Abstract

Structural changes in biological macromolecules are very fast at ambient temperature and often occur on submicrosecond time scale. Recent technological, instrumentation, and software developments allow us to investigate these fast structural changes in protein crystals on nanosecond and possibly subnanosecond time scales. These investigations require brilliant, focused, polychromatic, and pulsed x-ray sources, available today at third-generation synchrotrons; fast shutter systems¹ to isolate individual single or super-x-ray pulses; high- efficiency, low-noise area detectors¹ to record relatively weak diffraction patterns; uniform initiation of structural changes in crystals by laser pulses synchronized with x-ray pulses;¹ optical monitoring of crystals, in parallel to x-ray measurements, to quantify reaction initiation and progress;¹'² and novel data-pro-cessing methods for weak and mosaic Laue diffraction patterns.³ We are conducting nanosecond time-resolved crystallographic studies at the ID9 beamline of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France, on two heme proteins: sperm whale myoglobin (Mb) and dimeric hemoglobin (HbI) from the dam Scapharca in-aequivalvis. The goals of these studies are to understand the evolution of the photo-induced structural changes and their propagation from the active site through the protein and to determine the trajectories and the docking sites of the photo-dissociated ligand and how they are affected by mutations of side chains known to affect ligand binding properties. We use intense, focused, white, 150-ps and 1 - µs x-ray pulses in a pump-probe type of measurement to investigate structural changes in carbonmonoxy complexes of Mb and HbI. Changes are photo-initiated by 10-ns laser pulses. The complete reversibility of the reaction simplifies the use of the signal averaging (typically 10- to 30-fold) to improve the data quality and allows us to obtain complete and even multiple data sets from one crystal. Data are typically 70% complete to 1.7Å resolution, with R_merge≈12%. Departure of the CO ligand upon photolysis and its subsequent rebinding, and the iron atom displacement from the heme plane are clearly observed in both molecules. We also identify a possible docking site for the photo-dissociated CO molecule in the MbCO heme pocket.

© 1998 Optical Society of America