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Membrane proteins &Methods in Structural Biology

MMSF 2008, lecture 4

Human impression honeybee impression

Frequency Range of Hearing.

Animal frequency (Hz) low high

Humans 20 20,000Cats 100 32,000Dogs 40 46,000Horses 31 40,000Elephants 16 12,000Cattle 16 40,000Bats 1000 150,000Grasshoppers 100 50,000Rodents 1000 100,000Whales and dolphins 70 150,000Seals and sea lions 200 55,000

The senses come to us via receptors,that link via neurons to the brain.

The Five Senses

• Hear – mechanosensitive channels

• Feel– mechanosensitive channels

• Taste– ion channels, GPCR

• Smell– GPCR

• See– GPCR

What do membrane receptors do?

• Sense and communicate the environment– Physico-chemical environment

• salt, pH, nutrients, light, temperature, sound.....

– Biological environment• Hormones, pheromones• Odorants, food• Small signalling molecules, ions• Metabolic state of the cell• Proliferative state of the cell

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The fluid mosaic model

The lipid bilayer

• impermeable to most polar molecules, ions

• fast lateral diffusion

• very slow transverse diffusion (lipids do not flip spontaneously)

The biomembrane

• asymmetric structure - inside and outside are different

Membrane proteins are keycomponents of biosystems and any

biological activity• Photosynthesis, oxidative phosphorylation, hormone signalling, neurotransmission, mechanical work, transport

• About 30% of genes encode membrane proteins (pumps, transporters, receptors, channels, structural proteins)

• An estimated 2/3 of drugs have membraneproteins as molecular targets

• Only 0.2% of known protein structures are of membrane proteins - a major challenge inmolecular biology

Grouping membrane proteins on function• Transporters (up-hill transport)

– Primary transporters• Light-driven• Redox-driven• ATP-driven

– Secondary transporters• Symporters• Antiporters/Exchangers

• Channels (down-hill transport)– Passive pores– Ligand-gated channels– Voltage-gated channels

• Receptors– 7TM (G-protein coupled receptors)– Ligand-gated channels– Type 1 and type 2 single-pass receptors

Membrane protein types

Membraneproteins

Integral

Peripheral

Trans-membrane

Anchored

Single pass

Multi-passα or β

Lipid-chain

GPIGlycosylPhospha

tidylinositol

Type-1N out

Type-2C out

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Multi-pass membrane proteinsTwo types minimize desolvation penalty

α-helical β-barrel

Trp /Tyr/(Phe) residues form “paddles” at the interface

Characteristics of transmembrane segments• Hydrophobic side chains are predominant towards lipid phase• Trp, Tyr, (Phe) residues form “paddles” at the membrane interfaces• Polar interactions are satisfied by intramolecular or protein-protein interactions

• α-helical- Helices about 21 aa (+) long with high hydrophobic scores- Often capped by the large side chains at interfaces- Quite easy to identify from sequence (TMHMM algorithm)

• β-barrels:- Rigid pores- diameter dependent on beta strands and lining residues- Pore inside: hydrophobic- Pore outside: hydrophobic- Bacterial outer membrane, pores in inner membranes of eukaryotes- Difficult to depict from sequence

The K+-channel

Roderic MacKinnonNobel prize in Chemistry 2003

The selectivity filter at high resolution

Moving throughthe selectivity filter K+ stabilises the selectivity

filter

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Opening and closing of themechanosensitive channel

The channel adapts to membrane thickness. (Opening and closure can therefore be experimentally controlled by changing the composition of the lipid bilayer e.g. C20 C14 lipids)

GPCRs

• G-proteincoupledreceptors

• 7TM receptors• Large family of

receptorsinvolved in manydiseases, targetfor about half ofall medicinaldrugs

GPCR are major drug targets

Blockbuster examples:

• Anti-histamines (Claritin, Semprex)

• Beta-blockers (Inderal)

• H2-blockers (Zantac, Targamet)

• Opioids (morphine and derivatives)

• Bronchodilators (Ventoline, Bricanyl)

Datorövning 3

• Två olika kanaler: porin och aquaporin

• Generellt: hur känner mantransmembranproteiner

• Specifikt: kan vi se skillnader beroendepå vad de släpper igenom?

Methods overview

• X-ray crystallography (chapter 5)

• NMR (chapter 5)

• Single-particle cryo electron microscopy

• Function from sequence (Chapter 4)

• Protein engineering

Structures in theProtein Data Bank (PDB)

• X-ray crystallography 32073

• NMR 3975

• Electron diffraction 6

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X-ray crystallographyCrystal Concept

• In 1611 Kepler suggested thatsnowflakes derived from a regulararrangement of minute brick-likeunits.

