Protein-Protein Interactions as a Tool for Site-Specific Labeling of Proteins
Tech ID: 21720 / UC Case 2005-314-0
UCLA investigators have developed a method of utilizing protein-protein interactions to site-specifically label recombinantly expressed multi-Cys proteins. Being able to site-specifically label a donor and acceptor fluorophore is a critical component for the use of fluorescence resonance energy transfer analysis in monitoring protein folding reactions. This new invention accomplishes all of the necessary requirements for such labeling.
Fluorescence resonance energy transfer (FRET) between a single donor fluorophore and a complementary single acceptor fluorophore is a powerful and sensitive method for monitoring protein folding reactions at single molecule resolution. It can be used as a distance ruler to track intrachain-conformational dynamics in polypeptide chains in the 2 to 8 nm range.
However, a critical component in this experiment is the ability to label a polypeptide chain with a unique donor/acceptor fluorophore pair in a controlled and site-specific way. Current methods of labeling polypeptide chains have certain limitations. Chemical synthesis can be exploited to facilitate site-specific 2-color labeling, but run into difficulties for 3-color labeling or for proteins longer than 100 amino acids. Recombinant expression of proteins using Cys residues can be used with longer proteins, but is not strictly site-specific. This can lead to unwanted sample heterogeneity and preclude 3- or multi-color FRET experiments. Finally, a novel method of site-specific incorporation of non-natural amino acids into proteins in vivo using genetically modified orthogonal t-RNA/t-RNA synthetase pairs is powerful but not yet broadly available to the scientific community. Thus, there is a need for a new method of labeling a polypeptide chain without all of the limitations.
UCLA investigators have developed a novel method of utilizing protein-protein interactions to site-specifically label recombinantly expressed multi-Cys proteins. An engineered Cys side chain can be physically protected from conjugation with extrinsic fluorophores by the burial of surface area in the protein-protein interaction. Meanwhile, a second Cys remains solvent-exposed after complex formation and can be selectively labeled in the pre-assembled complex. By utilizing the surface area burial in protein-protein interactions, this invention can be performed in simple batch-mode without a need to separate singly-labeled proteins from non- or doubly-labeled side-products. Thus, it allows for simple site-specific labeling of recombinant proteins for FRET-based single molecule studies.
- Site-specific labeling of a polypeptide chain with a unique donor/acceptor fluorophore pair for FRET monitoring of protein folding reactions.
- Rapid optimization of dye pairs (i.e., to optimize the FRET-efficiencies for protein folding studies).
- Possible development into a general method for site-specific protein labeling.
- Allows for site-specific labeling and can be used with longer proteins.
- Can be performed in simple batch-mode.
- No need for time-consuming two-step chromatography to separate singly-labeled proteins from non- or doubly-labeled side-products.
- Can be used when sequential labeling does not work.
- Allows for 3-color labeling of recombinantly expressed proteins.
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