DEUTERATED CONCANAVALIN A STUDIED BY NEUTRON LAUE DIFFRACTION AT 2.4A RESOLUTION
Jarjis Habash1, James Raftery1, John R. Helliwell1, Clive Wilkinson2, A. Joseph Kalb(Gilboa)3
1Department of Chemistry, University
of Manchester, Manchester M13 9PL, UK
2EMBL, F38042, Grenoble Cedex,
France Mogens S. Lehmann ILL, F38042, Grenoble Cedex, France
3The Weizmann Institute of Science,
Rehovot, Israel
Biological structural studies by neutron diffraction permits elucidation of H/D exchange, which can yield vital information in understanding particular catalytic processes at the molecular level. This approach exploits the difference in the neutron scattering lengths between hydrogen and deuterium, which is not feasible via X-ray diffraction. However hydrogen atoms can be found from protein crystallography via ultra- high resolution data collection involving synchrotron radiation, cryoprotection of the sample and electronic detectors. Recently we have shown that both neutron and ultra-high resolution structural studies of the 25kDa protein concanavalin A are feasible [1,2]. The neutron data collection involved Laue geometry, which harnesses a broader band of neutron wavelengths than a monochromatic neutron beam, and a very large IP detector thus allowing data collection in a reasonable period. In the following, most recent, experiment we have studied a H2O/D2O exchanged concanavalin A crystal, soaked over a period of 2 months, rather than vapour exchange over a period of 21 days as used earlier [1]. The deuterated concanavalin A (space group I222, unit cell parameters a=88.7, b=86.5, c=62.5A) used to collect neutron Laue data at the ILL in Grenoble with a neutron wavelength range of 2.49-3.5A. The neutron diffraction data were then used to refine the model using X-PLOR to 2.4A resolution. The neutron density maps revealed that 399 hydrogen atoms along the main chain and side chain of the protein molecule have exchanged with deuterium. Also a total of 140 water molecules are defined as D2O. These results show then a more efficient exchange of hydrogen for deuterium than previously. This study is within a programme of work involving the detailed elucidation of protein saccharide interactions at the molecular level [eg see ref 3].