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

FIGURE 5.

FIGURE 5.

Evidence that mtHSP70 binds preferentially to the D-state of ISCU. A, two-dimensional 1H,15N HSQC spectra of U-15N-ISCU (left panel), U-15N-ISCU sample mixed with 0.2 eq of unlabeled mtHSP70 and diluted by a factor of 1.2 (middle panel), U-15N-ISCU mixed with 1.0 eq of mtHSP70 and diluted by a factor of 2 (right panel). B, expansions of the Trp-76 1H,15N cross-peaks from the spectra in A. C, %S calculated by FMLR analysis of the intensities of the Trp-76 cross-peaks assigned to S and D under the conditions indicated. D, two-dimensional 1H,15N TROSY-HSQC spectra of U-15N-ISCU(N90A) (left panel), U-15N-ISCU(N90A) plus 0.2 eq of unlabeled mtHSP70 and diluted by a factor of 1.2 (middle panel), U-15N-ISCU(N90A) mixed with 1.0 eq of mtHSP70 and diluted by a factor of 2 (right panel). E, expansions of the Trp-76 1H,15N cross-peaks from the spectra in D. F, %S calculated by FMLR analysis (44) of the intensities of the Trp-76 cross-peaks assigned S and D under the conditions indicated. G, two-dimensional 1H,15N TROSY-HSQC spectra of U-15N-ISCU(H105A) (left panel), U-15N-ISCU(H105A) mixed with 0.2 eq of unlabeled mtHSP70 and diluted by a factor of 1.2 (middle panel), U-15N-ISCU(H105A) plus 1.0 eq of unlabeled mtHSP70 and diluted by a factor of 2 (right panel). H, expansions of the Trp-76 1H,15N cross-peaks from the spectra in G. I, %S calculated by FMLR analysis of the intensities of the Trp-76 cross-peaks assigned S and D under the conditions indicated. All NMR spectra were collected at 600 MHz (1H) at 25 °C with samples at pH 8.0.