BioSAXS – special applications Martin A. Schroer EMBL Hamburg
Examples for bioSAXS experiments Disclaimer • Might not work for all samples • Might not make sense for all samples • You will likely need more sample solution however • can give fundamental new insights • In situ • Time-resolved • … • demand a synchrotron source Contact the beamline scientists! • Photon flux: weak signals, temporal resolution • Small beam sizes: spatial resolution • Energy tunability: Penetration 2
Types of sample environments Robotic sample changer • Capillary holder Microfluidics (e.g. for THz) • Temperature cell • in vacuum capillary • continuous flow • Cryo chamber (P12) • High pressure cells • Rheological cells • Heating stages • User setups • … SEC-SAXS/MALS Laser excitation Stopped Flow • Online purification and 28/02/2020 3 detection system
Non-standard bioSAXS • Biological macromolecules under external perturbations • Heat • Pressure • Shear stress • Laser light • THz-radiation • Examples for hierarchial samples • Radiation damage • Static • Time-resolved 4
In air operation In vacuum: • Quarz capillary • Removing in vacuum capillary / sample changer • Two sealing windows + BSA + buffer Air gap + buffer • Place sample cell Higher X-ray absorption by air Higher background signal (air + windows + sample cell ) In air Try to reduce the air gap as • Polystyrene cell much as possible! • Air • Kapton windows Use proper window material! + BSA + buffer + buffer 5
Heating stages (at P12) Sample changer temperature range: • 7 – 40 ° C • Keep samples stored at different T Temperature controlled capillary holder • Peltier element • Quartz capillaries • different T range • fast T changes User setup • highly viscous samples • e.g. Linkam heating stage • tricky samples (toxic, corrosive, dirty,..) • Apolar solvent • for non-liquid samples 6
Temperature SAXS studies • Unfolding of proteins • Gelation processes • Coil-to-globule transition (biopolymers) • Phase transitions in lipids, liquid crystals • … Example: • Biological relevant lipids • T-induced melting of lamellar structures F. A. Facchini et al. J. Med. Chem. 61, 2895 (2018). 7
High Pressure SAXS Allows to study protein stability • (pressure-) unfolded state smaller volume than folded state (~ 1% effect) • Pressure 1 – 7 kbar : effect on non-covalent bonds: Changes of the tertiary structure • Pressure > 10 kbar : effect on covalent bonds: changes of the secondary structures Need High pressure sample cells • Pressure range: 1 bar – 4....7 kbar • X-ray windows: two flat diamonds -> Higher X-ray energy! • Pressurizing medium: water 8 27/05/2020 C. Krywka et al. , ChemPhysChem 9 2809 (2008).
HP SAXS – SNase • 149 amino acids, M w = 16.8 kDa • Globular protein • No disulfidec bonds -> destabilized • Standard protein for high pressure studies High pressure effects • Decrease of I(0) ⇒ reduced constrast as water gets compressed • Increase of radius of gyration ⇒ unfolding Guinier plot G. Panick et al. , J. Mol. Biol. 275, 389 (1998). 9
SNase - (de-)stabilization by cosolvents C. Krywka et al. , ChemPhysChem 9 2809 (2008). Cosolvents can change the pressure – effect • Kosmotropic: stabilizing • Chaotropic: destabilizing Urea p [bar] • destabilizes proteins TMAO • Stabilizes, counteracts urea • Concentration in fishes increases with sea depth p [bar] Recently more studies (several protein, tRNA, microtubuli) but still more to be explored: terra incognita 10
Rheo-SAXS: Effect on shear • Shear can align or disrupt molecules; is present in joints and blood flow Hyaluronan: 750 kDa • Hyaluronan is a biopolymer and essential part of the extracelluar matrix (joints) 0 - 1500 1/s Rheo-SAXS: Hyaluronan chain network gets more ordered 11 D.C.F. Wieland et al. , J. Syn. Rad. 24, 646 (2017).
Light-induced reactions Light can induce different types of reactions • 360 – 500 nm: Structural changes of photosensitive proteins Opening of caged compounds • Infra red (1440 nm): Temperature jump by fast heating of water Example: • Caged ATP released after laser pulse • ATP induces dimerization of NBD • TR-SAXS/WAXS Tidow group (Hamburg) @ ID09, ESRF 12 I. Josts et al. IUCrJ 5, 667 (2018).
