Trapping membrane proteins with amphipols. St t d ti f b t i / hi l … · 2015. 6. 10. · • Bacteriorhodopsin: very sm all transmembrane movements; no or very limited effects

March 8, 2010 – Paris

Workshop: Applications of Amphipols to Membrane protein Studies

Trapping membrane proteins with amphipols.

St t d ti f b t i / hi l lStructure and properties of membrane protein/amphipol complexes

M l ZManuela ZoonensCIMR, University of Cambridge, UK

CNRS, Institut de Biologie Physico-Chimique, France

Contact: [email protected]@g

1) How to trap a membrane protein (MP) in amphipols (APols)?

• Direct extraction of MPs from the biological membrane has been observed in a very few cases



• Usually, MPs are extracted and purified in detergent and then they are transferred in APol



• Usually, MPs are extracted and purified in detergent and then they are transferred in APol

Detergent

Protein

Protein/detergentcomplexes

Detergent

AmphipolStep 1

Protein/detergent/amphipolternary complexes

100

FApol C8E4

Protein/amphipol

Bio-BeadsStep 2Dilution under the cmc

0

60

80

% O

D

complexes

SpinStep 3dilution/concentration cycles0 20 40 60 80 100 120

0

20

40%

dil/conc cycles

Protein/amphipolcomplexes

Time (min.)

1) How to define conditions for trapping a MP in APols?

• Estimation of the best [protein:APol] w/w ratio

before centri after centriTrapping after dilution under the cmc

Estimation of the best [protein:APol] w/w ratio

25

30

35

40

(a.u

.)

Measure the absorbance at 280nm

10

15

20

25

OD

at 2

80 n

m

High speed centrifugation

Measure the absorbance at 280nm

T + T - [1:0,5] [1:1] [1:2] [1:3] [1:4]0

5

OSamples

Ref -Ref +

absorbance at 280nm

Samples

best ratio

• Analyze the resulting complexes by size exclusion chromatography (SEC)a y e t e esu t g co p e es by s e e c us o c o atog ap y (S C)

2) What do MP/APol complexes look like after trapping?

1 MP/AP l l li htl l th MP/d t t l (SEC SANS AUC NMR)1. MP/APol complexes are slightly larger than MP/detergent complexes (SEC, SANS, AUC, NMR)

60

50

bc1/C12-M

SEC (bc1, 486 kDa)SEC (tOmpA, 19 kDa)

30

40

50

280

nm (m

DO

)

30

40

415

nm (a

.u.)

bc1/A8-35

in detergent in detergent

in A8-35

in A8-35

0

10

20

Abso

ranc

e at

10

20

OD

at 4

Ve Vt

in A8-35

Ve Vt

5 10 15 20

Volume (ml)

Zoonens et al Biochemistry 2007

00 5 10 15 20 25

Volume (mL)

Charvolin et al in preparation

in A8 35+ detergent

Zoonens et al., Biochemistry, 2007 Charvolin et al., in preparation


1 MP/AP l l li htl l th MP/d t t l

pH effect Presence of divalent cations (Ca2+)

1. MP/APol complexes are slightly larger than MP/detergent complexes 2. The monodispersity of the complexes depends on several factors

14

8

10

12

14

m (a

.u.)

pH 6,5 - 0h pH 6,5 - 24h pH 7,9

V

2

4

6

OD

at 2

80nm Ve

Vt

5 10 15 20-2

0

Volume (ml)

Picard et al., Biochemistry, 2006

Hypothesis:Because the solubility of A8 35 is conferred by its charges lowering pH or the

Zoonens et al., Biochemistry, 2007

Because the solubility of A8-35 is conferred by its charges, lowering pH or the presence of divalent cations reduces the electrostatic repulsions between particles.

Divalent cations could also link up particles together and lead to aggregation.


1 MP/AP l l li htl l th MP/d t t l1. MP/APol complexes are slightly larger than MP/detergent complexes 2. The monodispersity of the complexes depends on several factors

5

Separating the complexes from extra free APol

Hypothesis:initial sample

3

4

5

280

nm (a

.u.)

ypBecause A8-35 is poorly dissociating, it has to be present in excess to efficiently compete with

protein/protein interactions

initial samplewithout free APol+ free APol

1

2

bsor

banc

e at

2

Ve Vt

5 10 15 20

0Ab

Volume (ml)

monodisperse polydisperse

Zoonens et al., Biochemistry, 2007



1. MP/APol complexes are slightly larger than MP/detergent complexes2. The monodispersity of the complexes depends on several factors3. Amphipols form a compact layer (1.5-2 nm) around the transmembrane surface of the protein (no diffuse corona; SANS, NMR, AUC)

SANS (BR, 27 kDa)NMR (tOmpA, 19 kDa)

110

115

105

110

115

105

110

115

105

pm)

125

115

120

125

115

120

125

115

120

15N

(p

Gohon et al Biophys J 2008

130.130.130.

