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. We are using NMR techniques to characterize the interaction between the iron-sulfur cluster template protein IscU and the chaperone protein Hsc66 and possibly Hsc20. Hsc66 is an Hsp70-class molecular chaperone, that is important in the biogenesis of Fe-S proteins. Although the exact role of Hsc66 in this process is unknown, the importance of Hsc66 in Fe-S cluster assembly may arise from its interactions with the Fe-S cluster template protein IscU. Recent studies indicate that IscU behaves as a substrate for Hsc66, and this interactions may serve to regulate the type and stability of Fe-S clusters formed on IscU. Moreover, an increase in the ATPase activity of Hsc66, suggests that the interaction of IscU with Hsc66 is further enhanced by the co-chaperone Hsc20, which directly binds to both IscU and Hsc66. We have collected preliminary NMR spectra on unlabeled IscU and Hsc66 proteins alone in solution. One-dimensional 1H spectra and two-dimensional natural abundance 15N-HSQC and homonuclear NOESY spectra adquired on an apo form of unlabeled IscU, suggest that IscU alone in solution exists as a mixture of different species that are partially unfolded or disordered and are exchanging during the the time of the NMR experiment (as indicated by the presence of exchange peaks in NOESY spectra). It is possible that the interaction with Hsc66 and/or Hsc20 may favor and stabilize the structure of one of these IscU species that is biologically relevant. On the other hand, spectra adquired on unlabeled Hsc66 alone suggest that this chaperone protein is folded even in the absence of IscU. We are now in the process of obtaining 15N and/or 13C enriched forms of the proteins to better study their structure and characterize their interactions at atomic resolution by using heternonuclear NMR techniques.
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