This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.We are using NMR techniques to characterize the interaction between theiron-sulfur cluster template protein IscU and the chaperone protein Hsc66and possibly Hsc20.Hsc66 is an Hsp70-class molecular chaperone, that is important in thebiogenesis of Fe-S proteins. Although the exact role of Hsc66 in thisprocess is unknown, the importance of Hsc66 in Fe-S cluster assembly mayarise from its interactions with the Fe-S cluster template protein IscU.Recent studies indicate that IscU behaves as a substrate for Hsc66, andthis interactions may serve to regulate the type and stability of Fe-Sclusters formed on IscU. Moreover, an increase in the ATPase activity ofHsc66, suggests that the interaction of IscU with Hsc66 is furtherenhanced by the co-chaperone Hsc20, which directly binds to both IscU andHsc66. We have collected preliminary NMR spectra on unlabeled IscU and Hsc66proteins alone in solution. One-dimensional 1H spectra and two-dimensional natural abundance 15N-HSQCand homonuclear NOESY spectra adquired on an apo form of unlabeled IscU,suggest that IscU alone in solution exists as a mixture of differentspecies that are partially unfolded or disordered and are exchangingduring the the time of the NMR experiment (as indicated by the presence ofexchange peaks in NOESY spectra). It is possible that the interaction with Hsc66 and/or Hsc20 may favorand stabilize the structure of one of these IscU species that isbiologically relevant. On the other hand, spectra adquired on unlabeled Hsc66 alone suggest thatthis chaperone protein is folded even in the absence of IscU.We are now in the process of obtaining 15N and/or 13C enriched forms ofthe proteins to better study their structure and characterize theirinteractions at atomic resolution by using heternonuclear NMR techniques.
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