Abstract
Time-resolved Laue protein crystallography at the European Synchrotron Radiation Facility (ESRF) opened up the field of sub-nanosecond protein crystal structure analyses. There are a limited number of such time-resolved studies in the literature. Why is this? The X-ray laser now gives us femtosecond (fs) duration pulses, typically 10 fs up to ∼50 fs. Their use is attractive for the fastest time-resolved protein crystallography studies. It has been proposed that single molecules could even be studied with the advantage of being able to measure X-ray diffraction from a 'crystal lattice free' single molecule, with or without temporal resolved structural changes. This is altogether very challenging R&D. So as to assist this effort we have undertaken studies of metal clusters that bind to proteins, both 'fresh' and after repeated X-ray irradiation to assess their X-ray-photo-dynamics, namely Ta6Br12, K2PtI6 and K2PtBr6 bound to a test protein, hen egg white lysozyme. These metal complexes have the major advantage of being very recognisable shapes (pseudo spherical or octahedral) and thereby offer a start to (probably very difficult) single molecule electron density map interpretations, both static and dynamic. A further approach is to investigate the X-ray laser beam diffraction strength of a well scattering nano-cluster; an example from nature being the iron containing ferritin. Electron crystallography and single particle electron microscopy imaging offers alternatives to X-ray structural studies; our structural studies of crustacyanin, a 320 kDa protein carotenoid complex, can be extended either by electron based techniques or with the X-ray laser representing a fascinating range of options. General outlook remarks concerning X-ray, electron and neutron macromolecular crystallography as well as 'NMR crystallography' conclude the article.
Highlights
The advent of intense synchrotron X-ray sources has allowed time-resolved diffraction in general, and macromolecular crystallography in particular, to have wide applicability to study time dependent phenomena at the molecular level; as spearhead time-resolved Laue protein crystallography at the ESRF opened up the eld of sub-nanosecond crystal structure analyses.[1]
It has been proposed that single molecules could even be studied with the advantage of being able to measure X-ray diffraction from a ‘crystal lattice free’ single molecule, with or without temporal resolved structural changes
As to assist this effort we have undertaken studies of metal clusters that bind to proteins, both ‘fresh’ and after repeated X-ray irradiation to assess their Xray-photo-dynamics, namely Ta6Br12, K2PtI6 and K2PtBr6 bound to a test protein, hen egg white lysozyme
Summary
The advent of intense synchrotron X-ray sources has allowed time-resolved diffraction in general, and macromolecular crystallography in particular, to have wide applicability to study time dependent phenomena at the molecular level; as spearhead time-resolved Laue protein crystallography at the ESRF (http:// www.esrf.eu/UsersAndScience/Experiments/So Matter/ID09B) opened up the eld of sub-nanosecond crystal structure analyses.[1]. Where we can expect difficult to interpret electron density maps, and their time-resolved changes in biochemical reactions, these ‘marker well-shaped complexes’ will offer a start to electron density map interpretations.[9] The break-up of platinum hexabromide, even when cryocooled, upon prolonged X-ray irradiation, akin to the UV study,[11] was reported previously.[13] Here we report the HEWL with bound PtBr6 at room temperature upon prolonged X-ray irradiation. The X-ray-photo-dynamics behaviour of PtBr6 at room temperature when bound to hen egg white lysozyme and the stages of its stepwise removal of bromine atoms are shown in Fig. 4 which compares the metal complex at successive stages of X-ray irradiation, namely data sets 1, 3, 5 and 7 (data sets 2, 4 and 6 are not shown), i.e. with cumulatively increasing X-ray dose to the same crystal. Glebov et al.[11] in their UV-vis studies undertook ash photolysis and ‘steady state illumination’ experiments and kinetics analyses and from which we quote:-“The spectral curves obtained by steady state irradiation and pulse photolysis coincide, indicating that the rst photoaquation step occurred within the time
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