Methods and systems for detection of stoichiometry by forster resonance energy transfer

Abstract

Methods to detect stoichiometries of protein complexes and/or interactions between proteins based on detection and quantification of FRET and related systems and compositions.

What is claimed is: 
     
         1 . A method to detect one or more stoichiometries of a protein complex, the method comprising:
 providing theoretically calculated Förster resonance energy transfer (FRET) efficiencies for the one or more stoichiometries of the protein complex;   performing FRET imaging on a region of interest expressing fluorescently labeled oligomer subunits of the protein complex to provide an acquired FRET image, the acquired FRET image having a plurality of pixels each pixel having a FRET signal amplitude;   calculating mean FRET efficiencies from the FRET signals amplitudes; and   correlating the calculated mean FRET efficiencies with the theoretically calculated FRET efficiencies to detect the one or more stoichiometries of the protein complex.   
     
     
         2 . The method of  claim 1 , wherein the calculating is performed by compiling a FRET distribution of the identified FRET amplitudes and the correlating is performed by identifying in the FRET distribution, FRET distribution components based on the theoretically calculated FRET efficiencies. 
     
     
         3 . The method of  claim 1 , wherein the region of interest comprises a plurality of regions. 
     
     
         4 . A method to detect one or more stoichiometries of a protein complex, the method comprising:
 performing a Förster resonance energy transfer (FRET) imaging acquisition of a region of interest including said protein complex, thus providing an acquired FRET image comprising a plurality of pixels;   identifying for each pixel a FRET amplitude, thus providing a plurality of identified FRET amplitudes;   compiling a distribution of identified FRET amplitudes as a function of a number of pixels associated with each FRET amplitude to provide a FRET distribution;   identifying in the FRET distribution one or more predetermined FRET components, each component associated via its mean FRET amplitude to a predetermined stoichiometry; and   detecting one or more FRET amplitudes associated with the one or more stoichiometries based on the identified one or more predetermined FRET components.   
     
     
         5 . The method of  claim 4  wherein the one or more stoichiometries are quantified as a percentage of a total population of stoichiometries by dividing the area of the one or more components associated with the one or more stoichiometry by the area of the FRET distribution. 
     
     
         6 . The method of  claim 4  wherein said region of interest is a cell. 
     
     
         7 . The method of  claim 4  wherein said region of interest is a subcellular region in a single cell. 
     
     
         8 . The method of  claim 4  wherein said protein complex is a pentameric Cys-loop receptor. 
     
     
         9 . A method to analyze an acquired FRET image having a plurality of pixels, the method comprising:
 analyzing a signal amplitude of each pixel in the acquired FRET image to provide a FRET distribution comprising a plurality of FRET distribution components; and   calculating for each FRET distribution component a mean FRET amplitude, thus simultaneously providing a plurality of measured mean FRET amplitudes in the acquired FRET image.   
     
     
         10 . A method to analyze a Förster resonance energy transfer (FRET) imaging acquisition comprising:
 providing an acquired FRET image, the acquired FRET image comprising a plurality of pixels; 
 identifying for each pixel a FRET amplitude, thus providing a plurality of identified FRET amplitudes; 
 compiling a distribution of identified FRET amplitudes as a function of a number of pixels associated with each FRET amplitude; 
 identifying in the FRET distribution one or more components, each components associated with a predetermined entity; and 
 detecting one or more mean FRET amplitudes associated with the one or more components based on the identified one or more entities. 
 
     
     
         11 . The method of  claim 10 , wherein the one or more entity is quantified as a percentage of the total population of entities by dividing the area of the one or more components associated with the one or more entities by the area of the distribution. 
     
     
         12 . A method to identify a compound capable of regulating a plasma membrane protein complex, the method comprising
 detecting alternative stoichiometries of the protein complex in a region of interest with the method of  claim 1 ;   quantifying a ratio of the detected alternative stoichiometries of the protein complex in the region of interest;   contacting the protein complex in the region of interest with a candidate compound; and   quantitatively detecting changes in the quantified ratio of detected alternative stoichiometries of the protein complex in the region of interest following contact of the candidate compound.   
     
     
         13 . The method of  claim 12 , wherein the region of interest is formed by a plurality of regions and corresponding changes in various regions of interest are associated with a regulated status of the protein complex. 
     
