estructura de proteínas métodos experimentales para la determinación de la estructura 3d de...

Estructura de Proteínas

Métodos experimentales para la determinación de la estructura 3D

de proteínas

Métodos para la determinación de la estructura 3D de proteínas:

• Métodos experimentales– Cristalografía por rayos-X– Resonancia Magnética Nuclear (NMR)– Dicroísmo circular

• Métodos teóricos

Qué es la luz y cómo interactúa con la materia?

R-X

TEORÍA DE LA MATERIA

-PARTÍCULA (~m)

-MOLÉCULA (~A)

-ÁTOMO (~A)

-PROTÓN, NEUTRÓN, ELECTRÓN (fm)

-QUARK (am)

MODELO ATÓMICO DE BOHR

Werner Heisenberg Erwin Schrodinger

LA MECÁNICA CUÁNTICA Y LA NUEVA TEORÍA DEL ÁTOMO

LAS MOLÉCULAS PRESENTAN NIVELES DE ENERGÍA MÁS COMPLEJOS

ENERGÍA INTERMOLECULAR DEPENDIENTE DE LA DISTANCIA

LOUIS DE BROGLIE

LA MATERIA TIENE UNA NATURALEZA ‘DUAL’… A VECES SE COMPORTA COMO

PARTÍCULA, PERO OTRAS VECES SE COMPORTA COMO ONDA…

HIPÓTESIS DE DE’BROGLIE

h/mv

EXPERIMENTO DE DOBLE RENDIJA CON ELECTRONES

QUE ES LA MATERIA, PARTÍCULA U ONDA?

LAS DOS COSAS!

LA MATERIA TIENE UN COMPORTAMIENTO DUAL. A VECES SE

MANIFIESTA COMO PARTÍCULA, Y OTRAS VECES SE MANIFIESTA COMO

ONDA!

LA RADIACIÓN ELECTROMAGNÉTICA

-TEORÍA ONDULATORIA

-TEORÍA CORPUSCULAR

UNA ONDA ES LA PERTURBACIÓN DE UN CAMPO…

ONDAS LONGITUDINALES Y TRANSVERSALES

EN UNA ONDA NO HAY TRANSPORTE DE MATERIA, PERO SI SE TRANSPORTA ENERGÍA

A wave is a disturbance of a medium which transports energy through the medium without permanently transporting matter. In a wave, particles of the medium are temporily displaced and then return to their original position.

EL SONIDO ES UNA ONDA LONGITUDINAL

LA LUZ ES UNA ONDA ELECTROMAGNÉTICA

C =

REFLEXIÓN, REFRACCIÓN Y DIFRACCIÓN DE LA LUZ

POLARIZACIÓN DE LA LUZ

ESPECTRO DE LA RADIACIÓN ELECTROMAGNÉTICA

FUENTES DE LA RADIACIÓN E.M.

LA INTENSIDAD DE LA LUZ DEPENDE DEL NÚMERO DE FOTONES POR UNIDAD DE

TIEMPO POR AREA

LAS ONDAS ELECTROMAGNÉTICAS (FOTONES), PUEDEN PROPAGARSE EN

UN MEDIO MATERIAL ASÍ COMO EN EL VACÍO

CON LA ABSORCIÓN DE LA LUZ OCURRE UNA EXITACIÓN ELECTRÓNICA

ABSORCIÓN DE LA LUZ

LA MATERIA PUEDE SER ‘TRANSPARENTE’ A LA RADIACIÓN

Cómo explica Usted este fenómeno ?

Purified Protein

Solve the Phase Problem-MIR, MAD or MR (next class)

Proteins Can Form an Ordered Lattice

Celda unitaria tridimensional

Overview of X-ray Experiment

Each reflection has an hkl index

and a measured intensity

8 reflections selected from a 30,000 reflection data set shown to the right

2 0 3 1483.63 -1 -3 19999.9 3 -1 -2 6729.63 -1 -1 30067.13 -1 1 8227.03 -1 2 29901.53 -1 3 24487.53 -1 4 502.1

h k l I

X-Ray Crystallography

incoming x-ray

detector

diffractedx-ray

protein crystal

diffraction patterngoniometer controls crystal orientation

Crystal lattice

“Real Space”

Reflections

“Reciprocal Space”

Periodic

(x, y, z)

Discrete, complex

F (h, k, l)

