Analytical Ultracentrifugation for the characterization of ... · I. Analytical Ultracentrifugation and the characterization of solutions of solubilized MPs. Instrumentation and theory

Analytical Ultracentrifugationfor the characterization of

membrane proteinsbefore crystallization

Christine Ebel

Institut de Biologie Structurale,Laboratoire de Biophysique Moléculaire

UMR 5075 CEA-CNRS-UJF Grenoble France

SolvationStability

SolubilityProtein-protein interactions

Solvent, co-solvent, partner?

IFNg-Heparin

Halophilic proteins

•Molecular adaptation to high salt•Structural studies of complex systems

Membrane proteins

Properties of the solutions prior to macromoleculecrystallization?

For soluble proteins:

-Homogeneity

-Resulting for the presence of theprecipitating agent

=>weakly attractive attractionsbetween macromolecules

Complexity of solutions of membraneproteins

-composition of the solutions? homogeneity?Stability?

-Association state of the membrane proteinin crystallization condition.

-And about the weak interactions incrystallization conditions?

I. Analytical Ultracentrifugation and thecharacterization of solutions of solubilized MPs.Instrumentation and theory

II. Sedimentation velocity for sample homogeneity

III. AUC and particle composition:

IV. Weak interactions and AUC

Centrifugal field: F=mw2rw=60000 rpm; r=6-7cm =>300 000g

6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 7

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

abso

rban

ce [O

D]

Abs

orba

nce

radius5,8 5,9 6,0 6,1

Radius

Abso

rban

ce

Velocity: s , D :Mb/RH, RH Equilibrium: s / D :Mb=M(1-rv)

t: 24h

Recent data treatments are based on numerical solutions of the Lamm equationIf interacting particle: s and D are concentration dependant s;D=f(si;Di;ci)

t: 2h

s D QSedimentation Diffusion Chemicalcoefficient coefficient reaction

(¶c/¶t) = - 1/r . ¶/¶r [r(c s w2t - D. ¶c/¶r)] + Q

6.56.2 6.8

For an homogeneous ideal solute:

Transport equation: For each solute

fNvMs

A

)1( r-=

massbuoyant mass

Mb

D =RT / NA.f

f=6 p h RH

RH= f/f° R°

velocityof the particles

Parachutes : hppt://www.smm.org

s

spreadingfriction

particle distributionat equilibrium

relative density

shape, viscosity

0 2 4 6 8 10

0

2

4

6

8

10

12

BR-Dapol/bufferH s=1-10

s

c(s)

f72ip2 f72ra2l555 f72ra2l280

6,0 6,5 7,0Rayon

-0,50,00,51,01,52,0

Abs

orba

nce

Analytical ultracentrifugation


II. Sedimentation velocity for sample homogeneity1. BmrA; 2. Hupon; 3. BR/Apol

III. AUC and particle composition


BmrA de Bacillus subtilis

X Data

6.2 6.4 6.6 6.8 7.00.0

0.5

radius (cm)6.2 6.4 6.6 6.8 7.0

resi

dual

s

-0.040.000.04

A(2

80 n

m)

s20,w (S)0 10 20 30 40 50

c(s)

0.0

0.5

1.0

A

B

C

BmrA 0.25 mg/ml 0.02%C12E8

6.2 6.4 6.6 6.8 7.0

0.0

0.5

radius (cm)6.2 6.4 6.6 6.8 7.0

resi

dual

s

-0.040.000.04

5 10 15 20

c(s)

0.0

0.5

1.0

A(2

76 n

m)

s20,w (S)

A

B

C

BmrA, 0.05%DDM

0.13, 0.5, 1.2 mg/ml

Ravaud et al (2006) Biochem. J.

0

2 4 6 8s (S)

c(s)

6 µM HUPON17,29mM C12E8140mM NaCl

6 µM HUPON11.54 mM C12E8280 mM NaCl

6 µM HUPON 10.81mM C12E8,280 mM NaCl

18 µM HUPON10.36 mMC12E8*

6 µM HuPON10.36 mMC12E8*

C12E8 ↓ HuPON ↑

Human Paroxonase

Josse et al (2002) J. Biol. Chem.

s20,w (S)3 5 7

0

1

radius (cm)6.5 7.0

A28

0

0

1

s20,w (S)5 10 15 20

c(s)

/c(s

) max

0

1

Bacteriorhodopsin trapped in Amphipol

Gohon et al (under correction) Biophys. J.



