DETERGENTS AND THEIR PROPERTIES

BEN HANKAMER

INSTITUTE FOR MOLECULARBIOSCIENCEUNIVERSITY OF QUEENSLANDBRISBANE

b.hankamer@imb.uq.edu.au

FROM TISSUE TO PROTEIN

SEIBERT ET AL 1987 J CELL BIOL 105:2257

DETERGENT DEFINITION

DETERGENTS ARE AMPHIPATHIC MOLECULES THAT CONTAIN BOTH APOLAR (HEADGROUP) AND HYDROPHOBIC REGIONS (TAIL).

PURIFICATION STABILIZATION CRYSTALLIZATION

SEIBERT ET AL 1987 J CELL BIOL105:2257

PLAY AN IMPORTANT ROLE IN:

1. POLAR HEADGROUPS FORM HYDROGEN BONDS WITH WATER.

2. HYDROPHOBIC TAILS DO NOT FORM H-BONDS WITHH2O….THEREFORE AGGREGATE INTO MICELLES.

3. DETERGENTS REPLACE LIPIDS DURING SOLUBILIZATION ANDSTABILIZE TRANS-MEMBRANE DOMAINS IN SOLUTION.

MEMBRANE PROTEIN SOLUBILIZATION

PROTEIN: DETERGENT

MICELLE

BICELLE:PROTEIN COMPLEX

PROTEIN:DETERGENTCOMPLEX(NATIVE LIPID)

PROTEIN:DETERGENT

COMPLEX(ADDED LIPID)

MP EMBEDDED IN A PLANAR LIPIDDETERGENT BILAYER

SMALL VESICLES

DENATURED PROTEIN

MAIN CLASSES OF DETERGENTS

IONIC DETERGENTS1. -VE CHARGE = ANIONIC (E.G. SDS)2. +VE CHARGE = CATIONIC (CTAB)3. EFFECTIVE AT BREAKING PROTEIN: PROTEIN INTERACTIONS4. THEREFORE OFTEN DENATURING5. SIMILARLY CHARGED HEADGROUPS REPEL6. THEREFORE MICELLE SIZE DEPENDENT ON:

A. COUNTERION TYPE/CONC- NEUTRALIZATION = REDUCED CURVATURE = INCREASED MICELLE SIZE. B. CHAIN LENGTH & SHAPE OF HYDROPHOBIC REGION

BILE ACID SALTS1. ANIONIC DETERGENTS CONTAINING RIGID STEROIDAL RING2. NO CLEARLY DEFINED HEADGROUP (POLAR/APOLAR FACE)

DUE TO HYDROXYLATION OF THE STEROIDAL RINGS3. KIDNEY SHAPED MICELLES-SIZE DEPENDENT ON

COUNTERIONS4. DUE TO LOW pKa (5-6) AND LOW SOLUBILITY OF

UNCONJUGATED BILE SALTS ONLY USEFUL IN ALKALINERANGE. CONJUGATED BILE SALTS CAN BE USED OVER ABROADER pH RANGE.

NON-IONIC DETERGENTS1. NON-IONIC DETERGENTS ARE GOOD AT BREAKING LIPID:LIPID

AND LIPID:PROTEIN CONTACTS2. NOT SO GOOD AT BREAKING PROTEIN:PROTEIN CONTACTS3. THEREFORE PROTECT PROTEIN FUNCTION4. MICELLE SIZE NOT GREATLY EFFECTED BY COUNTERIONS5, MICELLE SIZE IS EFFECTED BY SALT CONCENTRATION

1. POLYOXYETHELENE HEADGROUPS (E.G. BRIJ)- CAN FORM RANDOM COIL STRUCTURES- SHORT CHAIN POLYOXYETHELENES FORM AGGREGATES AND

VISCOUS SOLUTIONS IN WATER AT ROOM TEMPERATURE-LONG CHAIN POLYOXYETHELENES DO NOT AGGREGATE

2. POLYOXYETHELENE HEADGROUPS COUPLED TO PHENOL RING(E.G. TRITON)- HIGH ABSORPTION AT 280NM- TRITON CAN OFTEN CONTAIN PEROXIDE CONTAMINATION- OFTEN USED DURING PURIFICATION (CHEAP, LOW CMC)