I (h, k, l) (h, k, l)

Fourier transform - FT

intensity phase

X-ray Equipment in Delaware

RU-H3RX-ray generator

Area Detector

Close-up of Cryo Crystal

Patrón de difracción

Protein Crystal X-ray Diffraction

Zoom into a single reflection

The Diffraction Condition

Reflections are the result of constructive interference

Geometry determines the constructive condition

Bragg’s Law: n = 2d sin

sin = AB / d

d sin = AB

= 2d sin or

n = 2d sin

Fase de la onda y la nterferencia constructiva/destructiva

Each Reflection Index Defines a Set of Parallel Planes that Slice Through the Crystal

Miller indices hkl Reciprocal

Space

Real

Space

h a

k b

l c

Example:draw parallel planesthat define the single reflection hkl 2 3 4

Reciprocal space has all reflections out to diffraction limit

h 0 to 30k 0 to 35 just a hypothetical examplel 0 to 43

Crystallography and Crystallization

• To obtain X-ray diffraction data:– First, need to grow a crystal – Field of macromolecular crystallization

• Large parameter space (22 or so)

• Effects change over time

• Little theory

• Protein purification costly

• Tedious experimentation

Cristalización

Vapor Diffusion Method

Experimentation to grow protein crystals

Trial-and-errorExperimentation

Partial Success

Failure

Trial 1Trial 1

Trial 2Trial 2

Trial 3Trial 3

Success

Control Parameters

Observables

3. CrystalsWhat defines a crystal?Atoms, lattice points, symmetry, space groupsDiffractionB-factorsR-factorsResolutionRefinementModeling!

CrystalsWhat defines a crystal?

3D periodicity: anything (atom/molecule/void) presentat some point in space, repeats at regular intervals,in three dimensions.

X-rays ‘see’ electrons (r) = (r+X)

(r): electron density at position rX: n1a + n2b + n3cn1, n2, n3: integersa, b, c: vectors

Crystals

What defines a crystal?

crystalprimary building block:

the unit celllattice:

set of points withidentical environment

Crystalsorganic versus inorganic

* lattice points need not coincide with atoms

* symmetry can be ‘low’

* unit cell dimensions: 5-50Å, 200-5000Å3

1 Å = 10-10 m = 0.1 nm

Crystals: X-ray diffractiondiffraction: scattering of X-rays by periodic electron densitydiffraction ~ reflection against lattice planes, if: 2dhklsin = n

~ 0.5--2.0ÅCu: 1.54Å

path: 2dhklsin

dhkl

X

Data set:list of intensities I

and angles

Crystalsinformation contained in diffraction data

* How well does the proposed structure correspond to the experimental data? R-factor

consider all (typically 5000) reflections, and comparecalculated structure factors to observed ones.

R = | Fhklobserved - Fhkl

calculated | Fhkl = Ihkl

Fhklobserved

OK if 0.02 < R < 0.06 (small molecules)

* Influence of movement due to temperature: atoms appear ‘smeared out’compared to the static model ADP’s (‘B-factors’).

The R-Factor: Measuring Convergence• To compare the generated electron density map and your

model, you have to use the R-factor.• The R-factor is a measure of convergence between the

intensities given off by your model and the observed intensities.

||Fobs| - |Fcalc||R= ------------------

|Fobs|R:

0.6-VERY BAD

0.5 -BAD

0.4-Recoverable

0.2-Good for Protein

0.05-Good for small organic models

0-PERFECT FIT

Cooling Protein Crystals

• Crystals are damaged by x-ray radiation, resulting in loss of diffraction resolution due to formation of ion radicals or breaking of bonds within the protein. By cooling the crystal to liquid nitrogen temperatures, most of the radiation damage is eliminated. Cryocrystallography is thus an important method in determining protein structures.

• The common method for cooling is flash cooling. The crystal is held in a millimeter diameter loop (see picture) and quickly immersed in liquid nitrogen, liquid propane, or placed under a nitrogen boil-off cold stream.

• Flash cooling helps to prevent ice formation. Stresses during ice formation damage fragile protein crystals. Chemical cryoprotectants added to the solvent further suppress ice formation, keeping the surrounding solution glassy or vitreous.

• Finding the proper cryoprotectant is tedious because it is protein specific. Trial-and-error is the main method, and some crystals don’t have useable cryoprotectants. A general method for cooling without needing crystal specific cryoprotectants would be useful.

