)

I. Grupos R no polares (b)

L-Prolina (Pro) :

L-Fenilalanina (Phe) :

L-Triptofano (Trp) :

C

C

C

C

C

C

H

H

N

H

H

H

H

H-

-

-

+2

2 2

2

2

2

N H

II. Grupos R polares sin carga

L-Serina (Ser) :

L-Treonina (Thr) :

L-Cisteína (Cys) :

L-Asparragina (Asn) :

L-Glutamina (Gln) :

L-Tirosina (Tyr) :

H

C

H

O

O

N

N

C

C O

C

C

C C

CH

C

C

O

H

S

H

H

H

H H

H

H

H H

HO

-

- -

-

- -

- -

- - -

-

3

2

2

2

2

2

2 2

2

III. Grupos R con carga negativa

L-Aspartato (Asp) :

L-Glutamato (Glu) :

C

C C

C

C

O

O

O

O

-

-

H

H H

-

- -

-

-

2

2 2

IV. Grupos R con carga positiva

L-Lisina (Lys) :

L-Arginina (Arg) :

L-Histidina (His) :

C

C

N

N

N

N

H

C C

NC

C

C

H

H

)

)

(

(

H

H

H

H

N

H

H

H

H

-

-

- 2

2

4

3

3

22

+

+

+

2

lys

phe

a la

a la

asp

C A D E N A P O LIP E P TÍD IC A P LA N A

ser

s s

s s

s s

21 aa

30 aa

S E C U E N C IA D E A M IN O Á C ID O S D E LA IN S U LIN A(e structura prim aria )

l e v o d e x t r o

A S I M E T R Í A D E L A- H É L I C E