Kinetic protein crystallography : a new approach to visualize reaction intermediates at the near-atomic scale
Kinetic crystallography consists in triggering biological function in the crystalline state of a protein, so as to capture functionally relevant conformational changes. The majority of protein structures presently solved by X-ray crystallography actually correspond to a static state, that represent only poorly the ensemble of conformations adopted in reality by a protein in action. “Kinetic protein crystallography” allows investigating structure in a dynamical fashion. See Bourgeois & Weik, Crystallography Rev. (2009) 15,87-118 and Bourgeois & 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, whose time-resolution reach 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 (for an example in the team: see 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 Raman spectroscopy). We currently apply these techniques to the case of fluorescent proteins, which are particularly well adapted to kinetic crystallography approaches.
Enzymatic mechanism of superoxyde reductase fom Desulfoarculus baarsii. A lysine residue imports a water molecule essential for H2O2 production into the SOR active site. (Katona et al., 2007 DOI: 10.1126/science.1138885).