Fig. 5.

Testing long-range allostery in the 20S CP via L81V mutations. (A) A pair of asymmetric proteasomes has been prepared, containing either an S95C (green frame) or an L81V+S95C (gray frame) α₇-ring that is not NMR active at one end, and an ILVM-¹³CH₃ M1I+M6A+S95C α₇-ring at the other end (*). An additional 20S CP (symmetric) was generated where both α₇-rings are NMR active (*) and contain the L81V substitution (red frame), as a control. (B) Spectral regions of a ¹³C–¹H HMQC dataset containing selected peaks derived from residues that are part of the allosteric pathway linking the α-subunits with the βT1 active sites. Shown with red, gray, and green contours are correlations derived from M1I+M6A+L81V+S95C symmetric (construct in red frame), M1I+M6A+S95C/L81V+S95C asymmetric (gray frame), and M1I+M6A+S95C/S95C asymmetric (green frame) CPs. The positions of these residues are indicated in the expanded structure shown in C, Right, by the blue spheres, with the red spheres denoting side-chain atoms of the mutated L81 residue. Surrounding α- and β-subunits are shown as yellow and white surfaces, respectively, as highlighted in the full structure on the Left.