3. SUGAR HEADGROUPS (E.G. MALTOSIDES, GLUCOSIDES)- HOMOGENEOUS STRUCTURE (but check α and β conformations)- SELECTION OF HYDROPHOBIC CHAIN LENGTHS (C6-C12)STRAIGHT, AS WELL AS CYCLIC) AND HEADGROUPS (MALTOSE,SUCROSE, GLUCOSE)

IMPORTANT PROPERTIES OF NONIONIC DETERGENTS: PURE, WELL DEFINED MICELLES, PROTECT AGAINST DENATURATION

ZWITTERIONIC DETERGENTSOFFER COMBINED PROPERTIES OF IONIC AND NON-IONICDETERGENTS.1. NO NET CHARGE = LACK CONDUCTIVITY, ELECTROPHORETIC

MOBILITY AND ABILITY TO BIND TO ION EXCHANGE RESIN.2. BUT LIKE IONIC DETERGENTS GOOD AT BREAKING

PROTEIN:PROTEIN CONTACTS.3. RIGID STEROID RING OF CHAPS THOUGHT TO MAKE IT LESS

DENATURING

BHAIRI, S.M. (2001) A GUIDE TO THE PROPERTIES AND USES OF DETERGENTS IN BIOLOGY ANDBIOCHEMISTRY.CALBIOCHEM WEBSITE

BHAIRI, S.M. (2001) A GUIDE TO THE PROPERTIES AND USES OF DETERGENTS IN BIOLOGY ANDBIOCHEMISTRY. CALBIOCHEM WEBSITE

BHAIRI, S.M. (2001) A GUIDE TO THE PROPERTIES AND USES OF DETERGENTS IN BIOLOGY ANDBIOCHEMISTRY. CALBIOCHEM WEBSITE

GLUCOPYRANOSIDESNON-IONIC DETERGENTS IN MORE DETAIL

ALKYL GLUCOPYRANOSIDES (THIO-GLYCOSIDES ARE LESS POLAR)

CMC=250mM

Low solubility

ALKYL MALTOPYRANOSIDES

OFTEN MILDER AND MORE EFFECTIVE THAN GLUCS. INCREASED SOLUBILITY

SUCROSE BASED DETERGENTS

MILD AND USEFUL FOR EXTRACTION BUT ESTERS …EASILY HYDROLYSED,ALSO A MIXTURE OF ISOMERS

ANATRACE CATALOGUE

NONIONIC DETERGENTS IN MORE DETAIL

MEGA SERIES: GLUCOSE DERIVATIVES WITH AMIDE LINKAGE

CHEAP, LOW SOLUBILITY EXCEPT FOR MEGA 8

ANATRACE CATALOGUE

HEGA SERIES: GLUCOSE DERIVATIVES WITH AMIDE LINKAGE, PLUS ADDITIONAL OH

277mM CMC108mM CMC 35 mM CMC 11 mM CMC

INCREASED SOLUBILITY

CYMAL

CYMAL: CYCLIC MALTOSIDE

HIGHER CMCCYCLOHEXYL TAILS PACK MORE HYDROPHOBICITY INTO A SHORTER EFFECTIVE CHAIN LENGTH.

HARLAN, GARAVITO ANATRACE CATALOGUE

HIGER CMC VALUES THAN CORRESPONDING LINEAR DETERGENT

ALTERNATIVE AMPHIPATHIC MOLECULES

P. Nollert 2005 Progress in Biophysics and Molecular Biology 88:339–357

SCHAFMEISTER ET AL 1993• A 24-AMINO ACID PEPTIDE DESIGNED TO SOLUBILIZE INTEGRAL MEMBRANE PROTEINS.• DESIGNED AS AN AMPHIPATHIC ALPHA HELIX WITH A "FLAT" HYDROPHOBIC SURFACE

THAT INTERACTS WITH A TRANSMEMBRANE DOMAIN AS A DETERGENT.• WHEN MIXED WITH PEPTIDE, 85 PERCENT OF BACTERIORHODOPSIN AND 60

PERCENT OF RHODOPSIN REMAINED IN SOLUTION OVER A PERIOD OF 2 DAYSIN THEIR NATIVE FORMS.