-The essential idea of a crystal.

X-rays

• Discovered by Rontgen 1895

• Cause of tremdous scientificexcitement.

• 1,500 scientific communicationswithin first twelve months.

• US scientists repeated experimentswithin four weeks.

Theory of Diffraction

• 1910 von Laue derived theory of diffraction from a lattice.

Bragg’s Law of diffraction 1912

• Diffraction observed if X-rays scattering from a plane add in phase.• Path difference ΔP = 2d sin θ.

- d is the spaceing between planes & θ is the angle of incidence.

• Scatter in phase if path difference is nλ

- n is an integer & λ is the X-ray wavelength.

2d sin θ = n λ

• First structure (NaCl) in 1912.

W. H. Bragg & W. L. Bragg

1953: Double helix structure of DNA

• Crick & Watson used X-ray diffraction to work out the way genes areencoded.

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Diffraction pattern.

• Crystals & diffraction pattern recorded byRosalind Franklin.

• Revealed the symmetry of the helix &pitch of helix.

First Protein Structure

• Myoglobin.

• Protein purified from whale blood.

• John Kendrew 1958.• Showed a 75% α-helical fold.

• 155 amino acids, ~ 17 kDa.

First Protein Complex

• Hemoglobin.• Two copies each of α & βchains of myoglobin in acomplex.

• Solved by Max Perutz.

Structure of TBSV

• First Virus structure, tomato bushy stunt virus, 1978.

• By Steve Harrison.

• Revealed icosohedral symmetry of a virus particle.

Crystal definition

A crystal is an object with translational symmetry:ρ(r) = ρ(r) + a·x + b·y + c·z

Has crystal symmetry Doesn’t have crystal symmetry

Proteins pack symmeterically within crystals

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Prerequisites for protein crystallisation.

• Need about 10 mg purified protein.

- Various forms of chromatography.

- Better than 95 % purity if possible.

• Must be homogeneous.

- Protein isoforms & microhetrogenityvery damaging to crystal growth.

• Typically concentrate to about 20 mg/ml.

• Must be stable throughout the experiment.

- Can take days, weeks or months togrow crystals.

Crystallisation concept

• Protein solubility affected by adding "precipation agents"

- eg. salt, polyethelene glycol etc.

• In a controlled way take protein to supersaturation.

- Adding percipitant.

- Drying out the drop.

- Exchanging the buffer (dialysis).

• Wait & regulatly observe the experiment under a microscope.

Precipitant solution

Protein + precipitantsolution

Vapourdiffusion

Vapour diffusion• Soluble protein placed in a drop (~5 µl) above a buffer with higherprecipitant agent concentration.

• Drop & reservoir equilibrate by exchanging water (vapour diffusion).

- The most popular method

- Hanging drop & sitting drop.

• Achieve supersaturation, nucleation & crystal growth.

X-ray source Diffractometer

• Freeze a crystal on a loop & mount in an X-ray beam.

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X-ray diffraction from a protein

• Large number of spots

• unit cell typically 30 to 300 Å.

Synchrotron Radiation

• Large international facilities.

- Brightest X-ray sources available.

• Cost about 1 billion Euros.

- Sweden has a cheap one in Lund.

• User communities of scientists travel to them.

Collecting data

• Must rotate the crystal over many degrees so as to sampleall angles.

• Typically ~ 100 X-ray diffraction images in a “data set”. • Process data

• Solve ”phase problem”

• Interpret electron densitymap (model building)

• Refinement: minimizedifference between modeland data.

• Validation: check structure

• Coordinate deposition

Experimental result: electron density map

Resolution - level of detail

~4 Å ~3 Å ~2 Å ~1 ÅPolypeptide chain Side chains water/ions, H-bonds atomicity

Fold Full chemistry

Nuclear magnetic resonance

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•Kärnspinn är en kvantmekanisk egenskap * Isotoper med jämnt masstal spinn = 0, 1, 2 ...

* Isotoper med udda masstal spinn 1/2, 3/2, 5/2 ...

* Jämnt antal protoner och jämt antal neutroner

spinn = 0

* Udda antal protoner och udda antal neutroner

spinn = > 0

Nukleär magnetism/kärnspinn

Spinn Förekomst(%)1H 1/2 99.982H 1 0.0213C 1/2 1.1115N 1/2 0.3614N 1 99.6417O 5/2 0.0419F 1/2 10023Na 3/2 10031P 1/2 100113Cd 1/2 12.26

Ur NMR-synvinkel är endast kärnor medspinnkvanttal I ≠ 0 intressanta

Biologiskt intressanta kärnor

Vad händer med en isolerad kärna(spinn =1/2) i ett magnetiskt fält?

