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. 2022 Apr 22;11:e76100. doi: 10.7554/eLife.76100

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© 2022, Du et al

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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Figure 5. — (A) Deuterium uptake plots showing how the F117A mutation affects hydrogen-deuterium exchange in a peptide corresponding to residues 115–125 located in the α5–6 motif of UCH37. For comparison, the uptake plots corresponding to a region located outside the α5–6 motif (residues 200–218) are also shown. The data on the top represents the rate of exchange with UCH37^C88A•RPN13^DEUBAD and the data on the bottom corresponds to UCH37^C88A/F117A•RPN13^DEUBAD. At least two replicates of each experiment were performed. (B)¹H¹³C-methyl TROSY NMR spectra of the Ile region of ILV-labeled K48 di-ubiquitin (Ub) proximal subunit (Ub^prox) free in solution (gray) and bound to UCH37^C88A•RPN13^DEUBAD (the C88A complex; green). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A•RPN13^DEUBAD and 30 μM K48 di-Ub. (C)¹H¹³C-methyl TROSY NMR spectra of the Ile region of ILV-labeled K48 di-Ub distal subunit (Ub^dist) free in solution (gray) and bound to the C88A complex (orange). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A •RPN13^DEUBAD and 30 μM K48 di-Ub. (D) ¹H¹³C-methyl TROSY NMR spectra of the Ile region of K48-linked Ub^prox free in solution (gray) and bound to UCH37^C88A/F117A•RPN13^DEUBAD (the F117A complex; green). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A/F117A•RPN13^DEUBAD and 30 μM K48 di-Ub. (E)¹H¹³C-methyl TROSY NMR spectra of the Ile region of K48-linked Ub^dist free in solution (gray) and bound to the F117A complex (orange). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A/F117A•RPN13^DEUBAD and 30 μM K48 di-Ub. (F)¹H¹³C-methyl TROSY NMR spectra of the Ile region of mono-Ub bound to the C88A complex (orange) and the F117A complex (blue). Ratio of UCH37 to mono-Ub is 1:1.5. Concentrations used: 45 μM UCH37 •RPN13^DEUBAD complex and 30 μM mono-Ub.

Figure 5—source data 1. Full NMR spectra of mono-Ub and K48 di-Ub in presence and absence of UCH37.

elife-76100-fig5-data1.pdf^{(2.7MB, pdf)}

Figure 5—figure supplement 1. — (A) Deuterium uptake plots showing how the F117A mutation affects hydrogen-deuterium exchange in a peptide corresponding to residues 115–125 located in the α5–6 motif of UCH37. For comparison, the uptake plots corresponding to a region located outside the α5–6 motif (residues 200–218) are also shown. The data on the top represents the rate of exchange with UCH37^C88A•RPN13^DEUBAD and the data on the bottom corresponds to UCH37^C88A/F117A•RPN13^DEUBAD. At least two replicates of each experiment were performed. (B)¹H¹³C-methyl TROSY NMR spectra of the Ile region of ILV-labeled K48 di-ubiquitin (Ub) proximal subunit (Ub^prox) free in solution (gray) and bound to UCH37^C88A•RPN13^DEUBAD (the C88A complex; green). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A•RPN13^DEUBAD and 30 μM K48 di-Ub. (C)¹H¹³C-methyl TROSY NMR spectra of the Ile region of ILV-labeled K48 di-Ub distal subunit (Ub^dist) free in solution (gray) and bound to the C88A complex (orange). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A •RPN13^DEUBAD and 30 μM K48 di-Ub. (D) ¹H¹³C-methyl TROSY NMR spectra of the Ile region of K48-linked Ub^prox free in solution (gray) and bound to UCH37^C88A/F117A•RPN13^DEUBAD (the F117A complex; green). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A/F117A•RPN13^DEUBAD and 30 μM K48 di-Ub. (E)¹H¹³C-methyl TROSY NMR spectra of the Ile region of K48-linked Ub^dist free in solution (gray) and bound to the F117A complex (orange). Ratio of UCH37 to K48 di-Ub is 1:1.5. Concentrations used: 45 μM UCH37^C88A/F117A•RPN13^DEUBAD and 30 μM K48 di-Ub. (F)¹H¹³C-methyl TROSY NMR spectra of the Ile region of mono-Ub bound to the C88A complex (orange) and the F117A complex (blue). Ratio of UCH37 to mono-Ub is 1:1.5. Concentrations used: 45 μM UCH37 •RPN13^DEUBAD complex and 30 μM mono-Ub.

Figure 5—source data 1. Full NMR spectra of mono-Ub and K48 di-Ub in presence and absence of UCH37.

elife-76100-fig5-data1.pdf^{(2.7MB, pdf)}