Group of protein kinetic crystallography

Kinetic protein crystallography : a new approach to visualize reaction intermediates at the near-atomic scale.

At the “Institut de Biologie Structurale” in Grenoble, and in very close collaboration with the European Synchrotron Radiation Facility (ESRF), our team works on “kinetic protein crystallography”.

What is “kinetic protein crystallography” ?
The majority of protein structures presently solved by X-ray crystallography actually correspond to a static state, that only poorly represents the ensemble of conformations adopted in reality by a protein in action. “Kinetic protein crystallography” allows investigating structure in a dynamical fashion, by visualizing at high resolution the conformational changes that are linked to the function of macromolecules (Bourgeois et Royant, Curr. Opin. Struc. Biol. (2005) 15, 1-10). In a few favorable cases (for example the case of myoglobin), ultra-fast methods such as based on Laue diffraction allow to record real-time movies of proteins in action, with time-resolutions reaching 100 ps. However, these methods are difficult to apply. Nevertheless, it is often possible to “trap” reaction intermediates within the crystal, and then to solve the structure of these intermediates with more standard data collection techniques (see the case of superoxyde reductase : Katona et al, Science, (2007), 316, 449-52). To this aim, we have developed several instruments and methodological approaches based on photo-activation of endogenous or exogenous chromophores, precise temperature control of the samples, and monitoring of the reactions by “in crystallo” spectroscopy (UV-visible absorption and fluorescence, fluorescence lifetime, and recently Raman spectroscopy). “In crystallo” spectroscopy constitutes an essential part of our scientific activity. The methodological developments are carried out at the “Cryobench” laboratory, located at the ESRF (Bourgeois et al, J. Appl. Cryst. 2002, 35, 319; Royant et al, J. Appl. Cryst. 2007, 40, 1105, Carpentier et al, J. Appl. Cryst. 2007, 40, 1113).

Our activity is largely multi-disciplinary, at the interface between biology, physics and chemistry. Through various collaborations, since 2001 we have studied the enzymatic mechanisms of several biologically and/or medically relevant proteins (thymidylate kinase from M. tuberculosis : Fioravanti et al, J. Mol. Biol. (2003) 327, 1077-1092, Fioravanti et al, Biochemistry (2005) 44, 130-137, superoxide reductase from D. baarsii : Adam et al, Structure (2004), 12, 1729-1740, Katona et al, Science.(2007) 316, 449-52; acetylcholinesterase from T. californica : Colletier et al, Acta. Cryst. D, (2007), 63, 1115-1128, Colletier et al, PNAS (2008), 105, 11742-47, protochlorophyllide oxido-reductase from T. elongatus, Durin et al, submitted, myoglobin : Bourgeois et al, PNAS (2003) 100, 8704-8709, Bourgeois et al, PNAS (2006), 103(13), 4924-4929 and Drosophila nuclear receptors : de Rosny et al, Biochemistry (2006).45(32), 9727-34, Vos et al. Biochemistry (2008).47(21), 5718-23).

Since 2006, one major theme has emerged in the team : the study of structural dynamics of fluorescent proteins.

Fluorescent proteins

Since the cloning of GFP (Green Fluorescent Protein) in 1992, fluorescent proteins (FPs) have become extremely valuable tools in life sciences, as biological markers. Indeed, these proteins may be fused to many targets of interest, allowing us to follow localization and movement in real time in living cells, as a function of the environmental conditions or under the influence of drugs. Our project consists in studying from a structural standpoint the mechanisms by which FPs fluoresce, change their fluorescence properties, or cease to fluoresce. In particular, we are interested in photochromism and photoactivation properties encountered in a new set of fluorescent proteins which for most of them originate from Anthozoa species (corals, anemones). From a better fundamental knowledge of these mechanisms, we will attempt to engineer new FPs with improved properties in a rational manner. Several fluorescent proteins are studied, combining several approaches (crystallography, spectroscopy, ab initio simulations): EosFP and IrisFP, Keima, KillerRed, ECFP and Cerulean, Venus and Citrine. Since 2008, this project is funded by the French « ANR blanc » program.

Composition of the team

Publications

• List of publications from the group of protein kinetic crystallography
• List of publications related to the Cryobench laboratory

Collaborations

The team relies on an internal, close collaboration between the IBS and the ESRF, which has started in 1993 and which has led to the construction of the “Cryobench” laboratory. This collaboration is essential to the success of the team, both scientifically and financially

At the IBS, our team is strengthened by transverse collaborations with:

• The « Laboratoire de Biophysique Moléculaire » (LBM : Martin Weik’s team), which brings a strong expertise in the field of protein dynamics, and carries out the investigations on cholinesterases.
• The « Laboratoire de Dynamique Moléculaire » (LDM, Martin Field’s team) which participates in the Fluorescent Proteins project by performing ab initio calculations (QM/MM).
• The « Laboratoire d’Ingénierie des Macromolecules » (LIM, Marjolaine Noirclerc-Savoye), which participates in the Fluorescent Proteins project (expression/purification).

The Fluorescent Proteins project is also run through external collaborations

• University of Ulm, Germany
• PCV, iRTSV, CEA, Grenoble (Laurent Blanchoin et al)

The globin project (real-time-resolved Laue cristallography) is run in the frame of external collaborations with:

• University of Rome,
• NIH, Bethesda, USA

Gallery

Absorption and fluorescence microspectrophotometer. Details of the experimental setup (Bourgeois et al., 2002).

TDFM spectroscopy. Photo of a flash-cooled lysozyme crystal soaked in fluorescein and mounted in a cryoloop at 100K. Fluorescence (green light) is observed upon continuous laser illumination with blue light (Weik et al., 2004).

Thymidylate kinase from Mycobacterium tuberculosis. View of the catalytic site in the active form of the enzyme (complexed with dTMP and a magnesium ion), and in its AZTMP-inhibited form (Fioravanti et al., in press).

Superoxide reductase from Desulfoarculus baarsii. Details of ferricyanide binding in the active site of the enzyme, causing partial inhibition of the activity (Adam et al., 2004).

Enzymatic mechanism of superoxide reductase fom Desulfoarculus baarsii. A lysine residue imports a water molecule essential for H2O2 production into the SOR active site. (Katona et al., 2007).

Structure of EosFP, a fluorescent photoconvertible protein (collaboration with Ulm University, Germany)

Fluorescent Protein IrisFP, a photoconvertible and photochromic fluorescent protein (collaboration with Ulm University, Germany)