Methods for structural analysis of proteins

Abstract

The invention provides methods and compositions for protein structure analysis, including substrate binding sites, sites of protein-protein interactions, three dimensional structure analysis, and stability, all with single amino acid resolution. In general, the subject methods involve introduction of cysteine residues, which serve as probes for physical analysis, into a protein by translational misincorporation in vivo. In many embodiments, proteins containing misincorporated cysteine residues are reacted with a crosslinking agent that covalently links misincorporated cysteine residues to a proximal amino acid in the folded protein. These methods, termed “MXLINK” methods, may be used for protein tertiary structure analysis. In other embodiments, cysteine-misincorporated proteins are used in protein footprinting methods, termed “MPAX” or “MSX” methods.

1 - 31 . (canceled) 
     
     
         32 . A composition having the molecular structure: 
       
         
           
           
               
               
           
         
         where R is a thiol-reactive group; 
         X is an affinity tag that binds to a solid support; 
         Y is a cross-linking moiety; and 
         L is a linking moiety that links X, Y and R. 
       
     
     
         33 . The composition of  claim 32 , wherein R comprises a haloalkyl acyl group that reacts with a cysteine residue of a protein. 
     
     
         34 . The composition of  claim 32 , wherein R comprises a maleimide or a thiosulfonyl group. 
     
     
         35 . The composition of  claim 32 , wherein X selectively binds a capture agent immobilized on a solid support. 
     
     
         36 . The composition of  claim 35 , wherein X selectively binds said capture agent reversibly. 
     
     
         37 . The composition of  claim 32 , wherein X is a biotin affinity tag. 
     
     
         38 . The composition of  claim 32 , wherein Y is reactive with a hydroxyl group, free amino group, carboxylic group or another reactive functional group other than a cysteine sylfhydryl group in a protein. 
     
     
         39 . The composition of  claim 43 , wherein the reactivity of Y is light activated. 
     
     
         40 . The composition of  claim 32 , wherein L is nitrogen. 
     
     
         41 . The composition of  claim 32 , where L is carbon. 
     
     
         42 . The composition of  claim 32 , wherein the molecular structure is: 
       
         
           
           
               
               
           
         
         and Y is halobenzyl group, and Z is a halide. 
       
     
     
         43 . The composition of  claim 42 , wherein the molecular structure is: 
       
         
           
           
               
               
           
         
         wherein Y is a parahalobenzyl and Z is a halide. 
       
     
     
         44 . The composition of  claim 32 , wherein said composition crosslinks a cysteine to an amino acid that distanced from said cysteine by 1 Å to 30 Å.