III. AUC and particle composition1: BmrA; 2: new protocols in AUC; 3: AAC; 4: AcrB


6.2 6.4 6.6 6.8 7.0

0.0

0.5

radius (cm)6.2 6.4 6.6 6.8 7.0

resi

dual

s

-0.040.000.04

5 10 15 20

c(s)

0.0

0.5

1.0

A(2

76 n

m)

s20,w (S)

A

B

C

SV s20,w=8.9S

R6N)v1(M

fNMs

HAA

b

phr-

==

TLCδphospholip. = 0.07 g/g

r2/2 (cm2)

Abs

(1cm

, 279

nm)

0

1

2

22 23 24 25

resi

dual

s

-0.04

0.00

0.04

A

B

SE Mb=50kDa

BmrA, 0.05%DDMChr omatography of sample E2

500

1000

1500

2000

2500

0 2 4 6 8 10 12 14

Elut ion t ime

-2,00E-02

0,00E+00

2,00E- 02

4,00E- 02

6,00E- 02

8,00E- 02

1,00E- 01

1,20E- 01

[14C]DDM

RH=5.6nm δdet= 1,5 g/g

SEC

A280

M =110 kDa

Dimer: M = 132 kDa

s + RH Mb=47 kDa

+ δdet, δlip

BmrA is an active dimer in solution

SE Mb=50 kDa

0.13, 0.5, 1.2 mg/ml

Ravaud et al (2006) Biochem. J.

6,0 6,2 6,4 6,6 6,8 7,0 7,2

0

2

4

6 2A

Inte

rfere

nce

fring

es

radius (cm)

6,0 6,2 6,4 6,6 6,8 7,0 7,2

-0,2

0,0

0,2 2B

Inte

rfere

nce

fring

es

radius (cm)

Ca2+ATPase

6,0 6,2 6,4 6,6 6,8 7,0 7,2

0,0

0,4

0,8

1,2 1A

Abs

orba

nce

at 2

80 n

m

radius (cm)

0

1

2

3

4

5

6

1 3 5 7 9 11 13 15

sedimentation coefficient (S)

conc

entra

tion

(µM

)

Ca++ATPase100 mol DDM

c(s) analysis Schuck (2000)Multil analysis Balbo (2005)

==> Ca++-ATPase is a monomer with 0.75 - 0.9 g/g DDM

-Combining SV obtained with differentoptics-Varying the solvent density in SV-Combining with results from SEC

(Rs; bound detergent)

New protocols in Analytical Ultracentrifugation

Salvay & Ebel, Prog. in Colloid and Polym. Sci. (2006)AUC for the characterization of detergent in solution.

Salvay et al.submitted

The mitochondrial ADP–ATP carrier

Up to 10% of mito.membrane proteins

Particularly enriched in energydemanding tissuese.g. cardiac muscle

P 21 21 2 - 100 mM salt

Too far awayno interaction

Crystallography

Pebay-Peyroula et al., Nature, 2003C 2 2 21 - low saltNury et al., Febs Letters, 2004

CATR bindingstoechiometry

AUC Crosslink

2D crystals3D atomicstructure

1978 1980 1982 1995 2000 2003 2006 2007

SANSChimericdimers

?

dimer

SESEC

Negativedominance

Differentialtagging

monomer

2007

Bamber, Kunji, et al.

2005

Pebay-Peyroula, Nury, et col.

1000 detergents (10g/g)

1: Coupled AUC and SANS experiments in LAPAO

Similar to:

Same purification protocol as for crystal growth

Same samples for both techniques

Block et al., BBRC, 1982 (SANS)

The mixture is complex1 protein

170 lipids (3g/g)

LAPAO-lipids mixtures

H2O buffer D2O buffer

18 hours at 42000 rpm3 mm path length cells20°C

s = 0 S s = -0.75 S at 20°C

Behaviour is similar with or without treatment with Biobeads

LAPAO/lipids: v̅d+l close to 1.00 mL/g

LAPAO : v̅ d = 1.002 mL/g. Compatible with globular micelles with Nagg= 125

A 280

A 280

H2O

radius (cm)0

0.5

1

0 1 4

A 280

nm

5 radius (cm)

s [-0.2 -0.1]

