Post on 04-Feb-2016
description
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