Photoaffinity crosslinking

Key words Surface plasmon resonance imaging technique. Small molecule arrays. Photoaffinity crosslinking, High-throughput screening. Sugar array. Signal normalization... 

Most recently, we reported small molecule arrays on photoaffinity crosslinker coated gold surfaces (17). The small molecule arrays were fabricated by photoreaction, and then analyzed by SPR imaging technique. The small molecules don t have to be modified chemically for immobilization. The small molecules, which can interact with a target protein, can be screened by this methodology. Therefore, the integration of photoaffinity small molecule array and SPR imaging technique can be the first step of reverse chemical genetics. 

A photoaffinity crosslinker modified gold chip and 96-well microplate with 15 sugar solutions was placed in a contact pin type automated spotter. 

Figure 2. Specificity of photoaffinity crosslinking of 125-I-p-aminobenzylcarazolol (1) to a purified beta-adrenergic receptor preparation. Arrows to the left indicate positions of iodlnated molecular weight standards. Taken with permission of the copyright owners from ref. 12.

Wooddell, C. I., and Burgess, R. R. (2000). Topology of yeast RNA polymerase II subunits in transcription elongation complexes studied by photoaffinity crosslinking [in process citation]. Biochemistry 39(44), 13405-13421. 

Palladino, D.E. Hopkins, J.L. Ingraham, R.H. Warren, T.C. Kapadia, S.R. Van Moffaert, G.J. Grob, P.M. Stevenson, J.M. Cohen, KA. High-performance liquid chromatography and photoaffinity crosslinking to explore the binding environment of nevirapine to reverse transcriptase of human immunodeficiency virus, J.Chromatogr, 1994, 676, 99-112. 

Figure 8.20 (A) Generic chemical structure of the y-secretase inhibitors described by Seiffert et al. (2000). (B) y-Secretase inhibitor incorporating a benzophenone photoaffinity label for crosslinking studies.

PNP-DTP has been used to photoaffinity label the thyroid hormone nuclear receptors in intact cells by preparing a derivative of 3,5,3 -triiodo-L-thyronine with the crosslinker (Pascual et al., 1982 Casanova et al., 1984). Effective photoreactive conjugation was found to occur after irradiation with UV light at 254 or 310 nm. 

Photochemically reactive molecules have often been used as labels for specific sites in proteins and nucleic acids. Psoralen derivatives serve as relatively nonspecific photochemically activated crosslinking agents for DNA and double-stranded RNA.195 Aryl azides are converted by light to aryl nitrenes, which react in a variety of ways including insertion into C-H bonds (Eq. 23-27).200 201 In some cases UV irradiation can be used to join natural substrates to enzymes or hormones to receptors. For example, progesterone, testosterone, and other steroids have been used for direct photoaffinity labeling of their receptors.202 Synthetic benzophenones have also been used widely as photoactivated probes.203... 

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