NANOSECOND TIME-RESOLVED MACRO-MOLECULAR CRYSTALLOGRAPHY: PHOTOLYSIS OF CARBONMONOXY MYOGLOBIN. V. Srajer^*, T.-Y. Teng^*, T. Ursby⁺ , C. Pradervand^*, Z. Ren^*, S. Adachi^[[section]], W. Schildkamp^*, D. Bourgeois^{+[[daggerdbl]]}, M. Wulff⁺ and K. Moffat^{* *}Department of Biochemistry and Molecular Biology and CARS, University of Chicago, Chicago, IL, USA ⁺ESRF, Grenoble, France ^{[[daggerdbl]]}UPR 9015/IBS, Grenoble, France ^[[section]]RIKEN, Saitama, Japan

The pulse structure and high brilliance of the focused white synchrotron radiation from the BL3 wundulator¹ at ESRF, Grenoble, France were utilized to explore structural changes in carbonmonoxy myoglobin (MbCO) crystals induced by a 10ns laser pulse. Several essential experimental features were implemented: a fast shutter system¹ to isolate individual single and super- pulses; a high efficiency, low-noise area detector¹ to record relatively weak diffraction patterns; uniform photolysis of crystals by ns laser pulses synchronized with x-ray pulses¹; parallel X-ray and optical measurements on crystals to quantify reaction initiation and progress²; and novel Laue data processing methods³. Complete Laue X-ray diffraction data were collected using either single, 60ps X-ray pulses or 940ns, "super-pulses" at laser/X-ray pulse time delays of 4ns, 1us, 7.5us, 50us, 350us and 1.9ms. Data were typically about 70% complete to 1.8Å resolution with R_merge~11% and yielded successful wavelength normalization and deconvolution of harmonic energy overlaps. Departure of the CO ligand upon photolysis and subsequent us rebinding are clearly observed as well as partial iron displacement from the heme plane and other smaller, consequent tertiary structural changes in the heme pocket and the F-helix. These experimental results are compared with the structural changes inferred from numerous spectroscopic experiments and molecular dynamics simulations. They establish the feasibility of nanosecond time-resolved macromolecular crystallography.

¹Bourgeois D. et al., J. Synchrotron Rad., in press (1996); ²Chen, Y., Srajer, V., Ng, K., LeGrand, A. and Moffat, K., Rev. Sci. Instrum. 65, 1506 (1994); ³Ren, Z. and Moffat, K., J. Synchrotron Rad. 1, 78 (1994); Ren, Z. and Moffat, K., J. Appl. Cryst. 28, 461 (1995).