W0043

Time-Resolved Crystallographic Analysis of the Allosteric Pathway in a Cooperative Dimeric Hemoglobin. W.E. Royer¹, J. E. Knapp¹, V. Srajer² and R. Pahl², ¹Dept. of Biochemistry and Molecular Pharmacology, Univ. of Massachusetts Medical School, Worcester, MA 01605, ²Dept. of Biochemistry and Molecular Biology, The Univ. of Chicago, 920 East 58^th St., Chicago, IL 60637 & Consortium for Advanced Radiation Sources, The Univ. of Chicago, 5640 South Ellis Ave, Chicago, IL 60637.

Allosteric changes in proteins regulate many fundamental processes in biology. While the structures of the endpoints for a number of allosteric proteins are known, knowledge about the pathway between them is quite limited. ith recent progress in time-resolved crystallographic methods extending into nanosecond time ranges, the opportunity exists to follow such pathways in real time. Particularly amenable protein for investigating allosteric behavior is the cooperative dimeric hemoglobin found in the mollusk, Scapharca inaequivalvis. As with other heme proteins, the CO complex of this hemoglobin can be efficiently photolyzed by a laser pulse to trigger a reaction that can be probed by subsequent short pulses of X-ray photons. Properties that make this particular system exceptionally well suited for an ultrafast time-resolved study include the relatively localized structural transitions mediating strong cooperativity, the diffracting power of its crystals and the full cooperative ligand binding exhibited within the crystal lattice. We have collected time-resolved crystallographic data for wild-type crystals with delay times between the laser pulse and x-ray data collection of 1ns to 25ns, along with data on a mutant (M37V) with delay times of 5ns to 800ns. These structures have revealed transient conformations likely to be important for the allosteric mechanism and provide insight into the energetic barriers that separate the R and T states. In addition, the structures are providing evidence that the pathway for ligand transit in this dimeric hemoglobin is quite different from that in monomeric myoglobin or mammalian teterameric hemoglobins.