(1) Department of Structural Biology, Weizmann Institute of Science, Rehovot, Israel (2) Utrecht University, Utrecht, The Netherlands (3) IBS, Grenoble, France (4) Universit Louis Pasteur, Strasbourg, France (5) University of Chicago, Chicago, USA (6) Department of Neurobiology, Weizmann Institute of Science, Rehovot, Israel (7) Biology Department, BNL, Upton, USA
In the last ten years it has become feasible to study dynamic processes in macromolecules at the atomic level. Most of these studies have used the Laue diffraction technique that permits data collection on a second to pico-second time scale by virtue of the polychromatic synchrotron radiation.
It is our aim to do time-resolved experiments on the enzyme acetylcholinesterase (AChE). AChE cleaves the neurotransmitter acetylcholine (ACh) in the synapse at a very high turnover rate. The enzyme has a strong dipole moment that draws the positively charged substrate towards the catalytic site, situated at the bottom of a narrow gorge lined with aromatic residues. One of the reaction products, choline, bears the same charge as the substrate, ACh, which raises the problem of its exit route from the active site. In order to attempt to observe the reaction process, photolabile precursors of choline and of carbamylcholine have been synthesized and characterized [1]. These tools for the time-resolved crystallographic studies will hopefully permit monitoring of the exit of choline from the active site.
To test the feasibility of the use of the Laue method, trigonal crystals of Torpedo californica AChE were used to collect data at the ESRF in Grenoble, beam line ID9 (BL3) on a CCD detector with an exposure time of 1 msec. The frames were processed using the program LaueView [2], yielding a data set to 2.7 resolution of 83% completeness with 23,403 unique reflections. Careful refinement of the native protein coordinates with X-PLOR gave electron density maps that are almost comparable in quality to maps made with monochromatic data.
1. Peng, L. & Goeldner, M. Synthesis and Characterization of Photolabile Choline Precursors as Reversible Inhibitors of Cholinesterases: Release of Choline in the Microsecond Time Range. Journal of Organic Chemistry 61, 185-191 (1996).
2. Ren, Z. & Moffat, K. Quantitative Analysis of Synchrotron Laue Diffraction Patterns in Macromolecular Crystallography. J. Appl. Cryst. 28, 461-481 (1995).