TRIBET ET AL 1996 PNAS 93:150471. AMPHIPOLS = AMPHIPHILIC POLYMERS2. HYDROPHILIC BACKBONE3. GRAFTED HYDROPHOBIC CHAINS4. FAVOURABLE PHASE TRANSITIONS FOR

CRYSTALLIZATION

ALTERNATIVE AMPHIPATHIC MOLECULES

REVIEW: P. Nollert 2005 Progress in Biophysics and Molecular Biology 88:339–357

articles YU ET AL 2000 PROT SCI 9:2518

1. POLAR PORTION CONTAIN AMIDE AND AMINE OXIDE2. DESIGNED TO DISPLAY GREATER RIGIDITY (TETRA-SUBSTITUTED C).3. INCREASED RIGIDITY PROPOSED TO INCREASE ABILITY TO

CRYSTALLIZE

articles

MCGREGOR ET AL 2003 NAT BIOTECH 21:171-1761. SYNTHETIC LIPOPROTEINS. 2. MIMIC BILAYER 3. SELF ASSEMBLE INTO A CYLINDRICAL MICELLE4. RIGID OUTER SHELL (PROTECTIVE)5. FLEXIBLE INNER LIPID COMPONENT6. DISPERSE PHOSPHLIPID MEMBRANES7. GENTLE (PRESERVE MEMBRANE PROTEIN

STRUCTURES)

INTRINSIC CURVATURE HYPOTHESIS (Gruner 1985)

According to the ‘intrinsic curvature hypothesis’ (Gruner, 1985) amphiphiles form supramolecularstructures in water due to the hydrophobic effect (Tanford, 1980) and their shape.

REFERENCES1. ISRAELACHVILI (1991)

INTERMOLECULAR AND SURFACEFORCES (2/E)

2. ISRAELACHVILI, MITCHELL & NINHAM1977 BBA470, 185.

3. GRUNER, 1985. INTRINSIC CURVATUREHYPOTHESIS FOR BIOMEMBRANELIPID COMPOSITION: A ROLE FORNONBILAYER LIPIDS. PNAS 82:3665

4. TANFORD, C., 1980. THEHYDROPHOBIC EFFECT. WILEY, NEWYORK.

1. Water forms a highly ordered network of intermolecular hydrogen bonds.

2. It is the strength of all of the hydrogen bonds combined that give water its liquid properties. 3. Polar or hydrophilic substances dissolve in water because they form hydrogen bonds and electrostatic interactions with water molecules.

4. Non-polar or hydrophobic substances are unable to form these interactions and consequently are immiscible with water.

5. Addition of non-polar substances to water disrupts intermolecular hydrogen bonds of water molecules and creates a cavity which is devoid of water. At the surface of the cavity, water molecules rearrange in an orderly manner.

WATER, AMPHIPHILES AND HYDROPHOBIC INTERACTIONS

6. This results in a thermodynamicallyunfavourable decrease in entropy(decrease in disorder).

7. To compensate for this loss of entropy,water molecules force the hydrophobicmolecules to cluster and thus occupythe minimum space.

8. This effect is known as the hydrophobiceffect and the forces betweenhydrophobic regions (e.g. membranespanning domains of membraneproteins) are called hydrophobicinteractions.

DETERGENT PHASE DIAGRAMS

1. AT LOW TEMPERATURE, DETERGENTS LARGELY IN CRYSTALLINE PHASE

2. AS TEMPERATURE RISES DETERGENT REACHES AN EQUILIBRIUM BETWEEN CRYSTALLINE AND MONOMER PHASE

3. AT THE KRAFT POINT, SOLID, MONOMER AND MICELLES ARE IN EQUILIBRIUM

4. CRITICAL MICELLE CONCENTRATION (CMC) THE LOWEST DETERGENT CONCENTRATION ABOVE WHICH MONOMERS CLUSTER INTO MICELLES.

5. CRITICAL MICELLE TEMPERATURE (CMT) THE TEMPERATURE AT WHICH MICELLES FORM FROM CRYSTALS.

THE PHASE TO BE IN: FOR PURIFICATION,STABILIZATION AND AT THE START OF 2D CRYSTALLIZATION.