Proton (vätekärna), I=1/2, i ettstatiskt magnetfält B0=14.1 T

Resonansfrekvens = 600 MHz[ω0=γ*B0 (rad/s); ν0= γ*B0/2! (Hz);

γ=26.75*107 T/s]

Elektroner runt kärnan skapar eget magnetisktfält (“shielding”)

Andra kärnor med magnetiskt moment skapar också påverkande magnetiska fält ( “Nuclear Overhauser Effect”)

Kemiska bindningar “kopplar” kärnornas spinn(skalär koppling - “J-coupling”)

NMR-prover består knappast avisolerade kärnor.

NOE ger lokal information 1D NMR-spektrum på ett protein

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Kemiska skift i peptider

Random coil carbon and proton shifts of amino acids

•Provberedning

•Datainsamling

•Analys av data (NMR-spektra) -

tilldelning/asssignment

•Samla avstånds- och geometrisk information

(“Conformational restraints”)

•Strukturberäkningar

Strukturbestämning av proteinermed NMR-metoder

NOE “distance restraints”

“lokal” information (=avståndmellan atomer) används till att“knyta” ihop en peptid-kedja.

För lite information för att ge en lösning“ensemble”Varje modell förklarar data (NOE) likabra.Kemiska skift, skalära kopplingar (dihedrala vinklar) används för att ökainformationsmängden.

+ makro-molekyler i lösning+ även dynamiska objekt kan studeras+ kan studera dynamik och “mappa” interaktioner

- vanligtvis långsammare- storleken begränsar valet av objekt (< 30 kDa)

NMR jämfört med kristallografi

Folding/unfoldingchemical reactions

10-15 10-12 10-9 10-6 10-3 time(s)

Bond vibrations

Tumbling

Allosteric transitions

Internal motions/side-chain rotations

NMR-tekniker Amide hydrogen exchange

R2ex, R1ρ R2, R1{15N-1H} NOE

DiffusionRörelser iproteiner

Single-particle cryo electronmicroscopy

1930s 1960s 1970s 2000s

Electron microscopes record two-dimensional projections of three-dimensional objects

We need proper tools toretrieve 3D information

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Very low Signal-to-Noise Ratio (SNR) (avoiding damage by electron beam)

GroEL, Ludtke et al. JSB 1999

How are theseprojections related?

Single particles inthin ice

Which ones are thesame?

Most general case: Single-particle reconstruction without symmetry. • current resolution limit ~10Å

Ribosome, Gabashvili et al., Cell 2000

Single-particle reconstruction with symmetry:Icosahedral viruses, rotational symmetry

• more units for averaging due to symmetry • alignment parameters for symmetries well determined • current resolution limit ~7Å

Hepatitis B, Bottcher et al. Nature 1997

Helical reconstruction:Natural filamentous structures, tubular crystals • single helix can provide all projections for reconstruction • alignment parameters for helical units well determined • current resolution limit ~4Å (Acetylcholine channel, N. Unwin)

Actomyosin ~21Å, Volkmann et al. NSB 2000

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Bacteriorhodopsin, Henderson et al. JMB 1990

Two-dimensional arrays: Electron crystallography • particles perfectly aligned in the crystal lattice • atomic resolution achievable (four cases)

Reference free class averages

Pick two perpendicular views, average

GroEL, Ludtke et al. JSB 1999

Kryo-EM

• liten materialåtgång

• måste kunna hitta partiklarna - kan inte studera småmakromolekyler

• kan sortera bort vissa orenheter i datasetet

• Begränsad upplösning - kan inte se vätebindningar.

Säkerhet

Olikakonformationer

Dynamik

Upplösning/detaljnivå

Kostnad

Tid fördatasamling

Tid för prov-beredning

Prov-kvalitet

Prov-mängd

Model-lering

Kryo-EMNMRRöntgen-kristallografi Exempel

• Ni har just kommit fram till en metod för att rena APC-komplexet(anaphase promoting complex) och på biokemisk väg kommitfram till en ungefärlig stökiometri. Komplexet består av sex olikapolypeptidkedjor.

• Ni känner till sekvenserna för alla proteinerna. Subenheterna ärvar och en klonade och kan överuttryckas.

• Det fullständiga komplexet kan renas från jästceller.Reningsprotokollet är krävande och utbytet lågt.

• Diskutera hur de fyra olika metoderna kan användas för att fåmesta möjliga strukturella information om komplexet så snartsom möjligt.