smFRET WITH MEMBRANE PROTEINS

Abstract

This disclosure is directed to methods of conducting dynamic single-molecule fluorescence studies such as sm-FRET on a membrane protein which permits observation and quantification of conformational dynamics of a membrane protein. Also disclosed herein are mutant membrane proteins in which one or more mutations have been introduced for affixing a fluorophore, as well as reagents and kits containing such mutant membrane proteins for conducting dynamic single-molecule fluorescence studies. The methods and compositions disclosed herein can be used in screening for compounds that enhance or reduce the activity of a membrane protein, useful for treating diseases associated with the malfunction of the membrane protein or alterations in membrane protein conformation.

What is claimed is: 
     
         1 . A method of conducting dynamic single-molecule fluorescence studies on a membrane protein comprising:
 a) obtaining a membrane protein which is labeled with fluorophore at one or more sites, and is placed in a membrane protein carrier;   b) immobilizing said membrane protein or said carrier onto a solid surface;   c) imaging the immobilized membrane protein to acquire fluorescence data over a period of time; and   d) correlating the fluorescence data with conformational changes in said membrane protein.   
     
     
         2 . The method of  claim 1  wherein the dynamic single-molecule fluorescence studies are dynamic single-molecule fluorescence resonance energy transfer (smFRET) studies. 
     
     
         3 . The method of  claim 2  wherein at least two mutations have been introduced into said membrane protein to permit affixation of a donor fluorophore and an acceptor fluorophore. 
     
     
         4 . The method of  claim 3  wherein the two mutations are introduced into sites that increase or decrease in distance from each other depending on the conformation of said membrane protein. 
     
     
         5 . The method of  claim 4  wherein the two mutations are introduced into amino acid positions that are separated from one another in the protein tertiary structure by a distance approximating the R 0  for the donor fluorophore and acceptor fluorophore. 
     
     
         6 . The method of  claim 3  wherein the two mutations are introduced into sites that show low conservation between members of the family of proteins of which said membrane protein is a member. 
     
     
         7 . The method of  claim 3  wherein the mutations are introduced into sites that are outside of the activity domains of said membrane protein and do not affect ligand binding or transport activity of said membrane protein. 
     
     
         8 . The method of  claim 3  wherein at least one additional mutation is introduced to a site within an activity domain of said membrane protein to generate a gain-of-function or loss-of-function mutant protein. 
     
     
         9 . The method of  claim 3  wherein the mutations are cysteine substitutions. 
     
     
         10 . The method of  claim 9  wherein the fluorophores are maleimide dyes. 
     
     
         11 . The method of  claim 1  wherein said carrier is selected from the group consisting of: detergents, lipids, nanoparticles, micelles, liposomes, and cells. 
     
     
         12 . The method of  claim 1  wherein said membrane protein is biotinylated and immobilized by the biotin-streptavidin interaction to said solid surface. 
     
     
         13 . The method of  claim 2 , wherein fluorescence data is acquired for an extended imaging time period sufficient to observe multiple conformations of said membrane proteins and transitions therebetween. 
     
     
         14 . The method of  claim 13 , wherein the acquired data permits the identification of distinct conformations. 
     
     
         15 . The method of  claim 14 , wherein the acquired data permits determination of the FRET value for each conformation, the dwell time for each conformation, the transition time from one conformation to another, and the distribution among different conformations. 
     
     
         16 . A mutant membrane protein comprising one or more mutations introduced for affixing a fluorophore. 
     
     
         17 . The mutant membrane protein of  claim 16  comprising two mutations for affixing a donor fluorophore and an acceptor fluorophore. 
     
     
         18 . The mutant membrane protein of  claim 16  further comprising at least one additional mutation which results in a gain of function or loss of function. 
     
     
         19 . The mutant membrane protein of  claim 16  wherein the protein is LeuT and the mutations are selected from the group consisting of cysteine substitutions for His 7 in the N terminus, Arg 86 in IL1 Arg 185 in IL2, Lys 271 in IL3 and Thr 515 at the cytoplasmic end of TM12, Lys 239 in EL3, and His 480 in EL6. 
     
     
         20 . The mutant membrane protein of  claim 18  wherein the protein is LeuT and the mutation is a substitution at one or more of R5, F252, F259, or L400. 
     
     
         21 . The mutant membrane protein of  claim 16  wherein the protein is a neurotransmitter: sodium symporter (NSS) or a G-protein coupled receptor (GPCR). 
     
     
         22 . The mutant membrane protein of  claim 21  wherein the protein is an NSS selected from the group consisting of the dopamine transporter, the serotonin transporter, and the norepinephrine transporter. 
     
     
         23 . The mutant membrane protein of  claim 19  wherein the mutations comprise cysteine substitutions at residues homologous to the following LeuT residues: His 7 in the N terminus, Arg 86 in IL1 Arg 185 in IL2, Lys 271 in IL3 and Thr 515 at the cytoplasmic end of TM12, Lys 239 in EL3, and His 480 in EL6. 
     
     
         24 . A composition comprising the mutant membrane protein of  claim 16  wherein said membrane protein is labeled with a donor fluorophore and an acceptor fluorophore, and has been incorporated into a membrane protein carrier. 
     
     
         25 . The composition of  claim 24  wherein said membrane protein or said protein carrier is immobilized onto a solid support. 
     
     
         26 . A method of screening for compounds that affect the conformational dynamics of a membrane protein, comprising:
 a) conducting dynamic single-molecule fluorescence studies on said membrane protein in the absence of a test compound according to the method of  claim 1 ;   b) conducting dynamic single-molecule fluorescence studies on said membrane protein in the presence of a test compound; and   c) comparing the fluorescence data of said membrane protein in the absence of said test compound with the fluorescence data of said membrane protein in the presence of said test compound, thereby determining whether the test compound affects the conformational dynamics of the membrane protein.   
     
     
         27 . The method of  claim 26 , wherein said dynamic single-molecule fluorescence studies are dynamic smFRET studies. 
     
     
         28 . The method of  claim 27 , wherein the FRET data acquired are used to identify the available conformations of said membrane protein, and determine the FRET value for one or more conformations, the dwell time for one or more conformations, the transition time from one conformation to another, and the relative population of one or more conformations. 
     
     
         29 . The method of  claim 27 , wherein the smFRET studies in a) were performed in the presence of a substrate or ligand of said membrane protein, and the FRET data obtained from identifies at least one specific conformation and its association with one activity of said membrane protein. 
     
     
         30 . The method of  claim 29 , wherein an increase in the relative population or dwell time of said specific conformation among available conformations in the presence of the test compound indicates that the test compound stimulates the activity of the membrane protein, and a decrease in the relative population or dwell time of said specific conformation in the presence of the test compound indicates that the test compound inhibits the activity of the membrane protein.