A PARTICULAR TEMPERATURE ABOVE THE CMT, AT WHICH NON-IONICDETERGENTS UNDERGO PHASE SEPARATION TO YIELD A DETERGENT RICH AND ADETERGENT POOR AQUEOUS PHASE.

CAUSED BY DECREASED HYDRATION OF THE HEADGROUP.

E.G.TRITON X-100 CLOUD POINT = 64oCTRITON X-114 CLOUD POINT = 22oC

USES: MEMBRANE PROTEINS CAN BE SOLUBILIZED AT 0oC, BEFORE BEINGPURIFIED FROM SOLUBLE PROTEINS BY WARMING TO 30oC, SO THAT TWO PHASESARE FORMED, WITH THE MP BEING ENRICHED IN THE DETERGENT RICH PHASE.

CLOUD POINT

2000.13C12980.1-0.6C12

--C11-0.59C11

--C10-1.6C10

-6.5C9--C9

8420-25C88423.4C8

-79C7--C7

-250C6--C6

AGGNO.

CMC(mM)

CHAINLENGTH

AGGNO.

CMC(mM)

CHAINLENGTH

GLUCOSIDESMALTOSIDES

EFFECT OF CHAIN LENGTH ON

MALTOSIDE CMC

0

5

10

15

20

25

8 9 10 11 12

CHAIN LENGTH

CM

C (

mM

)

EFFECT OF CHAIN LENGTH ON

GLUCOSIDE CMC

0

50

100

150

200

250

6 7 8 9 10 11 12

CHAIN LENGTH

CM

C (

mM

)

EFFECT OF CHAIN LENGTH ON CMC

1. CMC DECREASES WITH INCREASEDCHAIN LENGTH

2. CMC INCREASES WITH DESATURATION

3. ADDITIVES THAT BREAK UP WATERSTRUCTURE (EG. UREA) INCREASE CMC.

4. LOW CMC DESIREABLE FOR REDUCEDCOST OF PURIFICATION

5. HIGH CMC DESIRABLE FORDETERGENT REMOVAL

6. LONG CHAIN LENGTHS = PROTECTIVEBUT STERIC HINDRANCE IN 3D XTALS.

BUT CMC DOES VARY: USEFUL TO DEFINE IT ACCURATELY

AGGREGATION NUMBER = MICELLAR MOLECULAR WEIGHT

MONOMER MOLECULAR WEIGHT

AGGREGATION NUMBER

DYE

SO

LUB

ILIZ

ATI

ON

(DS)

AB

SOR

BA

NC

ELI

GH

T SC

ATT

ERIN

G (L

S)

SUR

FAC

E TE

NSI

ON

(DYN

E/C

M)

DETERGENT CONCENTRATION (mM)

DETERMINING CRITICAL MICELLE CONCENTRATION

[MICELLE] = ([DET]-CMC)/AGG. No.

BHAIRI, S.M. (2001) A GUIDE TO THE PROPERTIES ANDUSES OF DETERGENTS IN BIOLOGY AND BIOCHEMISTRY.CALBIOCHEM WEBSITE

A NOVEL METHOD FOR DETERGENT CONCENTRATION DETERMINATIONKAUFMANN ET AL 2006 BIOPHYS J. 90:1310-317 ($US 2500-4000)

PRINCIPLE:1. PUT DROPLET ON PARAFILM.2. DET. MOLECULES AT SURFACE LESS

ENERGETICALLY FAVOURABLE THANTHOSE IN INTERIOR.

3. THUS SYSTEM TRIES TO REDUCESURFACE AREA.

4. ADSORPTION OF DET TO SURFACEDISTURBS THE ORDERING OF WATER.

5. THIS LEADS TO A DROP IN SURFACETENSION AND A SPREADING OF THEDROPLET (20µl).

6. SYSTEM STABLE AFTER 30s.7. THIS SPREADING IS MEASURED BY THE

YOUNG CONTACT ANGLE.

