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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013
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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013
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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013
Main schematics of beamline SWING
• Structural Biology (macromolecular shapes /
low resolution structure)
• Soft Condensed Matter (crystal growth,
colloids, polymers, liquid crystals, hierarchical
systems, …)
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013
• 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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013
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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013
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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013
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 ?
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • Actin complexes with β-thymosin peptides
HPLC with no compound in the buffer
How to deal with low affinity complexes
Collab : Louis Renault (LEBS) and Pierre Roblin (LEBS INRA/SOLEIL)
- =
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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • Actin complexes with β-thymosin peptides
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
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
+ biochemistry shows sequestering activity + biochemistry shows profilin-like activity
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • Actin complexes with β-thymosin peptides
• 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.
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
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 ?
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • AQP0 : a transmembrane protein solubilized in a detergent solution
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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • AQP0 : a transmembrane protein solubilized in a detergent solution
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
NSRRC Annual Meeting, Hsinchu, Sept. 4-5, 2013 • AQP0 : a transmembrane protein solubilized in a detergent solution
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