Photo-reactive amino acid analogs are artificial analogs of natural amino acids that can be used for crosslinking of protein complexes. Photo-reactive amino acid analogs may be incorporated into proteins and peptides ''in vivo'' or in ''vitro''. Photo-reactive amino acid analogs in common use are photoreactive diazirine analogs to

leucine Leucine (symbol Leu or L) is an essential amino acid that is used in the biosynthesis of proteins. Leucine is an α-amino acid, meaning it contains an α- amino group (which is in the protonated −NH3+ form under biological conditions), an α- ...

and

methionine Methionine (symbol Met or M) () is an essential amino acid in humans. As the precursor of other amino acids such as cysteine and taurine, versatile compounds such as SAM-e, and the important antioxidant glutathione, methionine plays a critical ...

, and ''para''-benzoylphenylalanine. Upon exposure to

ultraviolet light Ultraviolet (UV) is a form of electromagnetic radiation with wavelength from 10 nm (with a corresponding frequency around 30 PHz) to 400 nm (750 THz), shorter than that of visible light, but longer than X-rays. UV radiatio ...

, they are activated and covalently bind to interacting proteins that are within a few

angstrom The angstromEntry "angstrom" in the Oxford online dictionary. Retrieved on 2019-03-02 from https://en.oxforddictionaries.com/definition/angstrom.Entry "angstrom" in the Merriam-Webster online dictionary. Retrieved on 2019-03-02 from https://www.m ...

s of the photo-reactive amino acid analog. L-Photo-leucine and L-photo-methionine are analogs of the naturally occurring L-

and L-

amino acids that are endogenously incorporated into the primary sequence of proteins during synthesis using the normal translation machinery. They are then ultraviolet light (UV)-activated to covalently crosslink proteins within protein–protein interaction domains in their native ''in-vivo'' environment. The method enables the determination and characterization of both stable and transient protein interactions in cells without the addition of chemical crosslinkers and associated solvents that can adversely affect the

cell biology Cell biology (also cellular biology or cytology) is a branch of biology that studies the structure, function, and behavior of cells. All living organisms are made of cells. A cell is the basic unit of life that is responsible for the living an ...

being studied in the experiment. When used in combination with limiting media that is devoid of leucine and methionine, the photo-activatable derivatives are treated like naturally occurring amino acids by the cellular

protein synthesis Protein biosynthesis (or protein synthesis) is a core biological process, occurring inside cells, balancing the loss of cellular proteins (via degradation or export) through the production of new proteins. Proteins perform a number of critical ...

machinery. As a result, they can be substituted for leucine or methionine in the primary structure of proteins. Photo-leucine and photo-methionine derivatives contain diazirine rings that are activated when exposed to UV light to become reactive intermediates that form covalent bonds with nearby protein side chains and backbones. Naturally interacting proteins within the cell can be instantly trapped by

photoactivation A photoswitch is a type of molecule that can change its structural geometry and chemical properties upon irradiation with electromagnetic radiation. Although often used interchangeably with the term molecular machine, a switch does not perform work ...

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 The western blot (sometimes called the protein immunoblot), or western blotting, is a widely used analytical technique in molecular biology and immunogenetics to detect specific proteins in a sample of tissue homogenate or extract. Besides detect ...

ting, size exclusion chromatography,

sucrose Sucrose, a disaccharide, is a sugar composed of glucose and fructose subunits. It is produced naturally in plants and is the main constituent of white sugar. It has the molecular formula . For human consumption, sucrose is extracted and refine ...

density gradient sedimentation or

mass spectrometry Mass spectrometry (MS) is an analytical technique that is used to measure the mass-to-charge ratio of ions. The results are presented as a '' mass spectrum'', a plot of intensity as a function of the mass-to-charge ratio. Mass spectrometry is u ...

References

* 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):8068–69. * Bomgarden, R. (2008). Studying Protein Interactions in Living Cells. Gen. Eng. News. Vol. 28, No. 7

* P.-O. Hétu, et al. (2008) Photo-crosslinking of proteins in intact cells reveals a dimeric structure of cyclooxygenase-2 and an inhibitor-sensitive oligomeric structure of microsomal prostaglandin E2 synthase-1. Arch. Biochem. Biophys. * Weaver, M.S., et al. (2008) The copper-binding domain of sparc mediates cell survival in vitro via interaction with integrin beta 1 and activation of integrin-linked kinase. J. Biol. Chem. {{doi, 10.1074/jbc.M706563200 Biochemistry methods Biotechnology Proteomics Protein–protein interaction assays