Fluorescent protein sensors of post-translational modifications

Abstract

The present invention includes a fluorescent compound that can detect an activity. such as an enzymatic activity, and exhibits quenching. The fluorescent compound can include a fluorescent protein, such as an Aequorea-related green fluorescent protein. The fluorescent compound can include a substrate site for an enzymatic activity such as a kinase activity, a phosphatase activity, a protease activity, and a glycosylase activity The fluorescent compound of the present invention can be used to detect such enzymatic activities in samples, such as biological samples, including cells. The present invention also includes nucleic acids that encode the fluorescent compounds of the present inventions, and cells that include such nucleic acids or fluorescent compounds.

I claim:  
     
         1 . A fluorescent compound for detecting an activity, comprising: 
 a fluorescent protein moiety, and    at least one exogenous substrate recognition motif for an activity,    wherein said fluorescent protein moiety can be converted from a first state to a second state in response to said activity,    further wherein said fluorescent compound exhibits at least one different fluorescent property in said first state and said second state under quenching conditions.    
     
     
         2 . The fluorescent compound of  claim 1 , 
 wherein said activity is an enzymatic activity.    
     
     
         3 . The fluorescent compound of  claim 2 , 
 wherein said enzymatic activity is selected from the group consisting of a kinase activity, a phosphatase activity, a protease activity, a glycosylation activity, and a farnesyl transferase activity.    
     
     
         4 . The fluorescent compound of  claim 1 , 
 wherein said fluorescent protein moiety comprises an Aequorea-related fluorescent protein.    
     
     
         5 . The fluorescent compound of  claim 1 , 
 wherein said fluorescent protein moiety comprises a phosphorylation recognition motif for a protein kinase is a serine/threonine specific protein kinase.    
     
     
         6 . The fluorescent compound of  claim 1 , 
 wherein said fluorescent protein moiety comprises a phosphorylation recognition motif for a protein kinase selected from the group consisting of protein kinase A, a cGMP-dependent protein kinase, protein kinase C,    Ca 2+ /calmodulin-dependent protein kinase I, Ca 2+ /calmodulin-dependent protein kinase II, and MAP kinase activated protein kinase.    
     
     
         7 . The fluorescent compound of  claim 4 , 
 wherein said Aequorea-related fluorescent protein moiety comprises the mutations in GFP mutant K8.    
     
     
         8 . The fluorescent compound of  claim 4 , 
 wherein said at least one exogenous substrate recognition motif for an activity is within the first 20 amino acids of the amino terminus of said Aequorea-related fluorescent protein moiety.    
     
     
         9 . The fluorescent compound of  claim 4 , 
 wherein said at least one exogenous substrate recognition motif for an enzymatic activity is within the first 10 amino acids of the amino terminus of said Aequorea-related fluorescent protein moiety.    
     
     
         10 . The fluorescent compound of  claim 1 , 
 wherein said quenching conditions is acid quenching.    
     
     
         11 . The fluorescent compound of  claim 4 , 
 wherein said Aequorea-related fluorescent protein moiety is membrane associated.    
     
     
         12 . The fluorescent compound of  claim 11 , 
 wherein said Aequorea-related fluorescent moiety comprises a poly-Lys region.    
     
     
         13 . The fluorescent compound of  claim 4 , 
 wherein said Aequorea-related fluorescent protein moiety comprises a protein-protein interaction domain.    
     
     
         14 . The fluorescent compound of  claim 4 , 
 wherein said Aequorea-related fluorescent moiety is membrane bound.    
     
     
         15 . A nucleic acid molecule coding for the expression of a fluorescent compound, 
 wherein said fluorescent compound comprises 
 a fluorescent protein moiety, and  
 at least one exogenous substrate motif for an activity,  
   further wherein said fluorescent protein moiety can be converted from a first state to a second state by an activity,    further wherein said first state and said second state can be differentiated under quenching conditions.    
     
     
         16 . A cell, comprising: 
 a nucleic acid molecule coding for the expression of a fluorescent compound, wherein said fluorescent compound comprises 
 a fluorescent protein moiety, and  
 at least one exogenous substrate motif for an activity,  
   wherein said fluorescent protein moiety can be converted from a first state to a second state by an activity,    further wherein said first state and said second state can be differentiated under quenching conditions.    
     
     
         17 . A method for determining whether a sample contains an activity, comprising: 
 contacting a sample with a fluorescent compound, 
 wherein said fluorescent compound comprises 
 a fluorescent protein moiety, and  
 at least one exogenous substrate motif for an activity,  
 
 further wherein said fluorescent protein moiety can be converted  
 from a first state to a second state by an activity,  
   exciting said fluorescent compound, and    measuring the amount of emission from said fluorescent compound.    
     
     
         18 . The method of  claim 17 , 
 further comprising the step of comparing the amount of emission measured from said fluorescent compound with the emission from a control sample.    
     
     
         19 . The method of  claim 18 , 
 wherein said enzymatic activity is selected from the group consisting of a kinase activity, a phosphatase activity, a protease activity, a glycosylase activity, and a farnsyl transferase activity.    
     
     
         20 . The method of  claim 19 , 
 wherein said quenching is acid quenching.    
     
     
         21 . A method for determining whether a cell exhibits an activity comprising the steps of: 
 exciting a transfected host cell comprising a recombinant nucleic acid molecule, 
 wherein said recombinant nucleic acid molecule comprises at least one expression control sequence operatively linked to a nucleic acid sequence coding for the expression of a fluorescent compound, 
 wherein said fluorescent compound comprises 
 a fluorescent protein moiety, and  
 a substrate motif for an activity,  
 
 
 further wherein said fluorescent compound can be converted from a first state to a second state in the presence of said activity,  
 further wherein said first state and said second state and be differentiated under quenching conditions, and  
   measuring the emission from said fluorescent compound.    
     
     
         22 . The method of  claim 21 , 
 wherein said enzymatic activity is selected from the group consisting of a kinase activity, a phosphatase activity, a protease activity, a glycosylase activity, and a farnsyl transferase activity.    
     
     
         23 . A method for determining whether a sample contains an enzymatic activity, comprising: 
 a) contacting a sample with a fluorescent compound, 
 wherein said fluorescent compound comprises 
 an Aequorea-related fluorescent protein moiety and  
 an exogenous substrate motif for an enzymatic activity,  
 
 further wherein said fluorescent compound can be converted from a first state to a second state by said enzymatic activity,  
 further wherein said first state and said second state can be differentiated under quenching conditions,  
   b) exciting said fluorescent compound, and    c) measuring the amount of fluorescence emitted from said sample, whereby the amount of quenching that is consistent with the presence of said enzyme activity indicates the presence of said enzyme activity in said sample.    
     
     
         24 . A compound identified by the method comprising the steps of: 
 a) contacting a sample with a fluorescent compound, 
 wherein said fluorescent compound comprises 
 a fluorescent protein moiety and  
 an exogenous substrate motif for an activity,  
 
 further wherein said fluorescent compound can be converted from a first state to a second state by said activity,  
 further wherein said first state and said second state can be differentiated under quenching conditions,  
   b) exciting said fluorescent compound, and    c) measuring the amount of fluorescence emitted from said sample.