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. 2013 Aug 12;288(40):28755–28770. doi: 10.1074/jbc.M113.482042

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© 2013 by The American Society for Biochemistry and Molecular Biology, Inc.

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FIGURE 4. — Evidence that NFS1 binds preferentially to the D-state of ISCU. A, top panel, two-dimensional ¹H,¹⁵N HSQC spectrum of U-¹⁵N-ISCU. Middle panel, overlay of the two-dimensional ¹H,¹⁵N TROSY-HSQC spectra of U-¹⁵N-ISCU alone (red) and U-¹⁵N-ISCU (blue) mixed with a stoichiometric amount of unlabeled NFS1. Bottom panel, overlay of two-dimensional ¹H,¹⁵N TROSY-HSQC spectra of U-¹⁵N-ISCU (red) and the same sample mixed with stoichiometric amounts of unlabeled NFS1 and ISD11 (green). B, expansions show the ¹H,¹⁵N peaks from the indole ring of Trp-76 of ISCU. Top panel, U-¹⁵N-ISCU. Middle panel, U-¹⁵N-ISCU mixed with equimolar unlabeled NFS1. Bottom panel, U-¹⁵N-ISCU mixed with equimolar unlabeled NFS1 and ISD11. C, top panel, two-dimensional ¹H,¹⁵N HSQC spectrum of U-¹⁵N-ISCU. Bottom panel, two-dimensional ¹H,¹⁵N HSQC spectrum of U-¹⁵N-ISCU mixed with equimolar unlabeled ISD11. All NMR spectra were collected at 600 MHz (¹H) at 25 °C with samples at pH 8.0. D and E, %S calculated by FMLR analysis of the intensities of Trp-76 cross-peaks assigned to the S- and D-states under the conditions indicated.