Responsable : Martin Weik

Research summery
The team studies the structure and dynamics of light sensitive proteins by static and time-resolved X-ray scattering and crystallography at synchrotrons and X-ray free electron lasers and the dynamics of proteins, their fibrous aggregates and their hydration water by neutron spectroscopy.

Martin Weik (researcher, CEA – DR)
Giorgio Schirò (researcher, CNRS – CR1)
Martin Byrdin (researcher, CEA – CR, 50%)
Ninon Zala (technician, CEA, 50%)
Sophia Kapetanaki (postdoc)
Ronald Rios Santacruz (PhD student)

Frédéric Beisson (CEA Cadarache, France)
Dominique Bourgeois (IBS, Grenoble, France)
Manfred Burghammer (ESRF, Grenoble, France)
Marco Cammarata (Rennes University, France)
Lucia Comez (CNR, Perugia, Italy)
Nicolas Coquelle (ILL, Grenoble, France)
Antonio Cupane (University of Palermo, Italy)
Isabelle Demachy (Lab Chimie Physique, Orsay, France)
David Eisenberg (UC Los Angeles, USA)
Brian Federici (UC Riverside, USA)
Vito Foderà (University Copenhagen, Denmark)
Elspeth Garman (Oxford University, UK)
Stanislas Gobec (University of Ljubljana, Slovenia)
Derren J. Heyes (University of Manchester, UK)
Stefan Jakobs (MPI Göttingen, Germany)
Diana Kirilovsky (IB2C, Paris, France)
Meytal Landau (Israel Institute of Technology (Technion), Haifa Israel)
Frans Mulder (Aarhus University, Denmark)
Florian Nachon (IRBA, Bretigny-sur-Orge, France)
Pierre-Yves Renard (University Rouen, France)
Michael Sawaya (UC Los Angeles, USA)
Ilme Schlichting (MPI, Heidelberg, Germany)
Tilo Seudel (ILL, Grenoble, France)
Israel Silman (Weizmann Institute, Israel)
Joel Sussman (Weizmann Institute, Israel)
Michel Sliwa (LASIR, Lille, France)
Douglas Tobias (UC Irvine, USA)
Bente Vestergaard (University Copenhagen, Denmark)
Mathias Winterhalter (Jacobs University, Bremen, Germany)
Giuseppe Zaccai (ILL, Grenoble, France)

Research activity

Structure and dynamics of light-sensitive proteins using time-resolved X-ray scattering and crystallography at synchrotrons and XFELs

(PIs Weik, Colletier, Schirò)

A description for the general public in French can be found HERE.

The recent advent of X-ray free electron lasers now allows studying proteins in action down to the sub-picosecond time-scale by time-resolved X-ray crystallography and solution scattering. Our team has established a structural biology research program based on XFELs and we have carried out experiments at the LCLS XFEL at Stanford (USA), the SACLA XFEL (Japan) and the European XFEL in Hamburg (Germany) in collaboration with the groups of Ilme Schlichting (MPI Heidelberg), of Marco Cammarata (U Rennes) and Michel Sliwa (U Lille). We aim at uncovering the structural dynamics of light-sensitive proteins from ultra-fast photochemical events around the chromophore to large-scale structural changes in the protein moiety on longer time-scales. A highlight has been the structure determination of a photoswitchable fluorescent protein in its excited state intermediate, 1 ps after photon absorption (see figure ; Coquelle, Sliwa, Woodhouse, Schiro, Adam et al (2018) Nature Chemistry). Based on the intermediate state structure, we were able to design mutants with enhanced photoswitching properties that might be of use in nanoscopic applications. This research has been carried out in collaboration with DYNAMOPS’ PIXEL team. A further highlight has been the description of a protein-quake in myoglobin after photolysis by time-resolved solution scattering at the LCLS XFEL (Levantino, Schiro et al. (2015) Nature Communications).

