Sample preparation and characterization around SAXS

Transcription

1 Sample preparation and characterization around SAXS Experimental verification and validation? Rob Meijers EMBL Hamburg

2 Garbage in?

3 The right stuff Molecular weight Oligomerization state Monodispersity Aggregation Protein concentration Protein folding state

4 SDS Page Quality control MW Concentration Mass spec MW Circular Dichroism Folding? Gel filtration Aggregation Oligomeric state DLS/SLS Monodispersity

5 A gel of a heterodimeric receptor Dilute Concentrated Non-reduced 1

6 A size exclusion profile 1 2

7 A second look Dilute Concentrated Non-reduced 1

8 Different gels SDS SDS non-reduced Native Iso-electric focussing Purity Disulphide bonds Post-translational modifications: Phosphorylation glycosylation

9 Protein concentration UV-Vis Bradford Refractive index Sample injected Sample provided

10 A capricious sample 7XCYS ORIGINAL SEQUENCE, WITH HIS-TAG MGSSHHHHHHSSGLVPRGSHMKICITVGHSILKSGACTSADGVVNEY QYNKSLAPVLADTFRKEGHKVDVIICPEKQFKTKNEEKSYKIPRVNSG GYDLLIELHLNASNGQGKGSEVLYYSNKGLEYATRICDKLGTVFKNR GAKLDKRLYILNSSKPTAVLIESFFCDNKEDYDKAKKLGHEGIAKLIVE GVLNKNINNEGVKQMYKHTIVYDGEVDKISATVVGWGYNDGKILICDI KDYVPGQTQNLYVVGGGACEKISSITKEKFIMIKGNDRFDTLYKALDFIN

11 Sizing profile

12 Cysteine point mutations

13 Sizing profile

14 Sizing profile, SLS twist

15 Raleigh scattering Short wavelength=more efficient

16 Rayleigh Equation: R θ M w Rayleigh Ratio at scattering angle θ weight-average molecular weight P θ c K A 2 particle scattering function R θ /R 0 concentration optical constant 2nd virial coefficient I θ Scattered Detector Laser V θ I 0 Incident Detector

17 Angular Dependence Radius [nm]

18 Radius of Gyration (Rg) Determination by Static Light Scattering 7 o High MW allows Rg determination Intercept = Mw Slope 0 for Rg < 15 nm 7 o 90 o R θ /KC Sin 2 (θ/2) Determination of radius of gyration not possible for proteins

19 Combined particle analysis UV Refractive index Viscosity Right angle light scattering Viscotek/Malvern

20 What are the detectors responding to? Refractometer = K RI dn/dc Conc UV-Detector = K UV da/dc Conc Viscometer = K Visc Intrinsic Viscosity Conc Light Scattering = K LS Mw (dn/dc) 2 Conc

21 Refractive Index Response (mv) 1472,0 1394,0 1312,0 1230,0 1148,0 1066,0 984,0 902,0 820,0 738,0 656,0 574,0 492,0 410,0 328,0 246,0 164,0 82,0 0,0 Viscometer DP Response (mv) 36,0 34,0 32,0 30,0 28,0 26,0 24,0 22,0 20,0 18,0 16,0 14,0 12,0 10,0 8,0 6,0 4,0 2,0 0,0 BSA High MW Aggregates (Mw > 1 Mio D) Trimer Trimer Dimer Dimer Monomer Monomer 5,30 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 10,00 Retention Volume (ml) 239,0 224,0 210,0 196,0 182,0 168,0 154,0 140,0 126,0 112,0 98,0 84,0 70,0 56,0 42,0 28,0 14,0 0,0 Right Angle Light Scattering Response (mv) 7,000 6,800 6,600 6,400 6,200 6,000 5,800 5,600 5,400 5,200 5,000 4,800 4,600 4,400 4,200 4,000 Log Molecular Weight Mw (D) IVw (dl/g) Rh (nm) Weight Fraction (%) Monomer ,056 3,88 87 Dimer ,071 5,32 11 Trimer ,095 6,69 1,5 Light Scattering Viscometer Refractive Index