10. 9.0 8.0 7.0 Gohon et al., Biophys. J., 2008

Zoonens et al., PNAS, 2005

0 1H (ppm)

Catoire et al., Eur Biophys J, 2010



1. MP/APol complexes are slightly larger than MP/detergent complexes 2. The monodispersity of the complexes depends on several factors3. Amphipols form a compact layer around the transmembrane surface of the protein4. Binding is non-covalent, but irreversible in the absence of a competing surfactant

FRET measurements after 1000-fold dilution in surfactant free buffer

Surface Plasmon Reasonancemeasurements upon extensive

washing

0C12-G

A8-35

surfactant-free buffer

in surfactant free buffer washing

NBD

280 nm

325 nm

533 nm

-20

-10

C8-G

C12-G

rbitr

ary

units

0 200 400 600-40

-30 C8-POE

Time(s)

ar

Zoonens et al., Biochemistry, 2007 Hong & Lakey; in Popot et al., CMLS, 2003



1. MP/APol complexes are slightly larger than MP/detergent complexes 2. The monodispersity of the complexes depends on several factors3. Amphipols form a compact layer around the transmembrane surface of the protein4. Binding is non-covalent, but irreversible in the absence of a competing surfactant5. Bound amphipols can be displaced by free amphipols, detergents, or lipids (Tribet et al., Langmuir, 1997; Nagy et al., FEBS Lett., 2001; Pocanschi et al., Biochemistry, 2006; Zoonens et al., Biochemistry, 2007; Tribet et al., Langmuir, 2009)

Displacement by detergentExchange for free amphipol

NBD NBD

Zoonens et al.,Biochemistry, 2007

3) How do MP/APol complexes behave in term of activity ?

1. Amphipols may affect the dynamics –and, thereby, the activity– of the proteins they bind to

• Nicotinic acetylcholine receptor: no transmembrane movement; allosteric transitions unaffected

In native membranes

Kinetics of binding

In native membranes

In detergent (CHAPS)

In A8-35

Martinez et al., FEBS Lett., 2002




• Bacteriorhodopsin: very small transmembrane movements; no or very limited effects on the photocycle

Photocycle of BR

Spectral changes

Neutze et al., BBA, 2002





Ground stateUV-visible spectra of BR

M intermediate

Gohon et al Biophys J 2008

DAS1 (ns)

DAS2 (μs)

DAS3 (μs)

DAS4 (ms)

DAS5 (ms)

PM 650 21 84 1.4 4.7

BR/OTG 375 4.3 23 0.53 9Gohon et al., Biophys. J., 2008

M’s rise M’s decay

BR/OTG 375 4.3 23 0.53 9

BR/A8-35 480 5.8 53 1.0 6.3





• Sarcoplasmic calcium ATPase: large-scale transmembrane rearrangements; ATP hydrolysis and Ca2+

release reversibly inhibited Champeil et al., JBC 2000; Picard et al., Biochemistry 2006

Hydrolytic activity of Ca2+ ATPase

C 2+ di i ti i tCa2+ dissociation experiment

Kühlbrandt, Nature Reviews Molecular Cell Biology, 2004





• Sarcoplasmic calcium ATPase: large-scale transmembrane rearrangements; ATP hydrolysis and Ca2+

release reversibly inhibited Champeil et al., JBC 2000; Picard et al., Biochemistry 2006

damping of large-scale transmembrane

movements (‘Gulliver effect’)?

free bound


1. Amphipols may affect the dynamics –and, thereby, the activity– of the proteins they bind to2. Damping of dynamics may contribute to membrane protein stabilization by amphipols

Bacteriorhodopsin Calcium ATPase

t0: chelation of Ca2+ by addition of EGTA

In APol

In detergent (OTG)

Tribet et al., (1996) PNAS 93, 15047

Leukotriene receptor BLT1 (GPCR)

Champeil et al., JBC 2000

Dahmane et al., Biochemistry, 2009


Could the 'Gulliver effect‘ contribute to stabilizing APol-trapped MPs against inactivation ?

1. Amphipols may affect the dynamics –and, thereby, the activity– of the proteins they bind to2. Damping of dynamics may contribute to membrane protein stabilization by amphipols

g pp g

Denaturation by detergents Stabilization by amphipols

Misfolded forms Misfolded forms

Opening of structure lesslikely ('Gulliver effect') Lesser dissociating

efficiency

↓(irreversible) (irreversible?)↓

Intermolecular associations Partially loosened Native form in Intermolecular associations Partially loosened APol-trapped

For discussion, see Popot et al., CMLS, 2003; Picard et al., Biochemistry, 2006.

→ aggregates (irreversible)form (reversible?)detergent solution → aggregates (irreversible?)form (reversible?)native form

In conclusion

• Protocol of trapping:

• Solution properties:

Usually, we transfer MPs from detergent solution to APols

MP/APol complexes are essentially homogeneous but the monodispersity p p

• Structural organization:

p y g p ydepends on the pH (higher than 7), the absence of divalent cations, and the presence of extra free APols

APols interact exclusively with the hydrophobic transmembrane surface of• Structural organization:

• Dynamcis of association:

APols interact exclusively with the hydrophobic transmembrane surface of MPs and form a compact layer of 1.5 to 2nm thickness

APols do not desorb from MPs but they exchange for other surfactants

• Activity:

(detergent, APols, or lipids)

It seems to depend on the amplitude of the transmembrane conformational changes occurring during the catalytic cycle of the protein of interest

• Stability:

changes occurring during the catalytic cycle of the protein of interest

MPs trapped in APols are generally more stable than in detergent solution

• Ligand binding: Generally unaffected by Apol trapping

Documents

Trapping membrane proteins with amphipols. St t d ti f b t i / hi l … · 2015. 6. 10. · • Bacteriorhodopsin: very sm all transmembrane movements; no or very limited effects