299
these 672 linker variants. Library construction was performed in
the context of the pDusk-DsRed reporter plasmid [16] that affords
rapid screening for functional hybrid variants as described in
Subheading 3.2 (see Fig. 3).
Key results from the analysis of the PATCHY library are summarized in the following and in Fig. 5a. Strikingly, light-regulated
activity in BsYtvA-BjFixL hybrids is supported by quite different
linker lengths ranging from 4 to 50 residues, whereas the original
YF1 possesses a linker of 28 residues. Assuming a continuous
α-helical coiled-coil structure of the linker elements, this translates
into distances of separation of ~6–75 Å between LOV photosensor and effector modules. In addition to hybrids with lightrepressed activity as in YF1, several variants showed an inverse
response to light, i.e., they had light-induced activity. Hybrid variants with alike responses to light usually displayed a pronounced
5
Count
Count
0
0
10
2
4
2
6
3
0
4
1
5
2
6
3
0
4
1
5
Helical register m
Helical register m
YF1
Count
0
2
4
2
6
3
0
4
1
5
Helical register m
H22P
a
b
c
D21V
Fig. 5 Linker-length analysis of light-regulated BsYtvA-BjFixL hybrid variants. (a) For YF1, light-repressed variants (filled circles) showed periodic linker lengths of 7n or 7n + 5 residues but light-activated variants (open
circles) mostly occurred at linker lengths of 7n + 1 residues. (b) Light-activated D21V variants had linkers of
7n or 7n + 1 residues. Two variants with linkers belonging to the 7n and 7n + 3 classes showed inverted,
light- repressed response. (c) For H22P, only light-activated variants were identified that predominantly possessed linkers of 7n residues
Generation of Hybrid Gene Libraries