Use of selective labeling to detect and characterize molecular interactions by nuclear magnetic resonance spectroscopy

Abstract

Methods of detecting interactions of a putative ligand with a selectively labeled target molecule, methods of screening for compounds which bind to a selectively labeled target molecule, methods for calculating the dissociation constant of a ligand that binds to a selectively labeled target molecule, and methods to determine the specific amino acids of a target molecule affected by the binding of a ligand, as well as compounds identified by these screening methods, are provided.

What is claimed is:  
     
         1 . A method of detecting binding between a putative ligand and a selectively labeled target molecule, wherein the target molecule comprises a plurality of amino acid moieties including at least one tryptophan moiety, and wherein the tryptophan moiety is labeled, which method comprises: a) generating a first NMR spectrum of said target molecule; b) forming a mixture of said target molecule with at least one putative ligand; c) generating a second NMR spectrum of the mixture of step (b); and d) comparing the first and second spectra.  
     
     
         2 . The method of  claim 1 , wherein the tryptophan moiety is labeled with a nuclei selected from the group consisting of  1 H,  13 C,  15 N,  19 F.  
     
     
         3 . The method of  claim 1 , wherein the selectively labeled target molecule is selected from the group consisting of lipoproteins, lipoprotein fragments, glycoproteins, glycoprotein fragments, proteins, protein fragments, and polypeptides.  
     
     
         4 . The method of  claim 3 , wherein the selectively labeled target molecule is selected from the group consisting of proteins, protein fragments, and polypeptides.  
     
     
         5 . A method of producing a selectively labeled target molecule comprising culturing a transformed cell line containing an expression vector comprising a polynucleotide encoding the target molecule in a medium comprising a labeled tryptophan precursor or a labeled tryptophan moiety.  
     
     
         6 . A method of producing a selectively labeled target molecule comprising culturing a transformed cell line containing an expression vector comprising a polynucleotide encoding the target molecule in a medium comprising a labeled tryptophan moiety and at least one inhibitor of tryptophan synthesis.  
     
     
         7 . The method of  claim 6 , wherein the labeled tryptophan moiety is labeled with a nuclei selected from the group consisting of  1 H,  13 C,  15 N,  19 F.  
     
     
         8 . The method of  claim 6 , wherein the selectively labeled target molecule is selected from the group consisting of lipoproteins, lipoprotein fragments, glycoproteins, glycoprotein fragments, proteins, protein fragments, and polypeptides.  
     
     
         9 . The method of  claim 8 , wherein the selectively labeled target molecule is selected from the group consisting of proteins, protein fragments, and polypeptides.