22 Online purification & QC Combine SEC and SAXS Add: Refractive index RALS Integrate in BioSAXS beamline

23 Online SEC-BC-SAXS Courtesy Melissa Graewert

24 Au UV ~ c ɛ UV LS ~ c (dn/dc)² Ri MW Ri ~ c dn/dc LS ml Courtesy Melissa Graewert

25 Courtesy Melissa Graewert I(O) Frame 1 Frame 501 Frame 1001 Frame 1501 Frame frames collected (1 sec each) while proteins eluted from superdex / ml/min

26 Courtesy Melissa Graewert

27 SAXS Rg/I(0) profile SEC elution profile Monomer 66 kda Monomer versus Dimer Dimer 134 kda Trimer 205 kda Courtesy Melissa Graewert

28 Courtesy Melissa Graewert

29 Integration of experimental data SAXS scattering curve Accurate protein concentration (UV-VIS +RI) Molecular weight (RALS) Radius of gyration? (MALS) Hydrodynamic radius (DLS)

30 Estimation of Particle Radius Rg II H 2 O H 2 O H 2 O H 2 O Rg H 2 O H 2 O 2 O Rg Rh

31 The micro-world

32 Stokes-Einstein relation D = Diffusion coefficient k = Boltzmann s coefficient T = Temperature η = Viscosity R = hydrodynamic radius

33 Dynamic light scattering

34 Dynamic Light Scattering scheme Diffused Light Sample Transmitted light Correlator Detector

35 Information from the correlation curve

36 Schematic drawing in situ DLS Courtesy XtalConcept

37 Sample optimization Reduced Gel problematic: change purification protocol NR Gel problematic: check cysteines Protein aggregation, folding stability: Size exclusion, light scattering, CD, NMR, thermofluor

38 Thermal stability Thermofluor Modified real-time PCR machine Add hydrophobic fluorescent probe When protein unfolds Fluorescence increases

39 Principle of Thermofluor assay 39 Adapted from : Pantoliano et al., (2001) J. Biomol Screening 6: Cummings et al., (2006) J. Biomol. Screening 11:

40 Thermal stability Check protein stability Additive/ligand screen Ericsson et al Analytical Biochemistry 357 (2006) 289

41 Thermofluor TM useful for structural biology Optimization of protein purification and characterization using Thermofluor screens Stephane Boivin, Sandra Kozak, Rob Meijers Protein Expression and Purification Vol91;2. p

42 Initial TF screen: buffer and ph optimisation Thermal denaturation assay of glucoisomerase. Best stabilizing condition is Tris-HCl ph

43 Initial TF screen: buffer and ph optimisation Thermal denaturation assay of glucoisomerase. Best stabilizing ph

44 HTP Thermofluor TM Buffer Screen ex: Buffer concentration ex: NaCl concentration 44

45 HTP Thermofluor TM Additive Screen 45 45

46 HTP Thermofluor TM Additive Screen ex: imidazole effect 46

47 Improving purification protocol Courtesy Stephane Boivin 47

48 Is Ni +2 suitable for your protein? 48

49 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

50 Thermophilic organisms Chaetomium Thermophilum Thermophilic fungus (eukaryote) BLAST & order synthetic gene

51 Additives? DTT (will affect OD 280nm) Glycerol (no more than 5 %) Detergents at less than 2xCMC 0.1% 1-s-Nonyl-β-D-thioglucoside 0.2% n-decanoylsucrose 0.3% n-nonyl-β-d-maltoside 0.4% DDAO 0.5% C8E5 0.8% FOS-Choline % FOS-Choline -9

52 Conclusions QC at home is crucial But we can do it for you at Some quality control methods can provide useful complementary data (In)direct validation of the SAXS models

53 Acknowledgements Malvern Instruments (Bernd Tartsch) Melissa Graewert (EMBL Hamburg) Stephane Boivin (EMBL Hamburg)