Fig. 3.
Comparison of biophysical characteristics of wt ISCU and M140 mutants, and their corresponding effect on complex activity. (A) Circular Dichroism was used to determine secondary structure of the two states of ISCU and compare the WT to the M140I, M140L, and M140V variants and found no difference between them and showed similarly that the zinc-bound state had a higher helical percentage and a lower disordered percentage than that of the zinc-depleted state. (B) Analytical gel filtration was used to determine if complex formation was intact for NFS-ISD11-ACP (SDA), NFS1-ISD11-ACP-ISCUWT (SDAUWT), NFS1-ISD11-ACP--ISCUM140I (SDAUM140I), NFS1-ISD11-ACP-ISCUM140L (SDAUM140L), and NFS1-ISD11-ACP-ISCUM140V (SDAUM140V). (C, D) The methylene blue assay was used to determine the effect of desulfurase activity on the two states of ISCU WT and variants using buffer supplemented with either 100 μM EDTA (C) or 50 μM ZnCl2 (D). When noted, concentration of NFS1-ISD11-ACP (SDA) was at 0.5 μM, ISCU (U) at 2.5 μM, and Frataxin (F) at 40 μM. For (C) and (D) error bars denote standard deviations determined from experimental replicates of n = 3.