D2O

0

1

2

0 1 4 5

0.06

-0.06

s = 1.06 S

D = 4.2 F

J/A = 7.23

δdet+lip=1.6 - 2.2 g/g

RH= 38 ± 2 Å

Mb= 6 ± 0.3 kDa

Calculated for Monomer

RH= 41 Å

Mb= 8.4 kDa

SV of AAC in H2O Solvent

If the shape of AAC is the same inH20 and D2O, (B,v) must be the

same in both solvents A28

0

AAC in H2O buffer AAC in D2O buffer

s=1.06S s [-0.2 -0.1]

H O2

D O2

Monomer

0.98 1.140.9

1.9

2.9

Bde

t+lip

(g/g

)

vdet+lip

AUC clearly indicates that AACin LAPAO is a monomer

incertitude on s1.25<f/fmin<1.5

Dimer

0.9

2.9

0.981.14

Bde

t+lip

(g/g

)

v

SANSRg ≈ 30 Å Mono: 20 Å Dim.: 30 ÅM ≈ 40 kDa Manon: 32 kDa Dim.: 65 kDa

calculated /estimated

SV in H2O and D2O solvents

6.0 6.2 6.4 6.6 6.8 7.00.0

0.5

radius (cm)6 7resi

dual

s

-0.03

0.03

s20,w (S)2 6 10

c(s)

0.0

0.5

A27

8

A

B

E

6.0 6.2 6.4 6.6 6.8 7.00.5

1.0

1.5

radius (cm)6 7resi

dual

s

-0.03

0.03

A27

8

C

D

6 70.0

0.5

1.0

radius (cm)

frin

ge s

hift

FA278fringe shift

Absorbance interference

Absorbance in TpD

Mean s= 5S

Calculated with f/fmin=1.25:monomer : s=2.6±0.1Sdimer : s=4.1±0.1Strimer : s=5.4±0.1Stetramer: : s=6.9±0.25S.

From J, A, sH, sD :BDet= 1.5 g/g; Blip=1 g/g

Compatible withBDet= 1.5 g/g and Blip=0.25 g/g

determined by Hackenberg (1980).

2: AAC/Triton X100

Hackenberg (1980)

simulation≈ same cond.: s=3.9; D=3.4 10-7 cm2.s-1

AUC clearly indicatesthat AAC in Triton X-

100 is a mixture ofmultimers

5.6 µM CusA, 6.7 mM FC14, 4°C

5.6 µM CusA, 2.1 mM DDM 6°C

4.9 µM AcrB, 0.7 mM DDM 6°C

Crystallizes

Does not crystallize

Stroebel, Sendra, Cannella, Helbig, Nies, Coves, BBA, 2007

CusA (E. coli) : Cu transport

homotrimeric AcrB

Does not crystallize

AcrB and CusA

Conclusion of Stroebel et al.

-Distribution of multimer rather insensitive to detergentconcentrationÞsoluble domains responsible for PP interactions?

Þ- Pseudo heterogeneity of the AcrB preparation isnot a contra-indication for crystallization.Þ-reach a protein concentration that allows theformation of multimer that could trigger the nucleation?

homotrimeric AcrB



III. AUC and particle composition

IV. Weak interactions and AUCConclusion: Weak interactions, membrane proteins, AUC and SANS

( )( ) kTrW

22

e)r(gdrr)r(g1A

-=ò -µ

functionncorrelatiopair:)r(g

potentialeractionintparticleparticlemean:)r(W -

Non ideality, Why andHow? Measurement of A2

•Osmotic pressure•Static light scattering•Small angle X-rays scattering•Small angle neutron scattering•Equilibrium sedimentation

2A2M» ks + kD•Sedimentation velocity

- sedimentation coefficient- diffusion coefficient

1/M* = 1/M + 2A2c + ...

s = s° / (1+ ks c + …)D = D° . (1+ kD c + …)

A2: ml.mol.g-2; 2A2M: ml.g-1

sedimentation velocity when cs

Repulsion between particles

In an intuitive way

Larger objects at higher concentration:

Attraction between particles

is similar to auto-association

s when c

M* , size , D when c

Non ideality, Why andHow?

Sedimentation velocityfor the characterization of non-ideal systems?

Local s and D introduced in the Lamm equationin a modification of the program Sedfit (Schuck, 1998)

Direct boundary modeling by Lamm equation solution

XLI available in the laboratory, rapid method,recoverable protein, complex solvent.

