Crystallographic structure of TcPRACA and uses therefor

Abstract

A method of identifying a substance that affects the biological activity of a Trypanosome cruzi proline racemase (TcPRAC) protein comprises providing a purified TcPRAC protein, providing a substance to be tested, combining the TcPRAC protein with the substance, and determining the effect of the substance on the biological activity of the TcPRAC protein.

1 - 7 . (canceled) 
     
     
         8 . A method of identifying a substance that affects the biological activity of a  Trypanosoma cruzi  proline racemase (TcPRAC) protein comprising:
 providing a purified TcPRAC protein;   providing a substance to be tested;   combining the TcPRAC protein with the substance; and   determining the effect of the substance on the biological activity of the TcPRAC protein.   
     
     
         9 . (canceled) 
     
     
         10 . The method of  claim 8 , wherein the method identifies a substance that affects the infectivity of  Trypanosoma cruzi.    
     
     
         11 . (canceled) 
     
     
         12 . The method of  claim 8 , wherein the method identifies a substance that affects the mitogenicity of the TcPRAC protein. 
     
     
         13 . The method of  claim 8 , wherein the method identifies a substance that affects the parasitic activity of  Trypanosoma cruzi.    
     
     
         14 - 19 . (canceled) 
     
     
         20 . The method of  claim 29 , wherein the method identifies a substance that interacts with residue Cys160 of the TcPRAC protein. 
     
     
         21 . (canceled) 
     
     
         22 . The method of  claim 29 , wherein said method identifies a substance that interacts with residue Cys330 of the TcPRAC protein. 
     
     
         23 . The method of  claim 8 , wherein determining the effect of the substance on the biological activity of the TcPRAC protein comprises:
 (A) combining the TcPRAC protein with the substance in the presence of an equimolar mixture of a L- and D-proline;   (B) oxidatively deaminating any D-proline generated in step (A) by adding a D-amino oxidase and a prosthetic group; and   (C) detecting the hydrogen peroxide generated by the oxidative deamination; wherein modulation of the amount of hydrogen peroxide produced in step (B) compared to the amount of hydrogen peroxide produced using an equal amount of the TcPRAC protein under the same conditions, but in the absence of the substance, is indicative of the capability of the substance to modulate TcPRAC protein activity.   
     
     
         24 . The method of  claim 23 , wherein said substance inhibits the TcPRAC protein racemase activity. 
     
     
         25 . (canceled) 
     
     
         26 . The method of  claim 24 , wherein the racemase activity of the TcPRAC protein in the presence of the substance is 20% or less compared to the activity of the TcPRAC protein in the absence of the substance. 
     
     
         27 . The method of  claim 23 , wherein said substance enhances the TcPRAC protein racemase activity. 
     
     
         28 . The method of  claim 27 , wherein the racemase activity of the TcPRAC protein is at least 80% greater than the activity of the TcPRAC protein in the absence of the substance. 
     
     
         29 . The method of  claim 8 , wherein the method identifies a substance that interacts with the active site of the TcPRAC protein. 
     
     
         30 . The method of  claim 8 , wherein the method identifies a substance that interacts with a site on TcPRAC protein that is not part of the active site. 
     
     
         31 . A crystal of  Trypanosoma cruzi  proline racemase (TcPRAC), wherein the crystal diffracts X-rays determinative of the structure of the TcPRAC protein. 
     
     
         32 . The crystal of  claim 31 , wherein the TcPRAC protein consists of the amino acid sequence of SEQ ID NO:3. 
     
     
         33 . The crystal of  claim 32 , wherein methionine is replaced with selenomethionine. 
     
     
         34 . The crystal of  claim 33 , having a wavelength of 0.9795 {acute over (Å)}, a D min  of 2.9 {acute over (Å)}, a completeness of 98%, a multiplicity of 6.3 and an R sym  of 8.9%. 
     
     
         35 . The crystal of  claim 31 , wherein the TcPRAC protein is complexed with pyrrole-2-carboxylic acid (PYC), and wherein the crystal diffracts X-rays determinative of the structure of the TcPRACA PYC complex. 
     
     
         36 . The crystal of  claim 35 , wherein the TcPRAC protein consists of the amino acid sequence of SEQ ID NO:3. 
     
     
         37 . The crystal of  claim 36 , having a wavelength of 1.0072 {acute over (Å)}, a D min  of 2.1 {acute over (Å)}, a completeness of 98.8%, a multiplicity of 3.7 and an R sym  of 6.9%.