Spatio-temporal control of protein interactions using phytochromes

摘要

The invention provides methods, materials and systems of regulating association between proteins of interest using light. In an aspect, the invention takes advantage of the ability of phytochromes to change conformation upon exposure to appropriate light conditions, and to bind in a conformation-dependent manner to cognate proteins called phytochrome-interacting factors. The invention comprises a method of regulating interaction between a first protein of interest and second protein within a cell by light. Such a method optionally comprises providing in the cell (1) a first protein construct which comprises the first protein, a phytochrome domain (PHD), and (2) providing in the cell a second protein construct which comprises the second protein and a phytochrome domain-interacting peptide (PIP) that can bind selectively to the Pfr state, but not to the Pr state, of the phytochrome domain.

主权项

1. A method of spatially and/or temporally regulating an interaction between a first protein sequence and a second protein sequence within a cell by light, comprising:
a) providing in the cell a first protein construct which comprises the first protein sequence and a phytochrome domain (PHD), wherein the phytochrome domain:
(i) can reversibly convert from a red-light absorbing conformer (Pr state) to a far-red light-absorbing conformer (Pfr state) upon exposure to red light, and(ii) can reversibly convert from the Pfr state to the Pr state upon exposure to infra-red light; b) providing in the cell a second protein construct which comprises the second protein sequence and a phytochrome domain-interacting peptide (PIP) that can bind selectively to the Pfr state, but not to the Pr state, of the phytochrome domain; and c) regulating the interaction between the first protein construct and the second protein construct by regulating cell exposure to red light and/or infra-red light, and d) detecting a change in the interaction between the first protein construct and the second protein construct within the cell, wherein:
(i) the PHD comprises a partial fragment of PhyB from Arabidopsis thaliana, and the PIP comprises the APB domain of PIF6; or(ii) the first protein construct or second protein construct can be optically detected, and the change in interaction is detected by optically detecting its spatial distribution; or(iii) the change in interaction can be detected within one minute of exposure to regulatory red or infra-red light; or(iv) a portion of the cell is exposed to regulatory red or infra-red light and the change in interaction is spatially localized within a few microns of the exposed portion.