Table 1.
Photoswitching effect of different scPvuII variants cross-linked with 4,4′-bis(maleimido)azobenzene
| Type of modification | Variant | Relativeactivitycis/trans |
| Unmodified | sc | 1.0 ± 0.1 |
| sc(C49 C62)2 | 1.0 ± 0.1 | |
| sc(C49 C62)2(F94)2 | 1.0 ± 0.0 | |
| One azomal modification | sc(C49 C62)Azo | 2.2 ± 0.0 |
| sc(C74 C110)Azo | 1.1 ± 0.2 | |
| sc(C66 C96)Azo | 2.3 ± 0.4 | |
| sc(C147 C151)Azo | 1.5 ± 0.0 | |
| sc(C89 C96)Azo | 1.1 ± 0.1 | |
| sc(C129 C133)Azo | 1.1 ± 0.0 | |
| Two azomal modifications | sc(C49 C62)2Azo | 7.0 ± 0.3 |
| sc(C74 C110)2Azo | 3.0 ± 1.0 | |
| sc(C66 C96)2Azo | 3.4 ± 1.0 | |
| sc(C147 C151)2Azo | 1.1 ± 0.2 | |
| Two azomal modifications and additional mutation(s) | sc(C49 C62)2(A83)Azo | 6.2 ± 0.5 |
| sc(C49 C62)2(A83)2Azo | 10.0 ± 2.9 | |
| sc(C49 C62)2(F94)Azo | 15.9 ± 2.3 | |
| sc(C49 C62)2(F94)2Azo | 15.5 ± 2.4 | |
| sc(C49 C62)2(A83)2(F94)Azo | 5.2 ± 1.1 | |
| sc(C49 C62)2(A83)2(F94)2Azo | 2.6 ± 0.2 | |
| One azomal modification involving the linker | sc(C61 L1)Azo | 3.2 ± 0.3 |
| sc(C62 L1)Azo | 1.0 ± 0.1 | |
| sc(C61 L2)Azo | 0.8 ± 0.2 | |
| sc(C61 L3)Azo | 1.9 ± 0.1 | |
| sc(C61 L4)Azo | 2.2 ± 0.4 | |
| Two azomal modifications, one involving the linker | sc(C61 L1)(C49∗ C62∗)Azo | 3.3 ± 0.4 |
| sc(C62 L1)(C49∗ C62∗)Azo | 3.6 ± 0.5 | |
| sc(C61 L2)(C49∗ C62∗)Azo | 1.8 ± 0.9 | |
| sc(C61 L3)(C49∗ C62∗)Azo | 4.0 ± 0.2 | |
| sc(C61 L4)(C49∗ C62∗)Azo | 6.3 ± 0.9 | |
| sc(C61 L4)(C49∗ C62∗)(F94)Azo | 15.7 ± 2.0 | |
| sc(C61 L4)(C49∗ C62∗)(F94)2Azo | 16.3 ± 1.3 | |
| Two modifications in the homodimer | wt(C49 C62)2Azo | 5.2 ± 0.5 |
| wt(C49 C62)2(F94)2Azo | 6.3 ± 0.2 |
All variants produced for this study are indicated with their cysteine substitutions (see Table S1 for further information). The ratio of activities of the azomal-modified proteins in the trans- and cis-state (± standard deviation) are given, as derived from plasmid cleavage assays after blue-light and UV-light preillumination, respectively, under otherwise identical conditions. The nomenclature of the variants is as follows: sc(C49 C62) carries cysteine residues in positions 49 and 62 in the N-terminal half of scPvuII, sc(C49* C62*) carries cysteine residues in the equivalent positions 49 and 62 in the C-terminal half of scPvuII, sc(C49 C62)2 carries cysteine residues in positions 49 and 62 in the N- and C-terminal halves, and sc(C49 C62)Azo is the sc(C49 C62) variant with an azomal cross-link between the two cysteine residues.