Ultra-fast movie of a light-sensitive protein in action. The fluorescent protein (green) is excited by a visible light laser (violet ray) and impacted by an X-ray pulse (white ray) generated by an X-ray free electron laser (Coquelle, Sliwa, Woodhouse, Schiro, Adam et al (2018) Nature Chemistry). One picosecond after excitation, the (blue) chromophore at the core of the protein can be observed midway between its stable forms (grey, green); its speed of movement (4.6 Angströms in 1 picosecond) is equivalent to that of a supersonic aircraft (460 m/s).

Key publications
Coquelle N, Sliwa M, Woodhouse J, Schirò G, Adam V, Aquila A, Barends T, Boutet S, Byrdin M, Carbajo S, De la Mora E, Doak B, Feliks M, Fieschi F, Foucar L, Guillon V, Hilpert M, Hunter M, Jakobs S, Koglin J, Kovacsova G, Lane TJ, Lévy B, Liang M, Nass K, Ridard J, Robinson J, Roome C, Ruckebusch C, Seaberg M, Thepaut M, Cammarata M, Demachy I, Field M, Shoeman R, Bourgeois D, Colletier J-P, Schlichting I, Weik M. (2018) Chromophore twisting in the excited state of a fluorescent protein captured by time-resolved serial femtosecond crystallography. Nature Chem 10, 31-37.

Heyes DJ, Hardman SJO, Pedersen MN, Woodhouse J, De La Mora E, Wulff M, Weik M, Cammarata M, Scrutton NS, & Schirò G (2018) Light-induced structural changes in a full-length cyanobacterial phytochrome probed by time-resolved X-ray scattering. Comms. Biol. in press

Colletier JP, Sliwa M, Gallat FX, Sugahara M, Guillon V, Schiro G, Coquelle N, Woodhouse J, Roux L, Gotthard G, Royant A, Uriarte LM, Ruckebusch C, Joti Y, Byrdin M, Mizohata E, Nango E, Tanaka T, Tono K, Yabashi M, Adam V, Cammarata M, Schlichting I, Bourgeois D, Weik M (2016) Serial femtosecond crystallography and ultrafast absorption spectroscopy of the photoswitchable fluorescent protein IrisFP. The journal of physical chemistry letters: 882-887.

Levantino M, Schirò G, Lemke HT, Cottone G, Glownia JM, Zhu D, Chollet M, Ihee H, Cupane A & Cammarata M (2015) Ultrafast myoglobin structural dynamics observed with an X-ray free-electron laser. Nat. Commun. 6: 6772

Serial nanocrystallography

(PI Colletier)
Serial X-ray crystallography is a new methodology that mitigates radiation damage to biological macromolecules by distributing the absorbed dose over tens of thousands of tiny protein microcrystals. Serial crystallography is rapidly expanding and offers unprecedented possibilities to the structural biologist by allowing room-temperature experiments on tiny nano- to microcrystals at synchrotrons and XFELs. Such crystals are perfectly adapted for time-resolved studies. Highlights have been one of the very first serial synchrotron crystallography experiments (Coquelle et al (2015) Acta Cryst D), structure determination of recombinant in vivo grown nanocrystals of a bacterial toxin at the LCLS XFEL (Colletier et al (2016) Nature), and structure determination of bacterial amyloids and membrane proteins involved in biofilm formation from microcrystals (Salinas et al., 2018, Nat Comm; El-Khatib et al., 2018, PNAS; Tayeb-Fligelman et al., 2017, Science). The serial crystallography preprocessing software NanoPeakCell is developed in the framework of this activity, in strong collaboration with Dr. Nicolas Coquelle from the ILL.

De novo structure determination from serial femtosecond crystallography data collected on naturally-occurring nanocrystals allowed shedding light on one of nature’s most potent mosquito killer (Colletier et al., 2016, Nature).

