what the beamline swing can bring to structural...

NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013

Javier Pérez

Synchrotron SOLEIL

SWING

What the beamline SWING can bring to

structural biology : A few (uncommon)

SAXS studies on macromolecular

complexes


Energy : 2,75 GeV

Periphery : 354,1m

Current : 430 mA (500 mA)

Vertical emittance : 37,3 pm x rad

Horizontal emittance : 3,73 nm x rad

24 Straight sections: 4 x L (12m); 12 x M (7 m); 8 x C (3.6 m)

Synchrotron SOLEIL

How it looks inside


I0, l

IT

Detected : I(Q)

ii ek

l

2

if kkQ

fk

2θ

Sample

C > 0.1 mg/ml

V > 10 µl 2D detector

Ln ( I )

Q=4π sinq / l

Radial average

(isotropic sample)

SAXS provides structural information about macromolecules in solution

• Limits

• spherically averaged information low resolution

• does not distinguish elements in a mixture

• non unicity of the solution

SAXS is at its best when complementary (structural) information is available

•Advantages

• solution ( no crystal ) kinetics, titration, T°, P

• relatively easy to carry experiments

• can be checked against atomic models

Principles of Bio-Small Angle X-ray Scattering in solution


Main schematics of beamline SWING

• Structural Biology (macromolecular shapes /

low resolution structure)

• Soft Condensed Matter (crystal growth,

colloids, polymers, liquid crystals, hierarchical

systems, …)


• Courtesy Laboratoire de Physique du Solide (Orsay, France)

Home made Thermostated Couette Cell with torque measurements

Thermostated Anton Paar Rheometer

• SOLEIL

Biologic SFM400 Stopped-Flow for chemistry (courtesy beamline ODE)

Biologic SFM400 Stopped-Flow for biology (courtesy Biology Lab)

In-vacuum Automated sampler with T° control

Linkam Hot stage (T < 600°C)

Circulation cell for proteins in solution, with Peltier T° control & UV-Vis Abs

On-line HPLC coupled with preparator and injector for protein solution samples

Several available sample environments


Set-up for SEC-SAXS at Beamline SWING

Online UV-Vis

Sample

circulation

RX

G. David and J. Pérez (2009), J. Appl. Cryst

Incident

Beam

Incident

Beam

Since 2008


I(0) and Rg determined for each SAXS frame during elution

I(0)

Rg

V0 ASNP elution profile, monitored by UV absoprtion at 280 nm

ASNP with HPLC

Rg = 25.7 Å

Q.Rgmin = 0.657 / Q.Rgmax = 1.09

ASNP direct injection

Rg = 29.1 Å

Q.Rgmin = 0.659 / Q.Rgmax = 1.19

Comparison between HPLC-purified and Direct injection curves

Q / A-1

Typical advantages from the coupled HPLC / SAXS device

Elution time (minutes)

Frame number ( ~ time)

• Mono disperse Solution

• Separation of aggregates

• Perfect background subtraction

HPLC-purified experimental curve

Curve calculated from crystal structure

Fitting the HPLC-purified experimental curve with the crystal structure

Q / A-1


Outline

• Actin complexes with β-thymosin peptides

• AQP0 : a transmembrane protein solubilized in a detergent

solution

NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • Actin complexes with β-thymosin peptides

Cib-D1 (profilin like) Tβ4 (sequestering) Pointed face

Barbed face

Sequestering vs polymerising (profilin-like) properties of peptides able to bind actin

Complexes between actine and β-thymosin peptides

Collab : Louis Renault (LEBS) and Pierre Roblin (LEBS INRA/SOLEIL)

Didry et al. (2012), EMBO J. 31, 1000-1013

In physiological conditions

Crystallography shows no Cter density map

C-ter ?

Hydrogen bound Salt bridge

Cib-D1 (profilin like) TB4 (sequestering)

N-ter helix

Technical problem : How to collect SAXS data on low affinity complexes (KD > µM) ?

Is there a difference in the average positioning of this mobile part that would be linked

with the activity, and can it be seen by SAXS ?


HPLC with no compound in the buffer

How to deal with low affinity complexes


- =

Aggregates

Complex A-B A Buffer

dissociation

Scattering curve with three contributions (complex, compoud A and B)

+

+

Gel filtration column equilibrated with buffer alone

In the capillary

B

Equilibrum strongly displaced to complexed form

Buffer +

Compound B

- =

HPLC with compound in the buffer

Complex maintained

Scattering curve of the complexed form alone

Aggregates

Complex A-B

+ +

Gel filtration column equilibrated with compound B in the buffer

In the capillary

Reviewed in J. Pérez and Y. Nishino (2012), COSB, 22:670–678


Low salt: G-Buffer (usual to avoid spontaneous polymerisation)

Tβ4 Low [salt]

Cib-D1 Low [salt]

High salt: F-Buffer (closer to physiological conditions)

Cib-D1 High [salt]

Tβ4 High [salt]

+ NMR shows low C-Ter mobility (C. van Heijenoort) + NMR shows high C-Ter mobility

Tβ4 Cib-D1

fit the data

Tβ4

fits the data

Cib-D1



Didry et al. (2012), EMBO J. 31, 1000-1013

+ biochemistry shows sequestering activity + biochemistry shows profilin-like activity


• The ionic strength plays a crucial role in the activity of actin-binding

peptides

• For profilin-like peptides :

• The screening of the electrostatic interactions leads to the

detachment of the C-terminal part from the actin pointed end:

barbed-end polymerisation

• For sequestering peptides :

• The salt bridge present in the T-beta-4 complex forces the C-

terminal part to the surface of actin whatever the ionic strength:

Inhibits polymerisation

Conclusion : The presence / absence of the salt bridge appears to direct the C-

terminal positioning, which seems to be a clue to determine the peptide function.



Didry et al. (2012), EMBO J. 31, 1000-1013

NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • AQP0 : a transmembrane protein solubilized in a detergent solution

Idea: apply SAXS to Membrane proteins

One conformation A

One conformation B

• Membrane proteins undergo conformational changes

• SAXS is good at monitoring conformation changes

• How can we use SAXS with a membrane protein of known structure ?

• How can we use SAXS to monitor membrane proteins

conformation changes ?


Optical properties of the

cystallin lens

1. Transparent to light

2. Biconvex Lens

3. Accommodation

Very peculiar cellular architecture

Our system : Aquaporin-0 from Eye Crystallin Lens

cornea

Crystallin lens


Straub et al., 2003

50 µm

Transport + Adhesion mediated through membrane proteins

Eye Crystallin Lens

No organelles, avascular tissue.

Two differenciated fibrillar cell structures

No cell elimination : cell compaction/maturation

Transport system : nutrient supply, cellular

waste products, volume control

Ions and water flux

core cortex

Donaldson et al., 2001


Crystalline lens (eye)

AQP0 (ex-MIP)

60 % of the membrane

protein content

Natively tetramer

Water transport across

cell membranes

Gonen et al., Nature 2004

Purification

• From bovine eye to lens membrane

• From lens membrane to AQP0 in solution

• Detergent:

Dodecyl-β-D-maltopyranoside (DDM)

Concentration until 4 mg/ml (2ml)

Aquaporin-0

Full AQP0, from cortex

Tetramer

Truncated AQP0, from core

Octamer : does it exist in

solution ??

Two types of known existing states

3D already obtained

2 problems for SAXS:

• Mixture

• Detergent belt

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