Evidence for dynamic in vivo interconversion of the conformational states of IscU during iron-sulfur cluster biosynthesis.

IscU is a central component of the ISC machinery and serves as a scaffold for de novo assembly of Fe-S clusters. The dedicated chaperone system composed of the Hsp70-chaperone HscA and the J-protein cochaperone HscB synergistically interacts with IscU and facilitates cluster transfer from IscU to recipient apo-proteins. Here, we report that the otherwise essential roles of HscA and HscB can be bypassed in vivo by a number of single amino acid substitutions in IscU. CD spectroscopic studies of the variant IscU proteins capable of this bypass activity revealed dynamic interconversion between two conformations: the denatured (D) and the structured (S) state in the absence and presence of Zn2+ , respectively, which was far more prominent than interconversion observed in wild-type IscU. Furthermore, we found that neither the S-shifted (more structured) variants of IscU nor the perpetually denatured variants could perform their in vivo role regardless of whether the chaperone system was present or not. The present study thus provides for the first time evidence that an in vivo D-state of IscU exists and implies that conformational interconversion between the S- and D-states of the scaffolding protein is a fundamental requirement for the assembly and transfer of the Fe-S cluster.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.