1 research outputs found
Tuning the Binding Affinities and Reversion Kinetics of a Light Inducible Dimer Allows Control of Transmembrane Protein Localization
Inducible
dimers are powerful tools for controlling biological
processes through colocalizing signaling molecules. To be effective,
an inducible system should have a dissociation constant in the “off”
state that is greater (i.e., weaker affinity) than the concentrations
of the molecules that are being controlled, and in the “on”
state a dissociation constant that is less (i.e., stronger affinity)
than the relevant protein concentrations. Here, we reengineer the
interaction between the light inducible dimer, iLID, and its binding
partner SspB, to better control proteins present at high effective
concentrations (5–100 μM). iLID contains a light-oxygen-voltage
(LOV) domain that undergoes a conformational change upon activation
with blue light and exposes a peptide motif, ssrA, that binds to SspB.
The new variant of the dimer system contains a single SspB point mutation
(A58V), and displays a 42-fold change in binding affinity when activated
with blue light (from 3 ± 2 μM to 125 ± 40 μM)
and allows for light-activated colocalization of transmembrane proteins
in neurons, where a higher affinity switch (0.8–47 μM)
was less effective because more colocalization was seen in the dark.
Additionally, with a point mutation in the LOV domain (N414L), we
lengthened the reversion half-life of iLID. This expanded suite of
light induced dimers increases the variety of cellular pathways that
can be targeted with light