In order to be biologically useful, protein molecules rely on structural transitions to carry out their function. Despite their importance, it is usually difficult to follow such conformational rearrangements as they occur. Recent advances in time-resolved crystallography have extended its capability into the nanosecond (ns) time ranges required to observe such structural transitions in real time. We will apply time-resolved crystallographic techniques to a cooperative dimeric hemoglobin from Scapharca inaequivalvis that has proven to be an excellent model system for studying allosteric protein behavior. This protein is exceptionally well suited for such an analysis due to properties that include the ability to observe cooperative ligand binding behavior within the crystalline state and the diffracting power of its crystals. We propose to use this technique to investigate structural changes induced by ligand photo-dissociation. Structural changes will be triggered by short (10 ns) laser pulses and probed by 150 picosecond (ps) to 15ns X-ray pulses. This type of experiment can be successfully conducted at third generation synchrotron sources such as the Advanced Photon Source (APS - Argonne, USA), European Synchrotron Radiation Facility (ESRF - Grenoble, France) and SPring8 (Tsukuba, Japan), as extensive experiments on myoglobin and photoactive yellow protein have demonstrated. We have conducted preliminary experiments on wildtype and mutants of this dimeric hemoglobin at ESRF and APS that clearly show the feasibility of this approach. Our results to date show the loss of bound ligand coupled with heme doming and movement of the protein atoms, suggesting important transient intermediates at several time points between l ns and 25ns following ligand release. Density for released ligand suggests it follows an alternate pathway in transit between the active site and solvent. Our goal is to combine time-resolved and ultrahigh resolution crystallography with mutagenesis, spectroscopy and molecular dynamics simulation to gain an unprecedented understanding of principles by which conformational rearrangements mediate allosteric regulation of biological processes. Understanding such principles will aid in the rational manipulation of a number of biological processes.
| Knapp, James E; Pahl, Reinhard; Cohen, Jordi et al. (2009) Ligand migration and cavities within Scapharca Dimeric HbI: studies by time-resolved crystallo-graphy, Xe binding, and computational analysis. Structure 17:1494-504 |
| Srajer, Vukica; Royer Jr, William E (2008) Time-resolved x-ray crystallography of heme proteins. Methods Enzymol 437:379-95 |
| Nienhaus, Karin; Knapp, James E; Palladino, Pasquale et al. (2007) Ligand migration and binding in the dimeric hemoglobin of Scapharca inaequivalvis. Biochemistry 46:14018-31 |
| Nichols, Jeffry C; Royer Jr, William E; Gibson, Quentin H (2006) An optical signal correlated with the allosteric transition in Scapharca inaequivalvis HbI. Biochemistry 45:15748-55 |
| Knapp, James E; Pahl, Reinhard; Srajer, Vukica et al. (2006) Allosteric action in real time: time-resolved crystallographic studies of a cooperative dimeric hemoglobin. Proc Natl Acad Sci U S A 103:7649-54 |
