spectrophotometry MX School "Getting the most from the ESRF MX beamlines" February 3 rd, 2009
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1 Off- and on-line spectrophotometry MX School "Getting the most from the ESRF MX beamlines" February 3 rd, 2009 dominique.bourgeois@ibs.fr
2 Basics Practical realization Applications The ESRF Cryobench lab Superoxide reductase
3 Basics
4 Excited state Ground state Absorption
5 Practical realization
6 Absorption spectrum measurement Measurement time ~ 50 ms Pb: optically thick crystals, light scattering effects, orientation ion dependance, polarization effects Reference spectrum Measured spectrum
7 Fluorescence spectrum measurement Measurement time ~ 1 ms (with laser) Pb: inner filtering, polarization effects 3000 intensity (counts) spectres tryptophane wavelength (nm)
8 Raman spectrum measurement 785 nm Wealth of information Measurement time ~ 15 min,, solvent contamination, orientation, polarization effects
9 In crystallo Raman spectroscopy Cys vibrations 510, 530 cm -1 S-S S stretching 725 cm -1 Cys C-S C S stretching Trp vibrations : 760, 880, 1010, 1350 and 1558 cm -1 Phe vibrations: 1005 and 1200 cm -1 Tyr vibrations: 840, 860 and 1210 cm -1 Main chain vibrations Amide I band : cm 1700cm -1 Contribution carbonyl C=0 stretching Backbone conformation and H-bonding H pattern Amide II band : cm 1580cm -1 Contribution: N-H N H bending and C-N C N stretching Exposure time 15mins Lysozyme crystal
10 Raman spectroscopy: advantages of crystals High concentration of proteins in crystals 10-50mM ( time more than solution): Higher Raman intensity Less contribution from solvent in crystals (50%): Lower background in Raman spectra Selection of conformations in crystals: narrower Raman bands in crystals => better Raman resolution
11 Non-resonant in crystallo Raman data Lysozyme Crystal Solution (100 mg/ml)
12 Applications
13 In crystallo spectroscopy? Then If a tool to validate the crystal structure. J. Mc Geehan, EMBL & Dr Xavi Carpena, Parc Cientific de Barcelona
14 Photoconversion of Alpha-phycoerythrocyanin crystals (absorption) a tool to check activity in the crystalline state. Marius Schmidt, University of Munich, Germany, Biochemistry 46, 416, 2007
15 Binding of nitrate to xylose isomerase (Raman) a tool identify bound ligands. Carpentier et al, J. Appl. Cryst. (2007), 40,
16 Binding of EtBr to DNA (fluorescence lifetime) Bound EtBr (solution) Bound EtBr (crystal) Unbound EtBr Time [ns] Royant et al, J. Appl. Cryst. (2007), 40,
17 Near-atomic atomic global view Sub-atomic local view a tool to complement crystal structure.
18 Redox state of NADP(H) (fluorescence) Fluorescence [Counts] Malate Deshydrogenase Reduced NADPH Wavelength [nm]
19 X-rays a tool to monitor chemistry induced by X-rays. X
20 In crystallo reaction triggering Fluorophore Structure of the intermediate Glu222 Identification of intermediate state a tool to follow reactions in crystals
21 Weik et al,biophys. J. 86: (2004) Temperature dependent fluorescence microspectrophotometry (TDFM): Observing glass transition in nano-volume samples... hν a tool to study dynamics in crystals
22 a tool for cryo-enzymology studies in solution
23 The ESRF Cryobench lab
24
25 Cryobench Fibre optics Electronics Sample prep ID MAD ID microfocus X-ray X-ray
26 Fiber from lasers (fluorescence) Objective 3 Videomicroscope Fiber from light source (absorption) Cryostream Objective 1 Objective 2 Offline Fiber to spectrometer Crystal on goniometer head
27 John McGeehan, Florent Cipriani and Raimond Ravelli, EMBL Online
28 On-Line Raman experiment Raman probe system Cryo Cryo-system spindle crystal
29 Case example: superoxide reductase
30 Superoxide reductase: O H + + SOR(Fe 2+ ) H 2 O 2 + SOR(Fe 3+ ) O H + + Fe 2+ Fe 3+ -OOH - Fe 3+ + H 2 O 2 + H + Katona et al, Science (2007), 316, 449.
31 Puzzling crystallographic data??? b c d Limited crystallographic resolution (1.95 Å) Multiple observations in the asymmetric unit Can these be relevant intermediate species?
32 In crystallo Raman Spectroscopy of SOR Signature of iron-peroxide intermediate
33 Comparison with solution state
34 Radiation damage Gy absorbed dose does not affect Raman peaks
35 Proposed mechanism b c d b c d
36 Take home message Growing importance of complementary techniques In crystallo spectroscopy of particular interest for studies of protein mechanisms and dynamics Small niche in the MX field Projects are generally difficult and require dedicated efforts over long term.
37 Acknowledgments Gergely Katona (IBS, Grenoble) Philippe Carpentier (IBS, Grenoble) Virgile Adam (ESRF, Grenoble) Vincent Nivière (CEA, Grenoble) Antoine Royant (IBS, Grenoble) Jérémy Ohana (IBS, Grenoble) Xavier Vernède (IBS, Grenoble) Nikolay Tsanov (IBS, Grenoble) Martin Weik (IBS, Grenoble) Raimond Ravelli (EMBL Grenoble)
2012 HORIBA Scientific. All rights reserved. 2012 HORIBA Scientific. All rights reserved.
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