Assay for identification of therapeutics targeting ternary complex formation in protein synthesis
The present invention provides a novel assay that allows high-throughput screening of chemical compounds for the inhibition of binding between EF-Tu and tRNA.
1. A method of identifying whether a test compound is a potential candidate having antimicrobial activity, said method comprising:
a) mixing an elongation factor Tu (EF-Tu) mutant protein with GTP and an aminoacylated transfer RNA (aa-tRNA) in the presence and absence of a test compound and mixing said EF-Tu mutant protein with GTP and said aa-tRNA in the presence of a control compound, thereby forming a first mixture comprising said EF-Tu mutant protein, GTP, said aa-tRNA and said test compound, a second mixture without said test compound comprising said EF-Tu mutant protein, GTP, and said aa-tRNA, and a third mixture comprising said EF-Tu mutant protein, GTP, said aa-tRNA and said control compound, wherein said EF-Tu mutant protein is operably linked to a first energy transfer pair member, which comprises a fluorescent dye, wherein said aa-tRNA is operably linked to a second energy transfer pair member, which comprises a fluorescent dye, and wherein said first energy transfer pair member and said second energy transfer pair member form a fluorescence resonance energy transfer (“FRET”) pair, wherein said first energy transfer pair member is a FRET donor of the FRET pair and said second energy transfer pair member is a FRET acceptor of the FRET pair or said first energy transfer pair member is a FRET acceptor of the FRET pair and said second energy transfer pair member is a FRET donor of the FRET pair;
b) detecting the fluorescence emitted from the FRET acceptor in each of said first mixture, said second mixture, and said third mixture and comparing the fluorescent level of the FRET acceptor in said first mixture to the fluorescent level of the FRET acceptor in said second mixture and the fluorescent level of the FRET acceptor in said third mixture; and
(c) identifying the potential candidate having antimicrobial activity based on results obtained from the comparing step, wherein a drop in the fluorescent level of the FRET acceptor fluorescence in the first mixture relative to the fluorescent level of the FRET acceptor fluorescence in the second mixture indicates that said test compound interferes with the formation of a ternary complex comprising said EF-Tu mutant protein, GTP, and said aa-tRNA complex, and is a potential candidate having antimicrobial activity; and
wherein said EF-Tu mutant protein has EF-Tu functions, comprises a protein sequence having 90% homology or more than 90% homology to SEQ ID NO:1, and comprises a cysteine at position 81, a non-cysteine residue at position 137, a non-cysteine residue at position 255, and a cysteine at position 348 linking to said first FRET energy transfer member.
2. The method of claim 1 , wherein the second energy pair member is operably linked to position 47 of said aa-tRNA.
3. The method of claim 1 , wherein said first energy pair member is Cy3 and said second energy pair member is Cy5.
4. The method of claim 1 , wherein said first energy pair member is Cy5 and said second energy pair member is Cy3.
5. The method of claim 1 , wherein said EF-Tu mutant protein has three substitutions: C137A, C255V, and E348C.
6. The method of claim 1 , wherein said EF-Tu mutant protein comprises an amino acid sequence having SEQ ID NO:1.
7. The method of claim 1 , wherein said EF-Tu mutant protein comprises sequence having 95% homology or more than 95% homology to SEQ ID NO:1.
8. A protein having EF-Tu functions and comprising a protein sequence having 90% homology or more than 90% homology to SEQ ID NO: 1, wherein the protein further comprises a cysteine at position 81, another cysteine at a position suitable for a FRET energy transfer member for measuring an interaction of the protein with an aa-tRNA by FRET, a non-cysteine residue at position 137, and a non-cysteine residue at position 255, wherein said another cysteine is modified with a fluorescent dye suitable for FRET and is located at position 348.
9. The protein of claim 8 , wherein said protein comprises three substitutions: C137A, C255V, and E348C.
10. The protein of claim 8 , wherein said protein comprises an amino acid sequence having SEQ ID NO: 1.
11. The protein of claim 8 , wherein the protein comprises a sequence having 95% homology to SEQ ID NO: 1.
12. A protein having EF-Tu functions and comprising a protein sequence having 90% homology or more than 90% homology to SEQ ID NO: 1, wherein the protein further comprises a cysteine at position 81, another cysteine at a position suitable for a FRET energy transfer member for measuring an interaction of the protein with an aa-tRNA by FRET, a non-cysteine residue at position 137, and a non-cysteine residue at position 255, and said another cysteine is located at position 348.