Figure 1. Cross‐section of a small portion of an E. coli cell. The cell wall, with two concentric membranes studded with transmembrane proteins, is shown in green. A large flagellar motor crosses the entire wall, turning the flagellum that extends upwards from the surface. The cytoplasmic area is colored blue and purple. The large purple molecules are ribosomes and the small, L‐shaped maroon molecules are tRNAs, and the white strands are mRNAs. Enzymes are shown in blue. The nucleoid region is shown in yellow and orange, with the long DNA circle shown in yellow, wrapped around HU protein (bacterial nucleosomes). In the center of the nucleoid region shown here, you might find a replication fork, with DNA polymerase (in red/orange) replicating new DNA. (Reproduced with permission. © 1999 David S. Goodsell.)

Figure 2. Ubiquitin–ligand complexes in E. coli. (a) Overlay of – HSQC spectra of E. coli after 3 h of overexpression of U‐‐ubiquitin, and 0 (black), 2 (red), and 3 h (blue) of overexpression of AUIM. Individual peaks exhibiting large chemical shifts are labeled with corresponding assignments. (b) Overlay of – HSQC spectra of free U‐‐ubiquitin (black) and U‐‐ubiquitin–AUIM complexes at a molar ratio of 1 : 1 (red) and 1 : 2 (blue). NMR spectra were acquired at 23 °C. Reproduced with permission from [ 58].

Figure 3. Flowchart to generate a matrix of samples for STINT‐NMR analysis. Reproduced with permission from [ 59].

Figure 4. (Top) Ternary FKBP–FRB‐ligand complexes in E. coli. (a) Overlay of – HSQC spectra of E. coli after 4 h of overexpression of U‐‐FKBP and 4 h of sequential overexpression of FRB in the absence (black) and presence (red) of 150 µM rapamycin. (b) – HSQC spectrum of E. coli after 4 h of overexpression of U‐‐FKBP and 4 h of sequential overexpression of FRB in the presence (red) of 5 mM A–E. NMR spectra were acquired at 23 °C. [ 80]. (Bottom) Yeast assay for biological activity of the dipeptide, A–E. Isogenic haploid yeast strains (S. cerevisiae) that express (FPR1 ⁺) or lack (frp1 ^–) the FKBP proline isomerase were grown for 3 days on yeast extract peptone dextrose (YPD) medium. (c) Control plate. (d) 100 µM rapamycin. Expression of FKBP allows the formation of a toxic FKBP–rapamycin–FRB biocomplex. (e) 5 mM Ala–Glu (A–E). Results indicate that the dipeptide induces formation of a biocomplex similar to that induced by rapamycin. The reduced growth in the frp1 ^– strain likely reflects the weaker affinity of A–E for FKBP. Reproduced with permission from [ 66].

Figure 5. Interaction surface maps of ubiquitin–ligand complexes. Interaction surface of ubiquitin mapped onto the three‐dimensional structure of ubiquitin (Protein Data Bank ID: 1D3Z). Individual residues exhibiting either a chemical shift change above 0.05 ppm or significant differential broadening are indicated in white. All perturbed residues lie on the ubiquitin surface and, therefore, reflect changes in the interaction surface of the molecule rather than changes in tertiary or quaternary structure. (a) STAM2–ubiquitin interaction; (b) HRS–ubiquitin interaction; (c) STAM2–HRS–ubiquitin interaction; (d) phosphorylated STAM2–ubiquitin interaction (YP‐STAM2); (e) phosphorylated HRS–ubiquitin interaction (YP‐Hrs); (f) phosphorylated STAM2–HRS–ubiquitin interaction (YP‐STAM2‐Hrs). Ubiquitin ligands are indicated in each panel. (Reproduced from [ 67].)