TABLE 1.
Elucidation of disulfide bonds by target function and intercysteine distances analysis
Characterization of bundles of the 40 best conformers of copsin with different disulfide topologies. The disulfide pattern of the final structure (Fig. 3B) is represented in the top row of the first column for the two methods. Different other conceivable disulfide patterns are listed on following lines, and deviations in cysteine connectivities from the favored one are indicated in bold. Target function, amount of violations, and weights based on averaged Cβ–Cβ distances are shown for the different cysteine pairings. Best target function and weight factors were obtained for the cysteine pairings denoted in the first row (see footnote e).
| Disulfide bond pattern | Target functiona | Violationsa (distancesb/VdWc) | Weight factord |
|---|---|---|---|
| 10–40; 18–48; 22–50; 35–54; 3–32; 25–57e | 0.14 | 0/1 | 0.0648 |
| 10–40; 18–48; 22–35; 50–54; 3–32; 25–57 | 0.18 | 0/1 | 0.0283 |
| 10–40; 18–48; 22–54; 35–50; 3–32; 25–57 | 0.60 | 5/1 | 5.3654e-7 |
| 10–48; 18–40; 22–50; 35–54; 3–32; 25–57 | 0.16 | 0/1 | 1.3644e-4 |
| 10–48; 18–40; 22–35; 50–54; 3–32; 25–57 | 0.20 | 0/1 | 5.9653e-4 |
| 10–48; 18–40; 22–54; 35–50; 3–32; 25–57 | 0.95 | 6/2 | 1.1294e-9 |
| 10–18; 48–40; 22–50; 35–54; 3–32; 25–57 | 0.15 | 0/1 | 0.0044 |
| 10–18; 48–40; 22–35; 50–54; 3–32; 25–57 | 0.22 | 0/1 | 0.0019 |
| 10–18; 48–40; 22–54; 35–50; 3–32; 25–57 | 0.82 | 8/1 | 3.6487e-8 |
| 10–40; 18–22; 48–50; 35–54; 3–32; 25–57 | 0.54 | 3/1 | 1.7643e-5 |
| 10–40; 18–50; 48–22; 35–54; 3–32; 25–57 | 1.29 | 9/2 | 3.1976e-10 |
| 10–40; 18–48; 22–50; 35–54; 3–25; 32–57 | 0.15 | 0/1 | 0.0091 |
| 10–40; 18–48; 22–50; 35–54; 3–57; 32–25 | 0.30 | 5/1 | 7.5591e-5 |
a In target function and violation analysis, for every topology a structure calculation was performed using CYANA (15). The disulfides were given as fixed constraints determined by the standard three upper and lower distance limits (see text). Target function and violations are shown averaged over the 40 selected best energy structures.
b Distance violations.
c van der Waals (VdW) violations.
d Characterization of disulfide bond patterns by cysteine Cβ–Cβ distance analysis as previously described (18). Averaged distances were extracted from the structure ensemble generated without any disulfide constraints. Subsequently weights were assigned to every disulfide pattern. The weight is directly proportional to the likelihood of certain disulfide bond patterns to be realized in the final structure.
e Disulfide bonding connectivity of the final structure as described under “Results.” Variations of single disulfide bonds from this assignment are shown in bold in the first column.