Analysis of the region between amino acids 30 and 42 of intact UmuD by a monocysteine approach

Regulation of Escherichia coli SOS mutagenesis by dimeric intrinsically disordered umuD gene products

“…Consistent with a flexible structure, cross-linking of single-cysteine derivatives of UmuD 2 by slow, gentle methods such as dialysis shows that most derivatives will cross-link to form covalent UmuD 2 , with only a few positions that react much more or less than average (17). However, certain amino acid positions are consistently more solvent-exposed than others, and faster methods of cross-linking can better distinguish residues that are near the homodimer interface, suggesting that UmuD 2 is likely to have a flexible but nonrandom structure in solution (16)(17)(18). The high-resolution structures of UmuDЈ 2 (13,14) both may have relevance to its structure in vivo, although inside the cell umuD gene products are likely to be surrounded by interaction partners that may influence their actual structure (Fig.…”

“…Little is known about the precise structures of IDPs, although efforts to further characterize them have begun (28). In the case of UmuD 2 and UmuDЈ 2 , a considerable amount of structural information is already available from solution studies at physiologically relevant concentrations (16)(17)(18)29). Consistent with a flexible structure, cross-linking of single-cysteine derivatives of UmuD 2 by slow, gentle methods such as dialysis shows that most derivatives will cross-link to form covalent UmuD 2 , with only a few positions that react much more or less than average (17).…”

“…In the case of UmuD 2 and UmuDЈ 2 , a considerable amount of structural information is already available from solution studies at physiologically relevant concentrations (16)(17)(18)29). Consistent with a flexible structure, cross-linking of single-cysteine derivatives of UmuD 2 by slow, gentle methods such as dialysis shows that most derivatives will cross-link to form covalent UmuD 2 , with only a few positions that react much more or less than average (17). However, certain amino acid positions are consistently more solvent-exposed than others, and faster methods of cross-linking can better distinguish residues that are near the homodimer interface, suggesting that UmuD 2 is likely to have a flexible but nonrandom structure in solution (16)(17)(18).…”

“…Additionally, previous single-cysteine studies show that both proteins have a nonrandom structure at physiological concentrations (16)(17)(18)29). Our results provide a rare opportunity to probe the actual structure of proteins that appear unfolded by CD spectroscopy.…”

“…Previous single-cysteine studies of UmuD 2 , which probed the structure of UmuD 2 in solution at physiologically relevant concentrations, have generally been consistent with our structural models. Single-cysteine derivatives of many amino acids that are predicted to be close to the dimer interface robustly cross-link to covalent dimers (16,17). Interestingly, some positions that are predicted to be far away from the dimer interface also cross-link (16,18).…”