Sample preparation and characterization around SAXS

Rob Meijers

EMBL Hamburg

Experimental verification and… validation?

Garbage in

?

The right stuff

• Molecular weight

• Oligomerization state

• Monodispersity

• Aggregation

• Protein concentration

• Protein folding state

Quality controlSDS Page

Mass spec

Gel filtrationDLS/SLS

Circular Dichroism

1 2 1 2ConcentratedDilute Non‐reduced 

1

A gel of a heterodimeric receptor

1 2

A size exclusion profile

1 2 1 2ConcentratedDilute Non‐reduced 

1

A second look

Different gels 

• SDS 

• SDS non‐reduced

• Native

• Iso‐electric focussing

• Purity

• Disulphide bonds

• Post‐translational modifications:– Phosphorylation

– glycosylation

Protein concentration

• UV‐Vis

• Bradford

• Refractive index

Sample injected

Sampleprovided

Quality controlSDS Page

Gel filtration

Mass spec

Mass spectrometry

• MALDI‐TOF or ESI

• Integral sample

• Proteolytic digest:– Trypsin

– Carboxypeptidase

– Aminopeptidase

Mass spectrometry

• MS/MS + Ion mobility :– Detailed folding state

– Protein‐protein interactions

– Whole protein size…

Ion mobility derived particle size

2012‐10‐18 13

Ruotolo et al Nature Protocols (2008) 3, 1139

in combination with SAXS…

Sizing profile, SLS twist

Intramolecular interference produces a disymmetry in the scattered light.

Size of molecule/particle must be significant compared to wavelength of light

Angular Dependence

small moleculesradius < 15 nm Pθ = 1 for all θ

large moleculesradius > 15 nm Pθ= 1 for θ = 0°

Normalised LS signals show no angular dependence for proteins

– Molecular weight requires only RALS

– Can not measure size by light scattering alone

0.0

119.5

12.0

24.0

36.0

48.0

60.0

72.0

84.0

96.0

108.0

Rig

ht A

ngle

Lig

ht S

catte

ring

Res

pons

e (m

V)

0.0

56.7

6.0

12.0

18.0

24.0

30.0

36.0

42.0

48.0

Low

Ang

le L

ight

Sca

tterin

g R

espo

nse

(mV)

10.50 16.60Retention Volume (mL)

11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5

RALSLALS

Combined particle analysis

• UV

• Refractive index

• Viscosity

• Right angle light scattering

Viscotek/Malvern

What are the detectors responding to?

Refractometer =  KRI × dn/dc × Conc

UV‐Detector =  KUV × dA/dc × Conc

Viscometer  =  KVisc × Intrinsic Viscosity × Conc

Light Scattering =  KLS ×Mw × (dn/dc)2 × Conc

MonomerDimer

Trimer

0,0

1472,0

82,0

164,0

246,0

328,0

410,0

492,0

574,0

656,0

738,0

820,0

902,0

984,0

1066,0

1148,0

1230,0

1312,0

1394,0

Ref

ract

ive

Inde

x R

espo

nse

(mV

)

4,000

7,000

4,200

4,400

4,600

4,800

5,000

5,200

5,400

5,600

5,800

6,000

6,200

6,400

6,600

6,800

Log

Mol

ecul

ar W

eigh

t

0,0

239,0

14,0

28,0

42,0

56,0

70,0

84,0

98,0

112,0

126,0

140,0

154,0

168,0

182,0

196,0

210,0

224,0

Rig

ht A

ngle

Lig

ht S

catte

ring

Res

pons

e (m

V)

0,0

36,0

2,0

4,0

6,0

8,0

10,0

12,0

14,0

16,0

18,0

20,0

22,0

24,0

26,0

28,0

30,0

32,0

34,0V

isco

met

er D

P R

espo

nse

(mV

)

5,30 10,00Retention Volume (mL)

5,6 5,8 6,0 6,2 6,4 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 8,2 8,4 8,6 8,8 9,0 9,2 9,4 9,6

Monomer

DimerTrimer

High MW Aggregates(Mw > 1 Mio D)

Mw (D) IVw (dl/g) Rh (nm) Weight Fraction (%)

Monomer 66.430 0,056 3,88 87

Dimer 133.000 0,071 5,32 11

Trimer 201.000 0,095 6,69 1,5

Light Scattering Viscometer Refractive Index

BSA

LALS (7º) detector

RALS (90º)

