Site-specific protein modification
View Patent ↗Site-specific modified proteins and method for producing site-specific modified proteins using amino acid analogs are disclosed. Methods for labeling proteins at a desired site in the presence of nucleophilic side chains, including lysine and cysteine side chains, are also disclosed. Methods for labeling the site-specific modified proteins are also disclosed.
1. A method for determining protein-RNA interactions under simulated physiological conditions, the method comprising:
(a) synthesizing a site specific modified protein, wherein the site specific modified protein comprises a protein modified by replacement of an amino acid, other than lysine or cysteine residues, with an analog of the amino acid, wherein the amino acid analog does not affect a biological activity of the protein;
(b) subsequently site-specifically labeling the site specific modified protein at the site of the amino acid analog with a donor dye molecule, wherein the site-specific labeling is capable of occurring in the absence of orthogonal protection of nucleophilic side chains of lysine and cysteine;
(c) providing a RNA molecule labeled with an acceptor dye molecule, wherein the acceptor dye molecule is capable of participating in fluorescence resonance energy transfer with the donor dye molecule;
(d) measuring the emission of the labeled protein and labeled RNA in (b) and (c) respectively;
(e) combining the labeled RNA in (c) with the labeled protein in (b) to form a mixture;
(f) measuring the emission of the mixture in (e); and,
(g) determining the proximity between the donor dye molecule and the acceptor dye molecule.
2. The method of claim 1 , wherein the dye pair is fluorescein-rhodamine.
3. The method of claim 1 , wherein the site-specific modified protein is an Acetyl-Tyr-Tat peptide having the following sequence: Xaa Gly Mg Lys Lys Mg Arg Gin Mg Mg (SEQ ID NO:3), wherein Xaa is Acetyl-Tyr, which is represented by the following structure:
4. The method of claim 3 , wherein the RNA is TAR RNA.
5. The method of claim 1 , further comprising comparing the emission measurements from (d) and (f) to determine if fluorescence resonance energy transfer has occurred.
6. The method of claim 1 , wherein the replaced amino acid is Tyrosine.
7. The method of claim 6 , wherein the amino acid analog has the following structure:
8. The method of claim 1 , wherein the protein is a Tat peptide represented by SEQ ID NO:2.
9. The method of claim 1 , wherein step (b) comprises conjugating the donor dye molecule to a functional group on the amino acid analog, which is selected from the group consisting of an acetyl group and a formyl group.
10. The method of claim 1 , further comprising examining fluorescence quenching of the labeled protein at different concentrations of labeled RNA.
11. The method of claim 10 , further comprising determining the binding affinity between the protein and the RNA.