This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. X-ray diffraction studies conducted by our group and others have revealed that the nitrogenase Fe protein can exist in a number of conformations;however, not all conformational states of the Fe protein of interest, in terms of defining the role of MgATP binding and hydrolysis, have been characterized. The key missing structure in defining nucleotide dependent conformation change in the Fe proteins is the Fe protein with bound MgATP. Determining the structure of a protein that undergoes nucleotide dependent conformational change in the presence of bound nucleoside triphosphates is challenging, partially attributable to the fact that nucleoside triphosphates in aqueous solutions are rapidly hydrolyzed under most circumstances over the course of the time it takes to obtain protein crystals. We therefore intend to conduct solution studies using SAXS to complement our X-ray diffraction studies. Simulations based on known structures reveal strikingly different scattering curves in the 1/q range of 0.15 and 0.25 for the Fe protein with MgADP bound and the Fe protein variant presumed to represent a mimic of the native MgATP bound state. This provides the basis for examining whether the conformation of the native Fe protein with MgATP bound resembles in conformation the Fe protein with MgADP bound or the Fe protein variant assigned as a putative MgATP bound mimic. Preliminary investigations at SSRL on Beam Line 4-2 have provided the proof-of-concept and demonstrated that the proposed studies are feasible. We are requesting approval for beamtime to further optimize our data collection strategies, examine nucleotide bound states directly.
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