rob meijers embl hamburg
TRANSCRIPT
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Sample preparation and characterization around SAXS
Rob Meijers
EMBL Hamburg
Experimental verification and… validation?
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Garbage in
?
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The right stuff
• Molecular weight
• Oligomerization state
• Monodispersity
• Aggregation
• Protein concentration
• Protein folding state
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Quality controlSDS Page
Mass spec
Gel filtrationDLS/SLS
Circular Dichroism
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1 2 1 2ConcentratedDilute Non‐reduced
1
A gel of a heterodimeric receptor
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1 2
A size exclusion profile
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1 2 1 2ConcentratedDilute Non‐reduced
1
A second look
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Different gels
• SDS
• SDS non‐reduced
• Native
• Iso‐electric focussing
• Purity
• Disulphide bonds
• Post‐translational modifications:– Phosphorylation
– glycosylation
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Protein concentration
• UV‐Vis
• Bradford
• Refractive index
Sample injected
Sampleprovided
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Quality controlSDS Page
Gel filtration
Mass spec
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Mass spectrometry
• MALDI‐TOF or ESI
• Integral sample
• Proteolytic digest:– Trypsin
– Carboxypeptidase
– Aminopeptidase
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Mass spectrometry
• MS/MS + Ion mobility :– Detailed folding state
– Protein‐protein interactions
– Whole protein size…
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Ion mobility derived particle size
2012‐10‐18 13
Ruotolo et al Nature Protocols (2008) 3, 1139
in combination with SAXS…
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Sizing profile, SLS twist
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Intramolecular interference produces a disymmetry in the scattered light.
Size of molecule/particle must be significant compared to wavelength of light
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Angular Dependence
small moleculesradius < 15 nm Pθ = 1 for all θ
large moleculesradius > 15 nm Pθ= 1 for θ = 0°
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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
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Combined particle analysis
• UV
• Refractive index
• Viscosity
• Right angle light scattering
Viscotek/Malvern
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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
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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
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LALS (7º) detector
RALS (90º)
RI detector
UV detector
GPC columns
Laser Light Scattering Detector, Refractive Index Detector and UV‐Cell
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SEC + SAXS
• SWING beamline at Soleil
• HPLC in FPLC mode
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Online purification & QC
• Combine SEC and SAXS
• Add:– Refractive index
– RALS
• Integrate in BioSAXSbeamline
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Courtesy Melissa Graewert
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UVRiLS
UV ~ c ɛLS ~ c (dn/dc)² MWRi ~ c dn/dc
ml
Au
Courtesy Melissa Graewert
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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
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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
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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
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Definedbuffer region
Courtesy Melissa Graewert
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Integration of experimental data
• SAXS scattering curve
• Accurate protein concentration (UV‐VIS +RI)
• Molecular weight (RALS)
• Radius of gyration? (MALS)
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Estimation of Particle Radius
H2O
H2OH2O
H2O
H2O
H2O
RgRh
Rg II
Rg
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The micro‐world
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Stokes‐Einstein relation
• D = Diffusion coefficient
• k = Boltzmann’s coefficient
• T = Temperature
• η = Viscosity
• R = hydrodynamic radius
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Dynamic light scattering
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Dynamic Light Scattering scheme
Sample
Detector
Correlator
Transmitted light
DiffusedLight
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Information from the correlation curve
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Integration of experimental data
• SAXS scattering curve
• Accurate protein concentration (UV‐VIS +RI)
• Molecular weight (RALS)
• Radius of gyration? (MALS)
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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
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Current technology
Jerabek‐Willemsen et al. 2011
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Sample optimization
• Reduced Gel problematic: change purification protocol
• NR Gel problematic: check cysteines
• Protein aggregation, folding stability:– Size exclusion, light scattering, CD, NMR, thermofluor
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Thermal stability
• Thermofluor
• Modified real‐time PCR machine– Add hydrophobic fluorescent probe
– When protein unfolds…
– Fluorescence increases
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Thermal stability
• Check protein stability
• Additive/ligand screen
Ericsson et al Analytical Biochemistry 357 (2006) 289
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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
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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
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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
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Acknowledgements
• Malvern Instruments (Bernd Tartsch)
• Melissa Graewert (EMBL Hamburg)
• Stefan Duhr (Nanotemper)
• Stephane Boivin (EMBL Hamburg)