SURFACE ACTIVE COMPOUNDS (GLYCEROL/PEG/LIPIDS) INTERFERE:CALIBRATE

A NOVEL METHOD FOR DETERGENT CONCENTRATION DETERMINATIONKAUFMANN ET AL 2006 BIOPHYS J. 90:1310-317

RAWIMAGES

THRESHOLDAPPLIED

DROPLETCONTOURS

CONTACTANGLES &

MEANVOLUME

ACCURACY OF CMC DETERMINATION

SURFACE TENSION RELATED TO HEADGROUP:

1. NON-IONIC DETERGENTS REDUCESURFACE TENSION MORE EFFECTIVELYTHAN CHARGED DETERGENTS

2. CHARGED DETERGENTS HAVE A LIMITEDCAPACITY TO BIND TO THE AIR:WATERINTERFACE (CHARGE REPULSION)

3. FOR LDAO AND OTHER IONIC DETERGENTSCMC IS DEPENDENT ON IONIC STRENGTH(AND pH). ELECTROSTATIC INTERACTIONOF COUNTERIONS.

4. THE BULKIER THE HEADGROUP, THE LESSTHEY REDUCE SURFACE TENSION.

5. INCREASED CHAIN REDUCES CMCCMC

CMC DETERMINATION

1. PROTEINS USUALLY PURIFIED IN EXCESS DETERGENT2. DETERGENTS WITH LARGE MICELLES (e.g. DDM) ENRICHED DURING PROTEIN

CONCENTRATION3. PROLONGED EXPOSURE REDUCES STABILITY4. HIGH CONCENTRATIONS INCREASE CRYSTALLIZATION TIME5. OVERSIZED MICELLES CAN INTERFERE WITH CRYSTAL CONTACTS

OPTIMIZING LIPID:PROTEIN RATIO

PROTEIN PURIFICATION: SUCROSE GRADIENT CENTRIFUGATION

IMPORTANT PROPERTIES OF DETERGENTSTO CONSIDER FOR PURIFICATION

1. PURITY (e.g. α and β DDM)2. ABILITY TO SOLUBILIZE LIPIDS3. PROTECT AGAINST DENATURATION4. LOW CONCENTRATIONS (COST)5. ABILITY TO EXCHANGE AND REMOVE

1 Fos-Fenth2 Nonopol-Fos3 Fos-choline4 Fos-choline5 Fos-choline6 Fos-choline7 C8-β-D-thioglucoside8 C7-β-D-glucoside9 C8-β-D-glucoside10 C9-β-D-glucoside11 C10-β-D-glucoside12 C8-β-D-thiomaltoside13 C8-β-D-maltoside14 C10-β-D-maltoside15 C11-β-D-maltoside16 C12-β-D-maltoside17 HEGA818 HEGA919 HEGA1020 HEGA1121 CYMAL322 CYMAL423 CYMAL524 CYMAL625 C8E426 C8E527 C10E428 C10E5

29 C12E430 C12E531 C12E1032 Brij 3533 Brij 7634 Brij 7835 Brij 9336 Brij 9637 CHAPS38 Big CHAPS39 DDAO 2640 Sodium cholate41 Sarkosyl42 Digitonin43 C7-β-D-thioglucoside45 C9-β-D-thioglucoside46 C10-β-D-thioglucoside48 C9-β-D-thiomaltoside49 C10-β-D-thiomaltoside50 C12-β-D-thiomaltoside51 Surfonyl 48552 Triton X-30553 Triton X-40554 Triton X-70555 Tween 2056 Brij 70057 PEG 4000 distearate58 PEG 6000 distearate

Relating Surfactant Properties to Activity and Solubilization of theHuman Adenosine A3 Receptor Berger BW et al 2005 Biophys J. 89: 452-462

Relating Surfactant Properties to Activity and Solubilization of theHuman Adenosine A3 Receptor Berger BW et al 2005 Biophys J. 89: 452-462

EFFICIENCY AND SELECTIVITY OF PURIFICATION LINKEDEFFICIENCY OF PURIFICATION LINKED TO CHAIN LENGTH

OGEG. TRITON’S

OTG

Relating Surfactant Properties to Activity and Solubilization of theHuman Adenosine A3 Receptor Berger BW et al 2005 Biophys J. 89: 452-462

11 C10-β-D-glucoside12 C8-β-D-thiomaltoside13 C8-β-D-maltoside14 C10-β-D-maltoside15 C11-β-D-maltoside16 C12-β-D-maltoside