     
         14 . A method to identify a compound capable of functionally regulating a plasma membrane protein complex, the method comprising:
 providing one or more stoichiometries of the plasma membrane protein complex;   incubating a cell expressing the plasma membrane protein complex with a candidate compound;   quantitatively detecting the one or more stoichiometries of the plasma membrane protein complex following the incubating, the quantification performed with the method of  claim 1 ;   comparing the one or more quantitatively detected plasma membrane protein complex stoichiometries with predetermined quantified stoichiometries associated with a regulation state of the plasma membrane protein complex; and   identifying the candidate compound capable of regulating a plasma membrane protein complex based on the association of the one or more quantitatively detected plasma membrane protein complex stoichiometries with the predetermined quantified stoichiometries associated with the regulation state of the plasma membrane protein complex.   
     
     
         15 . The method of  claim 14  wherein the quantitative detection of the one or more stoichiometries is repeated in increments of time. 
     
     
         16 . The method of  claim 14  wherein the calculating is performed by:
 compiling a distribution of identified FRET amplitudes as a function of a number of pixels associated with each FRET amplitude; 
 
       and the correlating is performed by
 identifying on the FRET distribution one or more components, each component associated with a predetermined stoichiometry; 
 detecting the one or more mean FRET amplitudes associated with the one or more stoichiometries based on the identified one or more components; and 
 quantifying the one or more stoichiometries as a percentage of the total population of stoichiometries by dividing the area of the one or more components associated with the one or more stoichiometry by the area of the distribution. 
 
     
     
         17 . The method of  claim 14  wherein said identified candidate compound is a compound identified as beneficial to the fields of human and veterinary medicine. 
     
     
         18 . The method of  claim 14  wherein said identified candidate compound is an effective smoking cessation therapies, a treatment for Parkinson's Disease or Autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE) or an antihelmintic that targets specific stoichiometries of nAChRs expressed in the nerves and muscle of parasitic nematodes that infect humans or livestock. 
     
     
         19 . A method to detect a functionally regulated plasma membrane protein complex, the method comprising
 identifying one or more stoichiometries of the plasma membrane protein complex;   quantitatively detecting a FRET efficiency of the one or more stoichiometries of the plasma membrane protein complex;   providing a theoretically calculated FRET efficiency of one or more stoichiometries of the protein complex, the one or more stoichiometry associated with functional regulation of the plasma membrane protein complex;   associating the quantitatively detected FRET efficiency of the one or more plasma membrane protein stoichiometries with the theoretically calculated FRET efficiency of the one or more stoichiometries associated with functional upregulation of the plasma membrane protein; and   detecting functional regulation of the plasma membrane protein based on the association between the quantitatively detected FRET efficiency and the theoretically calculated FRET efficiency.   
     
     
         20 . The method of  claim 19  wherein the quantitatively detecting is performed by:
 performing Förster resonance energy transfer (FRET) image acquisition, thus providing an acquired FRET image comprising a plurality of pixels; 
 identifying for each pixel an FRET amplitude, thus providing a plurality of identified NFRET amplitudes; 
 compiling a distribution of identified FRET amplitudes as a function of a number of pixels associated with each FRET amplitude; 
 identifying in the FRET distribution one or more components, each component associated with a predetermined stoichiometry; 
 detecting the one or more mean FRET efficiencies associated with the one or more stoichiometries based on the identified one or more components; and 
 quantifying the one or more stoichiometries as a percentage of the total population of oligomers/complexes by dividing the area of each component associated with the each stoichiometry type by the area of the distribution. 
 
     
     
         21 . A method to detect interactions of protein complexes with another protein, the method comprising:
 detecting one or more stoichiometries of the protein complex with the method of  claim 1 , wherein   identification of mean FRET efficiencies uncorrelated with the theoretically calculated FRET efficiencies indicates interaction with of the protein complex with the another protein.   
     
     
         22 . A computer-readable medium containing a set of instructions that causes a computer to perform the method recited in any one of  claim 1 ,  4 ,  9 ,  10  or  19 . 
     
     
         23 . A computer, comprising the computer-readable medium of  claim 22 . 
     
     
         24 . A system for detection of one or more stoichiometries in a protein complex is described. The system provides at least two of a cell line capable of expressing a protein complex of interest; an expression plasmid capable of expressing fluorescently labeled subunits of the protein complex of interest; a computer readable medium according to  claim 22  and confocal imaging elements.