Optical control of protein activity and localization by fusion to photochromic protein domains

Abstract

Engineered fusion proteins comprising photochromic protein domains are disclosed. In particular, the inventors have constructed fusion proteins containing photoswitchable photochromic fluorescent protein domains linked to selected proteins and shown that such fusion proteins can be used to control the activity or localization of selected proteins with light.

What is claimed is: 
     
         1 . A method for controlling the activity of a selected polypeptide of interest with light, the method comprising:
 a) preparing a fusion protein comprising at least two photochromic polypeptides connected to a selected polypeptide of interest, wherein a first photochromic polypeptide is connected to the N-terminus of the selected polypeptide of interest and a second photochromic polypeptide is connected to the C-terminus of the selected polypeptide of interest, wherein the first photochromic polypeptide and the second photochromic polypeptide are capable of associating with each other, wherein the oligomerization state of the first photochromic polypeptide and the second photochromic polypeptide is controllable with light, wherein at least one photochromic polypeptide is an rsTagRFP polypeptide or an mApple polypeptide; and   b) illuminating the fusion protein with light at a wavelength that induces intramolecular dimerization of the first photochromic polypeptide and the second photochromic polypeptide, such that the activity of the selected polypeptide of interest is inactivated.   
     
     
         2 . The method of  claim 1 , further comprising illuminating the fusion protein with light at a wavelength that induces dissociation of the first photochromic polypeptide from the second photochromic polypeptide, such that the activity of the selected polypeptide is restored. 
     
     
         3 . The method of  claim 1 , further comprising visualizing the localization of the selected polypeptide by detecting fluorescence of the fusion protein resulting from the dimerization of the first photochromic polypeptide and the second photochromic polypeptide. 
     
     
         4 . The method of  claim 1 , further comprising detecting inactivation of the selected polypeptide by measuring fluorescence from dimerization of the first photochromic polypeptide and the second photochromic polypeptide. 
     
     
         5 . The method of  claim 1 , further comprising detecting inactivation of the selected polypeptide by measuring the activity of the selected polypeptide. 
     
     
         6 . The method of  claim 1 , wherein fluorescence of the fusion protein is detected by a fluorimeter, a fluorescence microscope, a fluorescence microplate reader, a fluorometric imaging plate reader, or fluorescence-activated cell sorting. 
     
     
         7 . The method of  claim 1 , wherein the first photochromic polypeptide or the second photochromic polypeptide comprises:
 a) an amino acid sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:9; or   b) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:7 and SEQ ID NO:9, wherein the polypeptide has fluorescence and oligomerization characteristics.   
     
     
         8 . A method for controlling the localization of a selected polypeptide of interest with light, the method comprising:
 a) preparing a first fusion protein comprising a photochromic polypeptide connected to a targeting sequence;   b) preparing a second fusion protein comprising a photochromic polypeptide connected to the selected polypeptide of interest;   c) introducing the first fusion protein and the second fusion protein into a cell, wherein the localization sequence targets the first fusion protein to a particular subcellular location;   d) illuminating the fusion proteins with light at a wavelength that induces oligomerization of the photochromic polypeptide in the first fusion protein with the photochromic polypeptide in the second fusion protein, such that the selected polypeptide accumulates at the subcellular location.   
     
     
         9 . The method of  claim 8 , further comprising illuminating the fusion proteins with light at a wavelength that induces dissociation of the photochromic polypeptides, such that the selected polypeptide in the second fusion protein is released from the subcellular location. 
     
     
         10 . The method of  claim 8 , further comprising visualizing the localization of the selected polypeptide by detecting fluorescence of the fusion proteins resulting from the oligomerization of the photochromic polypeptides. 
     
     
         11 . The method of  claim 8 , wherein the targeting sequence is selected from the group consisting of a secretory protein signal sequence, a membrane protein signal sequence, a nuclear localization sequence, a nucleolar localization signal sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, and a protein binding motif sequence. 
     
     
         12 . The method of  claim 8 , wherein fluorescence of the fusion proteins are detected by a fluorometer, a fluorescence microscope, a fluorescence microplate reader, a fluorometric imaging plate reader, or fluorescence-activated cell sorting. 
     
     
         13 . The method of  claim 8 , wherein the photochromic polypeptide in the first fusion protein and the photochromic polypeptide in the second fusion protein are selected from the group consisting of a Dronpa polypeptide, a Padron polypeptide, an rsTagRFP polypeptide, and an mApple polypeptide. 
     
     
         14 . The method of  claim 13 , wherein the photochromic polypeptide of the first fusion protein or the second fusion protein comprises:
 a) an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9; or   b) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9.   
     
     
         15 . The method of  claim 13 , wherein the photochromic polypeptide in the first fusion protein is a Dronpa-145N polypeptide or a Padron-145N polypeptide. 
     
     
         16 . The method of  claim 13 , wherein the photochromic polypeptide in the first fusion protein is a Dronpa-145K polypeptide. 
     
     
         17 . The method of  claim 13 , wherein the photochromic polypeptide in the second fusion protein is a Dronpa-145N polypeptide or a Padron-145N polypeptide. 
     
     
         18 . The method of  claim 13 , wherein the photochromic polypeptide in the second fusion protein is a Dronpa-145K polypeptide. 
     
     
         19 . A method for controlling the localization of a selected polypeptide of interest with light, the method comprising:
 a) preparing a fusion protein comprising a photochromic polypeptide, a targeting sequence, and the selected polypeptide of interest;   b) introducing the fusion protein into a cell, wherein the localization sequence targets the fusion protein to a particular subcellular location;   c) illuminating the fusion protein with light at a wavelength that induces oligomerization of the photochromic polypeptide in the fusion protein with photochromic polypeptides in other fusion proteins of the same type, said fusion proteins comprising the selected polypeptide of interest, such that the selected polypeptide of interest accumulates at the subcellular location.   
     
     
         20 . The method of  claim 19 , further comprising illuminating the fusion protein with light at a wavelength that induces dissociation of the photochromic polypeptides, such that the selected polypeptide of interest in the fusion protein is released from the subcellular location. 
     
     
         21 . The method of  claim 19 , further comprising visualizing the localization of the selected polypeptide of interest by detecting fluorescence of the fusion protein resulting from the oligomerization with the photochromic polypeptides of the other fusion proteins. 
     
     
         22 . The method of  claim 19 , wherein the targeting sequence is selected from the group consisting of a secretory protein signal sequence, a membrane protein signal sequence, a nuclear localization sequence, a nucleolar localization signal sequence, an endoplasmic reticulum localization sequence, a peroxisome localization sequence, a mitochondrial localization sequence, and a protein binding motif sequence. 
     
     
         23 . The method of  claim 19 , wherein the photochromic polypeptide of the fusion protein comprises an amino acid sequence selected from the group consisting of a Dronpa polypeptide, a Padron polypeptide, an rsTagRFP polypeptide, and an mApple polypeptide. 
     
     
         24 . The method of  claim 23 , wherein the photochromic polypeptide of the fusion protein comprises:
 a) an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9; or   b) an amino acid sequence having at least 95% identity to an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 3, 5, 7, and 9, wherein the photochromic polypeptide has fluorescence and oligomerization characteristics.   
     
     
         25 . The method of  claim 23 , wherein the photochromic polypeptide of the fusion protein is a Dronpa-145N polypeptide or a Padron-145N polypeptide. 
     
     
         26 . The method of  claim 23 , wherein the photochromic polypeptide of the fusion protein is an mApple-162H-164A polypeptide.