Solovyova et al, Biophys. J. 2001

Non ideality, Why and How?

Fitting the exp. profiles of hMalDH 12.3 mg/ml in 4 M NaCl.

-0,2

0,0

0,2

resi

dual

s

-0,2

0,0

0,2

resi

dual

s

ks= kD= 0=>s=2.06S=> « D » =2.0 107cm2s-1

rmsd= 0.059 fringe

ks ¹ 0; kD¹ 0=>s°=2.37S=>D°=3.2 107cm2s-1

=>kS=12ml/g; kD=1ml/grmsd= 0.039 fringe

6,0 6,2 6,4 6,6 6,8 7,00

2

4

6

8

10(b)

frin

ges

radius (cm)

6,0 6,2 6,4 6,6 6,8 7,00

2

4

6

8

10(a)

frin

ges

radius (cm)

Non ideality, Why and How?

hMalDH from non-ideal fitting of sedimentation profiles and other methods

s° D° M(¶r¤¶c) M(¶r¤¶c) r° ks kD 2A2M 2A2Min NaCl (ks+kD) (SANS)

S 107cm2s-1 kg/mol kg/mol g/ml ml/g ml/g ml/g ml/g4 M NaCl

non-ideal fitting 2.36 3.0 19 21 1.153 9 2 11 9mean s and D 2.41 2.6 23 14 3 17

5 % MPD, 2 M NaClnon-ideal fitting 3.8 3.1 30 32 1.079 7 6 13 7mean s and D 3.8 8

30 % MPD, 1.5 M NaClnon-ideal fitting 1.74 1.1 39 39 1.047 -14 -4 -18 -26mean s and D 1.73 1.5 28 -13 -2 -15

Comparison of s° and ksobtained from linearextrapolation:

s = s° . (1 - ks c + …)1.6

2.4

3.2

4

0 2 4 6 8 10 12

s (S)

c (mg/ml)

4 M NaCl

30% MPD, 1.5 M NaCl

5% MPD, 2 M NaCl








Comparison of D° and kDobtained from QELS:

D = D° . (1 + kD c + …)

0

1

2

3

4

0 10 20 30 40 50

D (1

07 cm2 s-1

)

c (mg/ml)

4 M NaCl

30% MPD, 1.5 M NaCl








Caracterization of the weak interparticle interactions

•2A2M from SANS in the same order of magnitude (Costenaro et al., 2002)• In 4M NaCl, ks, kD and 2A2M compatible with excluded volume effects(RH=41Å, largest dimension of the crystal: 40Å)• In 30%MPD 1.5M NaCl, A2» -8 10-5 ml/mol.g-2, moderatly attractive conditions,nice crystals.• kD<<ks 2A2M≈ ks

0

5E-5

0 1 2 3 4 5

C3

Second virial coefficient :

: NaClO: MgCl2D: (NH4)2SO4

A2>0

A2<0A2= a22

(e) - a322/a33

?

(¶m3/¶m2)m, preferentialbinding parameter :

-200

-100

0

100

0 0,1m3/m1

Solvation and weak protein-protein interactions?

Conclusion

- discriminate P-Pand D-D

interactionsin crystallization

conditions

- SV and/or SANS

Acknowledgments

Gérard Brandolin - iRTSV GrenobleGuy Lauquin, Veronique Treguezet, Bertrand Arnoux - IBGC Bordeaux

Marc le Maire, Marie Jidenko Cea, Saclay

Stéphanie Ravaud, Attilio di Pietro, Richard Haser, Nushin Aghajari, IBCP Lyon

IBS Andres SalvayLBM Georgy Pavlov

Frank GabelAlexandra SolovyovaLionel Costenaro

Jean-Luc Popot, Yann Gohon, D. Charvolin, Tassadite Dahmane, F. Rappaport- IBPC ParisChristophe Tribet - ESPCI Paris R. Ruigrok - EMBL GrenobleP. Timmins - ILL Grenoble D. Engelman - Yale U. USA

IBS Jean-Michel Jault

Eva Pebay PeyroulaHugues Nury

David Stroebel, Denis Josse, CRESSA Grenoble

P Schuck - NIH Bethesda USA

Documents

Analytical Ultracentrifugation for the characterization of ... · I. Analytical Ultracentrifugation and the characterization of solutions of solubilized MPs. Instrumentation and theory