26 C8E531 C12E1034 Brij 7836 Brij 9648 C9-β-D-thiomaltoside49 C10-β-D-thiomaltoside

HYDROPHILE-LIPOPHILE BALANCE (HLB)

1. HLB IS A MEASURE OF HYDROPHILIC CHARACTER OF THE DETERGENT2. THE LARGER THE HLB, THE MORE HYDROPHILIC IS THE DETERGENT.3. HLB VALUE OF A DETERGENT CORRELATES WITH ITS ABILITY TO SOLUBILIZE MEMBRANE PROTEINS.4. DETERGENTS WITH A HLB OF 12 TO 20 ARE PREFERRED FOR NON-DENATURING SOLUBILIZATION OF MEMBRANEPROTEINS).5. DETERGENTS WITH HLBS IN THE UPPER END OF THE ABOVE RANGE ARE PREFERRED FOR SOLUBILIZATION OFEXTRINSIC MEMBRANE PROTEINS.

HLB = 20(1- ML/MT) where ML = MW of the hydrophobic part of the molecule and MT = total MW.

W. C. GRIFFIN. CLASSIFICATION OF SURFACE-ACTIVE AGENT BYHLB. J. SOC. COSMETIC CHEM., 1:311, 1949.

W. C. GRIFFIN. CALCULATION OF HLB VALUES OF NON-IONICSURFACTANTS. J. SOC. COSMETIC CHEM., 5:259, 1954.

DETERGENT-PROTEIN AND DETERGENT-LIPID INTERACTIONS: IMPLICATIONS FOR TWO-DIMENSIONAL CRYSTALLIZATION OF MEMBRANE PROTEINS AND DEVELOPMENT OF TOOLS FORHIGH THROUGHPUT CRYSTALLOGRAPHY, THOMAS KAUFMANN DISSERTATION

PURIFICATION: SUBUNIT COMPOSITION AND OLIGOMERIC STATE

DETERGENTS: PROTEIN STABILIZATION

PROTEIN: DETERGENT MICELLE

IMPORTANT PROPERTIES OF DETERGENTSTO CONSIDER FOR STABILIZATION

1. PURITY2. ABILITY TO PROTECT AGAINST SUBUNIT DISSOCIATION3. ABILITY TO PROTECT AGAINST SUBUNIT UNFOLDING4. PREVENT EXPOSURE OF TRANSMEMBRANE DOMAIN5. LOW CONCENTRATIONS (COST)6. EXCHANGABILITY

DETERGENT STATISTICS (MICHAEL LANDSBERG)140 PUBLICATIONS 51 PROTEINS OR COMPLEXES 79 STRUCTURES

MOST COMMONLY USED DETERGENTS

6Å OR BETTER: ACh receptor, AQP-0, AQP-1, LHC-II, AQP-2, AQP-4, hR, connexin, GST, bR and PhoE data not included yet.

Purification

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE

Purification

02468

101214

TDM

Twee

n 20

C12

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DM

Trito

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100

DD

SLD

AOD

HPC DM DS

deox

ycho

late NG

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EC

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OTG

chol

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Hec

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HTG

none

OG

Purification

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE

DM

Tween 20

DETERGENT EXCHANGE

1. HYDROPHOBIC ADSORPTION (EG. BIOBEADS, CALBIOSORB)2. GEL CHROMATOGRAPHY3. DIALYSIS4. ION EXCHANGE CHROMATOGRAPHY

METHODS REVIEWED IN: A GUIDE TO THE PROPERTIES AND USES OF DETERGENTS IN BIOLOGY AND BIOCHEMISTRY CALBIOCHEM.

DETERGENTS: CRYSTALLIZATION

IMPORTANT PROPERTIES OF DETERGENTS TO CONSIDER FOR CRYSTALLIZATION 1. PURITY2. EASE OF REMOVAL BY DILUTION, DIALYSIS OR BIOBEADS3. IN MONOLAYER CRYSTALLIZATION – EFFECT ON MONOLAYER STABILITY.4. EFFECT OF TEMPERATURE ON HEADGROUP BEHAVIOUR (E.G. TRITON X-114)5. EFFECT OF COUNTERIONS ON HEADGROUP (IONIC DETERGENTS)6. LONGER CHAIN LENGTHS CAN BE USED THAN FOR 3D

GUIDELINES FOR CHOOSING A DETERGENT1. CHECK LITERATURE.2. NON-IONIC DETERGENTS PERHAPS THE GENTLEST.3. ASSAY FOR ACTIVITY, STABILITY (SEE Yeh et al 2006 Acta Cryst D62:451-457).

