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. The clam, Scapharca inaequivalvis, possesses two hemoglobins that represent exceptional model system for the investigation of protein allostery. Both hemoglobins bind oxygen cooperatively using a structural mechanism that is very different from the more well studied human hemoglobin. The dimeric hemoglobin, termed HbI, is the simplest possible model system for allostery with two identical subunits. Time-resolved crystallographic analysis of this hemoglobin provided, for the first time, a detailed structural description of allosteric changes in real time (Knapp et. al. 2006, PNAS 103 7649-7654). The tetrameric hemoglobin, termed HbII, is formed from two heterodimers, each of which has a similar assembly to that of HbI. However, the presence of two different subunits will permit investigation of how one subunit impacts a second subunit, which is not possible in the two-fold symmetric HbI. Therefore, we propose to use time-resolved x-ray diffraction experiments to elucidate the kinetic structural pathway in the tetrameric HbII and specific mutants of HbII. Mutants will allow us to separate out the effects of one subunit type on the second subunit either by altering the geminate recombination kinetics, or by locking one subunit in a high affinity or low affinity state. Like HbI, but unlike human hemoglobin, we have recently shown that Scapharca HbII crystals can undergo the full allosteric transition within crystals. As a result, this system is well suited for time-resolved crystallographic experiments of allosteric protein function. Allosteric transition will be triggered by laser photolysis of CO-liganded hemoglobin crystals. At various time points ranging from 100 picoseconds to 100 microseconds, diffraction data will be collected by Laue methods. The structures obtained at these time points will reveal the kinetic pathways as the protein undergoes its allosteric transition from the liganded to the unliganded form.
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| Pfoh, Roland; Pai, Emil F; Saridakis, Vivian (2015) Nicotinamide mononucleotide adenylyltransferase displays alternate binding modes for nicotinamide nucleotides. Acta Crystallogr D Biol Crystallogr 71:2032-9 |
| Mariette, Céline; Guérin, Laurent; Rabiller, Philippe et al. (2015) The creation of modulated monoclinic aperiodic composites in n-alkane/urea compounds. Z Kristallogr Cryst Mater 230:5-11 |
| Yang, Xiaojing; Stojkovi?, Emina A; Ozarowski, Wesley B et al. (2015) Light Signaling Mechanism of Two Tandem Bacteriophytochromes. Structure 23:1179-89 |
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