P12 – laser system To fibre • Tuneable Nd:YAG – laser ( Ekspla , Lithuania) Direct • Wave lengths: • 335 – 500 nm & 1065 – 2500 nm (fibre port; to P12 hutch experiments) • Repetiton rate: 10 Hz • Pulse length: 6 ns Energy per pulse [mJ] 13
Studying the effect of THz on proteins THz radiation • Electromagnetic radiation • Can induce large molecular vibrations (collective) / low in energy • Strong absorption in water • Non-ionizing but thermal/ athermal effects -> possible risks are discussed in literature 14 L. Wei et al. Frontiers in Laboratory Medicine (2019).
THz-excitation of proteins Fröhlich‘s prediction • THz-radiation can excite collective motions within biological macromolecules by coupling to their dipole moment ( Fröhlich condensation ) Such collective vibrations ( normal modes ) may lead to long-range conformational changes . Such changes can be probed by SAXS. THz excitation & SAXS probe 15 A. Panjkovich, D.I. Svergun. PCCP 18, 5707 (2016).
THz-SAXS - Experiment Such a noval type of experiment needs • THz sources (cw + pulsed) -> Excite the sample • Dedicated microfluidic cell -> small channel width Setup I • Sample delievery system Setup II • Small, asymmetric X-ray beam (80 x 120 µm 2 ) • Precise positioning (sub-micron) (hexapod) • Synchronization (data collection) M. Roessle (TH Lübeck) 16 G. Katona (U Gothenburg)
THz-SAXS - microfluidic cell Combined THz-SAXS measurements demand dedicated sample environment Microfluidic chips: • • Flowing of sample → reduce radiation damage • Transparent for THz → enough sample excitation • Narrow channel (500 µm) as THz absorption in water is strong → enough sample excitation • Low X-ray background → record SAXS signal 3D printed Polystyrene cell M. Roessle (TH Lübeck) 17 S. Schewa, et al., submitted
Setup installed at P12 • THz beam passes set of mirrors THz • THz can be detected by receveiver • THz beam & X-ray beam are perpendicular X-ray The same stop of the sample is illuminated 18
SAXS on cells • Recently, SAXS / USAXS studies have been performed on cells • Example: E. coli modelled with a geometrical model • Application in screening studies for different antibiotics E.F Semeraro et al. , IUCrJ 4, 751 (2017). 19 A.R. v. Gundlach et al. , BBA - Biomembranes 1858, 918 (2016).
Scanning SAXS • Scan samples with a small X-ray beam Real space maps of scattering intensity • Examples: • SAXS from the inside of cells • Structure of bone (orientation of hydroxyapatite crystals) B. Weinhausen et al. , Phys. Rev. Lett. 112, 088102 (2014). 20 16/03/2018 D.C.F. Wieland et al. , Acta Biomaterialia 25, 339 (2015).
SAXS tensor tomography • Method to study anisotropically oriented nanostructures at 3D spatial resolution • Example: Orientation of collagen fibrils within a trabecular bone 21 M. Liebi et al. , Nature 527, 349 (2015).
Radiation damage in protein solutions 2 H 2 O + X-ray → H 3 O + + • OH + e - • Mechanism of radiation damage: aq Radiolysis of water (99 % of sample volume) • + - OH e - aq + O 2 → HO 2 → radical formation S.D. Maleknia et al., Anal. Biochem. → interactions with solvent accessible sites 289 , 103 (2001). → formation of large aggregates For SAXS: Aggregates spoil signal of undamaged proteins C.M. Jeffries et al., J. Syn. Rad. 22 , Radiation damage limits data collection of biological samples 273 (2015). 22
Reducing radiation damage Coflow N. Kirby et al., Acta Problem for SAXS: Damage not ( easy ) predictable Cryst. D 72 , 1254 (2016). Different schemes to reduce the effect of radiation damage Cryo-cooling Beam attenuation S.P. Meisburger et al., Biophys. • J. 104 , 227 (2013). Addition of „scavenger“ or stabilizer molecules • (DTT, ascorbic acid; glycerol) High flux Continous sample flow • Coflow • • Cryo-cooling • Outrunning radiation damage (High flux) Cell geometry • Sample cell geometry M.A.Schroer et al. , J. Synchrotron Rad. 25 , 1113 (2018) 23
TR BioSAXS Proteins are scattering weakly • need more flux • possible to follow a changing signal However: • Radiation damage is harder to determine: • Data comparsion does not work How to deal with this? 24
Example: Stopped Flow TR-SAXS • Reaction of MsbA Nucleotide binding domain with ATP • Rapid mixing • 35 ms frames collected • Expected: Monomer – dimer transition H. Tidow 25 Josts et al, Structure 28, 348 (2020).
• Start reaction and directly probe Continuous change of Rg 26
• Start reaction and directly probe Continuous change of Rg • Start reaction, wait (delay time), then probe Continuous change of Rg But NOT overlapping Radiation Damage 27
Recommend
More recommend