- SUGGESTS COUPLING PCR TEMP SYSTEM WITH A INTRINSIC FLUORESCENCE OFAROMATIC GROUPS (E.G. Phe, Tyr, Trp).

DETERGENT STATISTICS (MICHAEL LANDSBERG)140 PUBLICATIONS 51 PROTEINS OR COMPLEXES 79 STRUCTURES

MOST COMMONLY USED DETERGENTS

6Å OR BETTER: ACh receptor, AQP-0, AQP-1, LHC-II, AQP-2, AQP-4, hR, connexin, GST, bR and PhoE data not included yet.

Purification

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE

Crystallization

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE

Purification

02468

101214

TDM

Twee

n 20

C12

E8D

DM

Trito

n X-

100

DD

SLD

AOD

HPC DM DS

deox

ycho

late NG

octy

l-PO

EC

8E4

OTG

chol

ate

OG

Hec

ameg

HTG

none

Crystallisation

02468

10121416

TDM

Twee

n 20

C12

E8D

DM

Trito

n X-

100

DD

SLD

AOD

HPC DM DS

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late NG

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l-PO

EC

8E4

OTG

chol

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OG

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HTG

none

OG

Purification

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE

Crystallization

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE

DM

Tween 20

OG

DM

NG

OGEG. TRITON’S

OTG

11 C10-β-D-glucoside12 C8-β-D-thiomaltoside13 C8-β-D-maltoside14 C10-β-D-maltoside15 C11-β-D-maltoside16 C12-β-D-maltoside

26 C8E531 C12E1034 Brij 7836 Brij 9648 C9-β-D-thiomaltoside49 C10-β-D-thiomaltoside

TYPICAL FACTORS OPTIMIZED:DETERGENT

HTG

1.Important properties of detergents for membrane protein crystallization

PIC OF SMALL VESICLES AND BIG SHEETS FROM MICHAEL

BERGER ET AL (2005) THE ROLE OFPROTEIN AND SURFACTANTINTERACTIONS IN MEMBRANE-PROTEINCRYSTALLIZATION. ACTA CRYST D61:724

USED SELF INTERACTIONCHROMATOGRAPHY TO CALCULATEB22

1 Fos-Fenth2 Nonopol-Fos3 Fos-choline4 Fos-choline5 Fos-choline6 Fos-choline7 C8-β-D-thioglucoside8 C7-β-D-glucoside9 C8-β-D-glucoside10 C9-β-D-glucoside11 C10-β-D-glucoside12 C8-β-D-thiomaltoside13 C8-β-D-maltoside14 C10-β-D-maltoside15 C11-β-D-maltoside16 C12-β-D-maltoside17 HEGA818 HEGA919 HEGA1020 HEGA1121 CYMAL322 CYMAL423 CYMAL524 CYMAL625 C8E426 C8E527 C10E428 C10E5

29 C12E430 C12E531 C12E1032 Brij 3533 Brij 7634 Brij 7835 Brij 9336 Brij 9637 CHAPS38 Big CHAPS39 DDAO 2640 Sodium cholate41 Sarkosyl42 Digitonin43 C7-β-D-thioglucoside45 C9-β-D-thioglucoside46 C10-β-D-thioglucoside48 C9-β-D-thiomaltoside49 C10-β-D-thiomaltoside50 C12-β-D-thiomaltoside51 Surfonyl 48552 Triton X-30553 Triton X-40554 Triton X-70555 Tween 2056 Brij 70057 PEG 4000 distearate58 PEG 6000 distearate

Relating Surfactant Properties to Activity and Solubilization of theHuman Adenosine A3 Receptor Berger BW et al 2005 Biophys J. 89: 452-462

Purification

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE

Crystallization

0

2

4

6

Twee

n 20

Trito

n X-

100

DH

PC DM NG OG

NO

NE