Site-specific protein modification

• US 7,029,846 B2
• Filed: 10/04/2001
• Issued: 04/18/2006
• Est. Priority Date: 10/04/2000
• Status: Expired due to Fees

• Current Assignee: Rutgers University
• Original Assignee: University of Medicine and Dentistry of New Jersey (State of New Jersey)
• Inventors: Rana, Tariq M., Tamilarasu, Natarajan
• Primary Examiner(s): Campell, Bruce R.
• Assistant Examiner(s): Lukton, David
• Application Number:
  US09/972,016
• Publication Number:
  US 20020106767A1
• Time in Patent Office: 1,657 Days
• Field of Search: 435/6, 514/2, 436/172, 530/345
• US Class Current: 435/6.13
• CPC Class Codes: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C07K 1/113 without change of the primary structure
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C12N 2740/16322 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Abstract

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.

Claims

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.