Key publications

El-Khatib M, Nasrallah C, Lopes J, Tran QT, Tetreau G, Basbous H, Fenel D, Gallet B, Lethier M, Bolla JM, Pages JM, Vivaudou M, Weik M, Winterhalter M, Colletier JP (2018) Porin self-association enables cell-to-cell contact in Providencia stuartii floating communities. Proc Natl Acad Sci U S A 115: E2220-E2228

Tayeb-Fligelman E, Tabachnikov O, Moshe M, Goldshmidt-Tran O, Sawaya MR, Coquelle N, Colletier J-P, Landau M (2016) The Cytotoxic Staphylococcus aureus PSMα3 Reveals a Novel Cross-alpha Amyloid-like Fibril. Science 355: 831-833.

Colletier JP, Sawaya MR, Gingery M, Rodriguez JA, Cascio D, Brewster AS, Michels-Clark T, Hice RH, Coquelle N, Boutet S, Williams GJ, Messerschmidt M, DePonte DP, Sierra RG, Laksmono H, Koglin JE, Hunter MS, Park HW, Uervirojnangkoorn M, Bideshi DK, Brunger AT, Federici BA, Sauter NK, Eisenberg DS. (2016) A potent binary mosquito larvicide revealed by de novo phasing with an X-ray free-electron laser. Nature 539: 43-47.

Coquelle N, Brewster AS, Kapp U, Shilova A, Weinhausen B, Burghammer M, Colletier J-P (2015) Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams. Acta Crystallogr D Biol Crystallogr 71: 1184–1196.

Coupling of protein and hydration water dynamics by neutron spectroscopy

(PIs Weik, Schiro)
What is the molecular secret behind water’s role as the matrix of life ? We address the molecular aspect of this question by studying the dynamical give-and-take of protein and hydration-water by neutron spectroscopy. We discovered water-translational diffusion on a protein surface as being the central mechanism enabling macromolecular activity by promoting internal dynamics of the biological solute (Schiro, Fichou, Gallat et al. (2015) Nature Communications). We started focusing our attention to studying the molecular mechanisms of pathological protein aggregation, in particular the changes in the dynamics of proteins and their hydration water that might drive aggregation. We studied hydration water-mobility on the surface of the intrinsically disordered human protein tau in its monomeric form and in its amyloid-fiber form in collaboration with Douglas Tobias (UC Irvine). It has been found that hydration water mobility is enhanced around tau amyloid fibers, a finding that identifies hydration water entropy as a potentially universal driving force behind amyloid-fiber formation (Fichou et al (2015) PNAS).

Water molecules undergo rotational (blue arrows) and translational (red arrows) diffusion on the surface of a soluble protein (green). It is the translational motion that is the central mechanism enabling protein activity by promoting internal dynamics of the biological solute (Schiro, Fichou, Gallat et al (2015) Nature Communications).

Key publications
Fichou Y, Heyden M, Zaccai G, Weik M, Tobias DJ (2015) Molecular dynamics simulations of a powder model of the intrinsically disordered protein tau. J Phys Chem B 119, 12580.

Fichou, Y, Gallat, FX, Schiro, G, Laguri, C, Moulin, M, Combet, J, Zampouni, M, Härtlein, M, Picart, C, Forsyth, T, Lortat-Jacob, H, Colletier, JP Tobias, D. & Weik, M. (2015) Hydration water mobility is enhanced around tau amyloid fibers. PNAS 112, 6365.

Schiro, G, Fichou, Y, Gallat, FX, Wood, K, Gabel, F, Moulin, M, Härtlein, M, Heyden, M, Colletier, JP, Orecchini, A, Paciaroni, A, Wuttke, J, Tobias, D & Weik, M. (2015) Translational diffusion of hydration water correlates with functional motions in both folded and intrinsically disordered proteins. Nat Commun 6: 6490.

Cupane A, Fomina M, Piazza I, Peters J & Schirò G (2014) Experimental evidence for a liquid-liquid crossover in deeply cooled confined water. Phys. Rev. Lett. 113: 215701