Computational method for designing enzymes for incorporation of amino acid analogs into proteins

Abstract

The instant invention provides methods, reagents, and computational tools for designing non-natural substrate analogs for enzymes, especially for designing unnatural amino acid analogs for aminoacyl tRNA Synthetases (AARSs), such as the Phe tRNA Synthetase. The instant invention also provides methods to incorporate unnatural amino acid analogs, especially those with interesting functional groups, into protein products to generate proteins of modified or novel functions.

1 . A method for generating a mutant aminoacyl tRNA synthetase (AARS), comprising: 
 (i) providing the coordinates for a plurality of different rotamers of an amino acid analog resulting from varying torsional angles;    (ii) providing a set of structure coordinates for amino acid residues that define a binding pocket for an aminoacyl tRNA synthetase;    (iii) modeling interactions of said rotamers with said binding pocket and identifying non-bond interactions between said residues of said binding pocket and said rotamers;    (iv) altering one or more amino acid residues in said binding pocket to produce one or more sets of structure coordinates that define altered binding pockets for mutants of said aminoacyl tRNA synthetases;    (v) modeling interactions of said rotamers with said one or more altered binding pockets; and,    (vi) generating a set of optimized aminoacyl tRNA synthetases sequences having favorable interactions with said amino acid analog.    
     
     
         2 . The method of  claim 1 , wherein the step generating a set of optimized aminoacyl tRNA synthetases sequences includes a Dead-End Elimination (DEE) computation to remove rotamers from said rotamer library and use in said modeling steps.  
     
     
         3 . The method of  claim 1 , where the amino acid analog has a non-naturally occurring sidechain.  
     
     
         4 . The method of  claim 1 , where the amino acid analog is a  D -enantiomer.  
     
     
         5 . A method for generating a mutant aminoacyl tRNA synthetase (AARS), comprising: 
 (i) providing a set of structure coordinates for amino acid residues that define a binding pocket for a mutant aminoacyl tRNA synthetase, which binding pocket varies by at least one residue from the sequence of the wild-type form of said aminoacyl tRNA synthetase;    (ii) providing a rotamer library of a plurality of amino acid analogs which represents the coordinates for a plurality of different conformations of each of said analogs resulting from varying torsional angles;    (iii) modeling interactions of said rotamers with said binding pocket and identifying non-bond interactions between residues of the binding pocket and the amino acid analogs;    (iv) identifying amino acid analogs that have favorable interactions with said mutant aminoacyl tRNA synthetase.    
     
     
         6 . An apparatus for generating a mutant aminoacyl tRNA synthetase (AARS), said apparatus comprising: 
 (i) means for providing the coordinates for a plurality of different rotamers of an amino acid analog resulting from varying torsional angles;    (ii) means for providing a set of structure coordinates for amino acid residues that define a binding pocket for an aminoacyl tRNA synthetase;    (iii) means for modeling interactions of said rotamers with said binding pocket and identifying non-bond interactions between said residues of said binding pocket and said rotamers;    (iv) means for altering one or more amino acid residues in said binding pocket to produce one or more sets of structure coordinates that define altered binding pockets for mutants of said aminoacyl tRNA synthetases;    (v) means for modeling interactions of said rotamers with said one or more altered binding pockets; and,    (vi) means for generating a set of optimized aminoacyl tRNA synthetases sequences having favorable interactions with said amino acid analog.    
     
     
         7 . A computer system for use in generating a mutant aminoacyl tRNA synthetase (AARS), said computer system comprising computer instructions for: 
 (i) providing the coordinates for a plurality of different rotamers of an amino acid analog resulting from varying torsional angles;    (ii) providing a set of structure coordinates for amino acid residues that define a binding pocket for an aminoacyl tRNA synthetase;    (iii) modeling interactions of said rotamers with said binding pocket and identifying non-bond interactions between said residues of said binding pocket and said rotamers;    (iv) altering one or more amino acid residues in said binding pocket to produce one or more sets of structure coordinates that define altered binding pockets for mutants of said aminoacyl tRNA synthetases;    (v) modeling interactions of said rotamers with said one or more altered binding pockets; and,    (vi) generating a set of optimized aminoacyl tRNA synthetases sequences having favorable interactions with said amino acid analog.    
     
     
         8 . A computer-readable medium storing a computer program executable by a plurality of server computers, the computer program comprising computer instructions for: 
 (i) providing the coordinates for a plurality of different rotamers of an amino acid analog resulting from varying torsional angles;    (ii) providing a set of structure coordinates for amino acid residues that define a binding pocket for an aminoacyl tRNA synthetase;    (iii) modeling interactions of said rotamers with said binding pocket and identifying non-bond interactions between said residues of said binding pocket and said rotamers;    (iv) altering one or more amino acid residues in said binding pocket to produce one or more sets of structure coordinates that define altered binding pockets for mutants of said aminoacyl tRNA synthetases;    (v) modeling interactions of said rotamers with said one or more altered binding pockets; and,    (vi) generating a set of optimized aminoacyl tRNA synthetases sequences having favorable interactions with said amino acid analog.    
     
     
         9 . A computer data signal embodied in a carrier wave, comprising computer instructions for: 
 (i) providing the coordinates for a plurality of different rotamers of an amino acid analog resulting from varying torsional angles;    (ii) providing a set of structure coordinates for amino acid residues that define a binding pocket for an aminoacyl tRNA synthetase;    (iii) modeling interactions of said rotamers with said binding pocket and identifying non-bond interactions between said residues of said binding pocket and said rotamers;    (iv) altering one or more amino acid residues in said binding pocket to produce one or more sets of structure coordinates that define altered binding pockets for mutants of said aminoacyl tRNA synthetases;    (v) modeling interactions of said rotamers with said one or more altered binding pockets; and,    (vi) generating a set of optimized aminoacyl tRNA synthetases sequences having favorable interactions with said amino acid analog.    
     
     
         10 . An apparatus comprising a computer readable storage medium having instructions stored thereon for: 
 (i) accessing a datafile representative of the coordinates for a plurality of different rotamers of an amino acid analog resulting from varying torsional angles;    (ii) accessing a datafile representative of a set of structure coordinates for amino acid residues that define a binding pocket for an aminoacyl tRNA synthetase;    (iii) a set of modeling routines for 
 (a) calculating interactions of said rotamers with said binding pocket and identifying non-bond interactions between residues of the binding pocket and the amino acid analog;  
 (b) altering one or more amino acid residues in the binding pocket for the aminoacyl tRNA synthetase to produce one or more sets of structure coordinates that define altered binding pockets for mutants of said aminoacyl tRNA synthetases;  
 (c) calculating interactions of said rotamers with said one or more altered binding pockets; and  
 (d) generating a list representative of optimized aminoacyl tRNA synthetases sequences having favorable interactions with said amino acid analog.