Flash cooled crystal embedded in

vitreous solvent.

Pressure and Proteins

• The lab uses pressure to elucidate the structural basis for pressure effects on proteins and to develop a novel method for cooling protein crystals. Pressures used in the lab are a few thousand atmospheres.

• EFFECTS ON PROTEIN STRUCTURE

• Fluctuations and the internal arrangements of atoms plays a crucial in protein function. Both can be probed with pressure. Pressure response of proteins is not compressive and is highly anisotropic. There are internal structural rearrangements.

• EFFECT ON CRYSTAL COOLING

• Pressure is known to slow kinetics of ice formation. Pressure also makes accessible other ice phases which contracts unlike normal hexagonal ice. Thus cooling under pressure might be a general method without needing chemical cryoprotectants.

Alpha helices in myoglobin rearrange when pressurized to 1500 atm. (yellow, 1 atm; green, 1500 atm)

Phase diagram of water.

Various High Pressure Techniques in the Lab

pressure generator

CELL FOR COOLING CRYSTALS UNDER PRESSURE

liquid pressurizing medium

protein crystal

beryllium pressure cell

protein crystal

pressurizingmedium

CELL FOR DIRECT X-RAY CRYSTALLOGRAPHY

UNDER PRESSUREat room temperature

Pressurised gas (blue arrow) is applied from a pump (not

shown).

protein crystal held on a loop using surface tension of solvent

CELL FOR COOLING CRYSTALS UNDER PRESSURE

gas pressurizing medium, cooling direclty onto loop

The Raw Data

Every atom in a unit cell contributes to every reflection

in the diffraction pattern.

Two Pieces of Data• The position of a reflection

point on the reciprocal lattice, given by coordinates h,k,l. Determined by the direction reflected.

• The intensity of the reflection.

From diffraction to electron density map

To get from the diffraction pattern to the electron density, you have to use a Fourier Transform.

Fourier Transform

Once you have an electron density map, you can begin to fit models to it.

Resolution• Resolution: another

measure of how good your model is.

• Resolution gives the size of the smallest molecule you can see or resolve.

• Dependent on the amount of data ultimately phased and used in structure determination.

Limitations and Difficulties, Besides the Phase Problem

• Crystallizing Protein:– Fragile

– Requires a crystal with shortest side 0.2 mm

• Flaws of Crystallization:– Disorder in Unit Cell

– Vibrations of molecules

– Distortion in Crystallization

Fix-Its:

Cryogenic Cooling

Steps of Protein X-ray Crystallography:

Crystallize your protein.

Cryo-freeze your protein.

Do an X-ray diffraction.

Make a heavy atom derivative of protein.

Take X-ray diffraction of the derivative.

Do a Fourier Transform (or let a computer do it).

Create models.

Check R-Factor of models.

Conclusión

Protein x-ray crystallography- practical point of view

A) cloning B) expression

6.5

14.4

21.5

31.0

45.0

66.2 kDa

1

pET3aTBPS1595153bp

TBP

NdeI (4088)

BamHI (4639)

Expression vector: a plasmid carrying the gene of interest

Protein SDS PAGE gel: each band corresponds to one protein

TBP

D) crystallization E) solving the structureC) purification

A protein crystalSDS PAGE showing a purified protein

Ribbon representation of a protein structure (violet) bound to DNA

Protein x-ray crystallography- practical point of view

Some important recent structures and what can we learn from them

K. Luger et al, Nature 1997.

The nucleosome core particle

Aquaporin

(water channel)

H. Sui et al, Nature 2001.

Some important recent structures and what can we learn from them

The ribosome

(large subunit)

N. Ban et al, Science 2000.

Some important recent structures and what can we learn from them

But it sometimes happens …

Rod Casey (Norwich): crystalloid in GM wheat with soya protein

Hydroxyapatite is the primary structural component of bone. As its formula

suggests, it consists of Ca2+ ions

surrounded by PO42– and OH– ions.

Estructura cristalina de la hidroxiapatita

http://www.rcsb.org/pdb/

PDB Protein Data Bank

• Currently contains about 13762 structures of macromolecules – proteins, nucleic acids, protein-DNA complexes and carbohydrates

• 11232 – X-Ray Diffraction & Other

• 2138 – NMR

• 302 – Theoretical Modeling

PDB: Growth

Unidad Asimétrica