RI detector

UV detector

GPC columns

Laser Light Scattering Detector, Refractive Index Detector and UV‐Cell

SEC + SAXS

• SWING beamline at Soleil

• HPLC in FPLC mode

Online purification & QC

• Combine SEC and SAXS

• Add:– Refractive index

– RALS

• Integrate in BioSAXSbeamline

Courtesy Melissa Graewert

UVRiLS

UV ~  c ɛLS ~ c (dn/dc)² MWRi ~ c dn/dc 

ml

Au

Courtesy Melissa Graewert

Frame 1 Frame 501

Frame 1001 Frame 1501 Frame 2001

I(O)

2500 frames collected (1 sec each) while proteins eluted from superdex 200 10/300 column @ 0.3 ml/min

Courtesy Melissa Graewert

Automation – how to handle > 2000 frames

Conventional pipe line (Autognom, Dammif, etc)

Detector images

Defining buffer region

Buffer substration

AutoRadius of Gyration

Peakdetection

Scaling and averaging

Radial averaging

Courtesy Melissa Graewert

Courtesy Melissa Graewert

0.0

197.0

20.0

40.0

60.0

80.0

100.0

120.0

140.0

160.0

180.0

Ref

ract

ive

Inde

x R

espo

nse

(mV

)

4.000

6.000

4.200

4.400

4.600

4.800

5.000

5.200

5.400

5.600

5.800

Log

Mol

ecul

ar W

eigh

t

10.50 16.60Retention Volume (mL)

11.5 12.0 12.5 13.0 13.5 14.0 14.5 15.0 15.5

Data File: 2005-08-04_12;51;19_BSA_01.vdt Method: Proteins SEC-0002.vcm

Dimer134 kDa

Trimer205 kDa

Monomer66 kDa

SAXS Rg/I(0) profile 

SEC elution profile

Monomer versus Dimer

Definedbuffer region

Courtesy Melissa Graewert

Integration of experimental data

• SAXS scattering curve

• Accurate protein concentration (UV‐VIS +RI)

• Molecular weight (RALS)

• Radius of gyration? (MALS)

Estimation of Particle Radius

H2O

H2OH2O

H2O

H2O

H2O

RgRh

Rg II

Rg

The micro‐world

Stokes‐Einstein relation

• D = Diffusion coefficient

• k = Boltzmann’s coefficient

• T = Temperature

• η = Viscosity

• R = hydrodynamic radius

Dynamic light scattering

Dynamic Light Scattering scheme

Sample

Detector

Correlator

Transmitted light

DiffusedLight

Information from the correlation curve

Integration of experimental data

• SAXS scattering curve

• Accurate protein concentration (UV‐VIS +RI)

• Molecular weight (RALS)

• Radius of gyration? (MALS)

Thermophoresis

• Heat sample by 2K

• Molecules dissipate

• When fluorescently labelled

• Equilibrate depending on size of hydration shell, charge distribution

Duhr and Braun (2006) PNAS 103, pp19678

Current technology

Jerabek‐Willemsen et al. 2011

Sample optimization

• Reduced Gel problematic: change purification protocol

• NR Gel problematic: check cysteines

• Protein aggregation, folding stability:– Size exclusion, light scattering, CD, NMR, thermofluor

Thermal stability

• Thermofluor

• Modified real‐time PCR machine– Add hydrophobic fluorescent probe

– When protein unfolds…

– Fluorescence increases

Thermal stability

• Check protein stability

• Additive/ligand screen

Ericsson et al Analytical Biochemistry 357 (2006) 289

Sample optimization

• Reduced Gel problematic: change purification protocol

• NR Gel problematic: check cysteines

• Protein aggregation, folding stability:– Size exclusion, light scattering, CD, NMR, thermofluor

• Modify buffers, additives

• If nothing works: change construct

Additives?

• DTT (will affect OD 280nm)

• Glycerol (no more than 5 %)

• Detergents at less than 2xCMC0.1% 1‐s‐Nonyl‐β‐D‐thioglucoside0.2% n‐Decanoylsucrose0.3% n‐Nonyl‐β‐D‐maltoside0.4% DDAO0.5% C8E50.8% FOS‐Choline®‐101.1% FOS‐Choline®‐9

Conclusions

• QC at home is crucial

• But we can do it for you at EMBL@PETRA3

• Some quality control methods can provide useful complementary data

• (In)direct validation of the SAXS models

Acknowledgements

• Malvern Instruments (Bernd Tartsch)

• Melissa Graewert (EMBL Hamburg)

• Stefan Duhr (Nanotemper)

• Stephane Boivin (EMBL Hamburg)