Diazirine-based amino acid analogs for real-time crosslinking of protein interactions in living cells.

Thermo Scientific L-Photo-Leucine and L-Photo-Methionine are analogs of L-Leucine and L-Methionine amino acids that can be endogenously incorporated into the primary sequence of proteins during synthesis and then ultraviolet light (UV)-activated to covalently crosslink proteins within protein-protein interaction domains in their native environment within live cells. The powerful method enables both stable and transient protein interactions in cells to be studied and characterized without the use of completely foreign chemical crosslinkers and associated reagent solvents during the interaction experiment that can adversely aspects of the cell biology being studied.

Structures of L-Photo-Leucine and L-Photo-Methionine and their natural analogs.

Highlights of Diazirine Amino Acids:

• In vivo labeling – incorporate photo-reactive group into proteins using normal cellular machinery of live cells
• In vivo crosslinking – find interacting proteins in the native cellular environment
• Increased specificity – crosslink interacting proteins correctly positioned at their interfaces within protein interaction domains
• Efficient recovery – 90% protein recovery in cell lysates after crosslinking
• Compatible – crosslink proteins expressed in a wide variety of cell lines, including HeLa, 293T, COS7, U2OS, A549, A431, HepG2, NIH 3T3 and C6
• Easy to use – reagents are photo-stable under normal laboratory lighting, eliminating the need to work in the dark

How Photo-Amino Acid Crosslinking Works:

Protocol summary for protein interaction experiments with Photo-activated Amino Acids.

When used in combination with specially formulated limiting media that is devoid of leucine and methionine, the photo-activatable derivatives are treated like naturally occurring amino acids by the protein synthesis machinery within the cell. As a result, they can be substituted for leucine or methionine in the primary sequence of proteins during catabolism and growth. Photo-Leucine and Photo-Methionine derivatives contain diazirine rings that activate when exposed to UV light to become reactive intermediates that form covalent bonds with nearby protein side chains and backbones. Naturally associating binding partners within the cell can be instantly trapped by photoactivation of the diazirine-containing proteins in the cultured cells. Crosslinked protein complexes can be detected by decreased mobility on SDS-PAGE followed by Western blot detection, size exclusion chromatography, sucrose density gradient sedimentation or mass spectrometry.

Photo-reactive amino acid crosslinking and formaldehyde crosslinking are complementary techniques for protein interaction analysis. HeLa cells were mocktreated (Lane 1), treated with 1% formaldehyde for 10 minutes (Lane 2), or treated with Photo-Methionine and Photo-Leucine followed by UV treatment (Lane 3). Cells were lysed and 10 mg of each was analyzed by SDS-PAGE and Western blotting with antibodies against HSP90, STAT3, Ku70 and Ku86. Lower panels are b-actin (upper band) and GAPDH (lower band), which were blotted as loading controls.

Related Products and Resources:

Photoreactive Amino Acid FAQ
Formaldehyde, methanol-free
Crosslinkers
Label Transfer Reagents
UV Lamps for Photo-Crosslinkers

References:

1. Bomgarden R.D., et al. (2007). In vivo crosslinking strategies to identify and characterize transcription factor protein complexes.[abstract] In: Proceedings of the 100th Annual Meeting of the American Association for Cancer Research; 2007 Apr 14-18; Los Angeles, CA. Philadelphia (PA): AACR; 2007. p 141. Abstract nr 1192.
2. Suchanek, M., et al. (2005). Photo-leucine and photo-methionine allow identification of protein-protein interactions. Nat. Methods 2(4):261-267.
3. Vila-Perello, M., et al. (2007). Covalent capture of phospho-dependent protein oligomerization by site-specific incorporation of a diazirine photo-cross-linker. J. Am. Chem. Soc., 129(26):8068 -8069.

Note: All four of the following reagents are required for the procedure.