MULTIPLE ANOMALOUS DISPERSION AT THE K-ABSORPTION EDGE OF SULFUR WITH BOVINE TRYPSIN. Sigrid Stuhrmann, Klaus S. Bartels, Heinrich B. Stuhrmann, GKSS-Research Center, D-21502 Geesthacht, Germany
Bovine trypsin is a serine protease which has six cystines and two methionines. The biochemistry and the structure of the protease is well known. It is therefore a good candidate for a first more rigourous application of MAD at the K-absorption edge of sulfur. The diffraction data were collected at three different wavelengths near the K-absorption egde of the sulfur containing aminoacids (5.02 ~) at the beamline A1 of HASYLAB (Hamburg). The anomalous dispersion is not obscured by the absorption due the sulfate ions of the mother liquor. The feasibility of protein crystallographic studies at wavelengths near the K-absorption edge of sulfur had first been shown with hen egg white lysozyme by M. Lehmann in 1991[1].
The crystallization method adapted from Bartunik et al. [2] was further improved for cryocooling under special conditions. The best cryoprotectant for the trypsin crystal was a buffer containing 80% of a synthetic sugar (Phytohistol) and 10% ethylenglycol. A special sample holder was developed for maintaining the humid atmosphere of the protein crystal at temperature of -80deg. C in an evacuated environment.
The bovine trypsin crystals have the orthorhombic unit cell a=54.9 Å, b=58.5 Å, c=67.6 Å and the space group P212121 [3]. The completeness of the data set is 90% at 5 Å resolution and 15% in the resolution shell of 5 to 3 Å. Considerable changes had to be made in the program FILM in order to index reflections collected on four area detectors. The difference patterson map based on 1000 unique reflections shows many of the vectors connecting the sulfur atoms. In the first step towards phasing the Bragg reflections it was observed that anomalous dispersion of the disulfide bridges is anisotrophic.
[1] M.S. Lehmann, H.-H. Müller, H.B. Stuhrmann, Acta Cryst. D49, 308-310 (1993)
[2] H.D. Bartunik, L.J. Summers, H.H. Bartsch, J. Mol. Biol. 210, 813-828 (1989)
[3] M. Marquardt, J. Walter, J. Deisenhofer, W. Bode, R. Huber, Acta Cryst. B39,480-490 (1983)