Kinase and ubiquination assays

Abstract

Compositions, including antibodies, polypeptides, and organic molecules, kits, and methods for probing molecular interactions (e.g., deubiquination, ubiquination and kinase activity), e.g., using resonance energy transfer (RET) are provided.

1 . A method for measuring kinase activity of at least one compound, the method comprising: 
 a) contacting the at least one compound and at least one fusion protein to form a test sample, wherein the fusion protein comprises a fluorescent polypeptide and a kinase substrate polypeptide;    b) incubating the test sample under conditions suitable for the kinase activity;    c) contacting the test sample, before, during or after (b), with a binding molecule comprising a label, wherein the binding molecule binds with specificity to the at least one fusion protein containing either an unphosphorylated or phosphorylated substrate and wherein the fluorescent polypeptide and the label are a RET pair;    d) exposing the test sample to at least one wavelength of light; and    e) measuring fluorescence emission from the test sample.    
     
     
         2 . A method for measuring de-ubiquinating activity of at least one compound, the method comprising: 
 a) contacting the compound and at least one fusion protein to form a test sample, wherein the at least one fusion protein comprises: 
 i) a fluorescent polypeptide;  
 ii) a de-ubiquinating enzyme polypeptide substrate; and  
 iii) a label, 
 wherein the fluorescent polypeptide and the label are a RET pair and wherein upon cleavage of the de-ubiquinating enzyme polypeptide substrate, resonance energy transfer between (i) and (iii) is decreased;  
 
   c) exposing the test sample to at least one wavelength of light; and    d) measuring fluorescence emission from the test sample.    
     
     
         3 . A method for identifying a modulator of kinase activity, the method comprising: 
 the method of  claim 2  wherein the compound is a kinase capable of phosphorylating the kinase substrate polypeptide and wherein the test sample further comprises at least one potential modulator of the kinase activity.    
     
     
         4 . A method for identifying a modulator of a de-ubiquinating activity, the method comprising: 
 the method of  claim 2 , wherein the compound has de-ubiquinating activity and is capable of cleaving the de-ubiquinating enzyme polypeptide substrate and wherein the test sample further comprises at least one potential modulator of the de-ubiquinating activity.    
     
     
         5 . An article of manufacture comprising a packaging material, the at least one fusion protein and the at least one binding molecule of  claim 1 .  
     
     
         6 . An article of manufacture comprising a packaging material and the at least one fusion protein of  claim 2 .  
     
     
         7 . The fusion protein of  claim 2 .  
     
     
         8 . A method for measuring ubiquitinating or ubiquitinating-like activity of at least one compound, the method comprising: 
 a) contacting the at least one compound with at least one protein and at least two populations of ubiquitons to form a test sample, wherein one population of ubiquitons comprises an acceptor moiety for a RET pair and a second population is labeled with a donor moiety for a RET pair;    b) exposing the test sample to at least one wavelength of light; and    c) measuring the fluorescence emission from the test sample.    
     
     
         9 . A method for identifying a modulator of a ubiquitinating or ubiquitinating-like activity, the method comprising: 
 the method of  claim 8 , wherein the compound has the ubiquitinating or ubiquitinating-like activity and is capable of ubiquitinating the at least one protein and wherein the test sample further comprises at least one potential modulator of the ubiquitinating or ubiquitinating-like activity.    
     
     
         10 . An article of manufacture comprising: 
 a packaging material and the at least two populations of ubiquitons of  claim 9 .    
     
     
         11 . A method for measuring ubiquitinating activity of at least one compound, the method comprising: 
 a) contacting i) the at least one compound with ii) a ubiquiton and iii) a protein to form a test sample, wherein the protein comprises a ubiquitination substrate and a first moiety of a RET pair;    b) incubating the test sample under conditions suitable for ubiquitination;    c) contacting the test sample either before, during or after (b) with a binding molecule that binds the ubiquiton, wherein the binding molecule is labeled with a second moiety of a FRET pair;    d) exposing the test sample to at least one wavelength of light; and    e) measuring the fluorescence emission from the test sample.    
     
     
         12 . A method for identifying a modulator of a ubiquitinating activity, the method comprising: 
 the method of  claim 11 , wherein the at least one compound has the ubiquitinating activity and is capable of ubiquitinating the at least one protein and wherein the test sample further comprises at least one potential modulator of the ubiquitinating activity.    
     
     
         13 . A method for detecting at least one substrate for at least one ubiquitination or ubiquitination-like enzyme, the method comprising: 
 a) contacting i) the at least one ubiquitination or ubiquitination-like enzyme with ii) polypeptides immobilized on a substrate and iii) at least one ubiquiton comprising a detectable moiety,    b) incubating (a) under conditions to allow for ubiquitination or ubiquitination-like activity,    c) detecting the detectable moiety associated with any of the polypeptides on the substrate.    
     
     
         14 . A method for identifying deubiquitinating activity of a sample, the method comprising: 
 a) contacting i) the sample with ii) at least one or a plurality of polypeptides immobilized on a substrate, wherein the at least one or plurality of polypeptides comprise a ubiquitin or ubiquitin-like protein associated with a detectable moiety, wherein the deubiquitinating activity causes the dissociation of the detectable moiety from the substrate,    b) incubating (a) under conditions to suitable for de-ubiquitination activity,    c) detecting the detectable moiety associated with any of the polypeptides on the substrate.    
     
     
         15 . A method of detecting phosphodiesterase activity, the method comprising: 
 a) contacting i) a sample with ii) a substrate for a phosphodiesterase wherein the substrate comprises a first member of a RET pair to form a test sample;    b) incubating (a) under conditions to suitable for the phosphodiesterase activity;    c) contacting the test sample, either before, during or after (b), with a binding molecule with specificity of a cleavage product of the phosphodiesterase,    wherein the binding molecule comprises a second member of the RET pair,    wherein the cleavage product comprises the first member of a the RET pair;    d) exposing the test sample to at least one wavelength of light; and    e) measuring the fluorescence emission from the test sample.