Abstract
Protein X-ray structures are determined with ionizing radiation that damages the protein at high X-ray doses. As a result, diffraction patterns deteriorate with the increased absorbed dose. Several strategies such as sample freezing or scavenging of X-ray-generated free radicals are currently employed to minimize this damage. However, little is known about how the absorbed X-ray dose affects time-resolved Laue data collected at physiological temperatures where the protein is fully functional in the crystal, and how the kinetic analysis of such data depends on the absorbed dose. Here, direct evidence for the impact of radiation damage on the function of a protein is presented using time-resolved macromolecular crystallography. The effect of radiation damage on the kinetic analysis of time-resolved X-ray data is also explored.
Highlights
Time-resolved macromolecular crystallography (Moffat, 1989) is a unique method that is able to determine atomic structure and chemical kinetics at the same time (Schmidt, 2008)
Little direct information is available on whether and how the protein kinetics is affected by radiation damage
Synchrotron beamlines, which are specialized in time-resolved crystallography (Graber et al, 2011), provide very specific experimental capabilities for the collection of time-resolved X-ray data: the beam size is much smaller than the crystal size typically used for time-resolved data collection, reciprocal space is covered at random to avoid orientational preferences, the crystal is translated along its axis after the collection of a diffraction pattern to expose a fresh crystal volume to the X-rays, and the X-ray beam impinges the crystal as close as possible to the surface where the extent of reaction initiation by a laser pulse is at a maximum
Summary
Time-resolved macromolecular crystallography (Moffat, 1989) is a unique method that is able to determine atomic structure and chemical kinetics at the same time (Schmidt, 2008). Techniques like cryo-cooling (Kuzay et al, 2001; Nicholson et al, 2001) and free-radical scavengers (Murray & Garman, 2002) are used in macromolecular crystallography to reduce radiation damage. Little direct information is available on whether and how the protein kinetics is affected by radiation damage. Extremely intense and ultra-short pulses of polychromatic narrow-bandwidth (pink) X-ray radiation are employed. All of these experimental details characteristic of time-resolved studies have never been taken into account in the calculations of the absorbed dose. To address the impact of radiation damage on the protein kinetics a reaction needs to be selected that can be readily investigated
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