Figure 5.

Addition of Ub alters the position of the proximal Ub^G76C upon chain elongation. (A) Expanded regions for ¹H-¹⁵N HSQC spectra of Ub^G76C (black) and labeled Ub^G76C in the UBE2K-Ub^G76C-Ub^K48R conjugate (magenta) are shown. Selected signals that are visible at lower contour levels in the UBE2K-Ub^G76C-Ub^K48R conjugate are boxed. Signals that are absent in the UBE2K-Ub^G76C conjugate but are visible in UBE2K-Ub^G76C-Ub^K48R are shown in italics. (B) A cartoon structure is given, showing residues that undergo significant chemical shift changes (average + 1 SD) in Ub^G76C (pink) and residues that “reappear” in the ¹H-¹⁵N HSQC spectrum of UBE2K-Ub^G76C-Ub^K48R that were absent because of conformational exchange in UBE2K-Ub^G76C (purple). The site of the K48 linkage in Ub^G76C with the distal Ub^K48R in UBE2K-Ub^G76C-Ub^K48R is indicated (blue). (C) Expanded regions of ¹H-¹⁵N HSQC spectra of the proximal Ub^G76C in Ub^G76C-Ub^K48R (blue) and UBE2K-Ub^G76C-Ub^K48R (magenta) are shown. Signals with significant shifts (average + 1 SD) are indicated. Boxes indicate the position of a signal that is visible at lower contour levels. (D) A cartoon representation of Ub showing significantly shifted residues from (C) is given.